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You are an expert at summarizing long articles. Proceed to summarize the following text: there were 504 participants of the parenteral and oral insulin dpt-1 trials included in the analysis . for certain analyses , subgroups of that cohort . estimated 5-year risks of > 50 and 2650% were required for entry into the parenteral and oral insulin trials , respectively . a > 50% 5-year risk estimate was based on a first - phase insulin response from an intravenous glucose tolerance test below a defined threshold and/or the presence of an ogtt abnormality other than diabetes . if those metabolic criteria were not present , but there were insulin autoantibodies , individuals were characterized as having a 2650% 5-year risk . there was no overall effect from the intervention in either trial . the interventions for the parenteral and oral insulin trials were recombinant human ultralente insulin and recombinant human insulin crystals , respectively . fasting samples were obtained before oral glucose administration ( 1.75 g / kg ; maximum 75 g carbohydrate ) and then at 30 , 60 , 90 , and 120 min . if ogtts were in the diabetic range , participants were asked to return for confirmation with another ogtt ( unless contraindicated ) . these included measurements of icas by indirect immunofluorescence and insulin autoantibodies by competitive fluid - phase radioassay . the interassay coefficient of variation for the c - peptide assay was 6.9% in a reference pool with relatively high values and 7.8% in a reference pool with relatively low values . fasting c - peptide values in the undetectable range ( < 0.2 ng / ml ) were assigned a value of 0.1 ng / ml for the analyses . for group and glucose tolerance abnormalities were defined as follows : impaired fasting glucose = fasting glucose value 100125 mg / dl ; indeterminate = 30- , 60- , and/or 90-min glucose value 200 mg / dl ; and impaired glucose tolerance = 2-h glucose value 140199 mg / dl . the thresholds for diabetes were a fasting glucose value 126 mg / dl and/or a 2-h glucose value 200 mg / dl . the sum of c - peptide levels after 30 min was calculated by subtracting the 30-min values from the 60- , 90- , and 120-min values and adding the sum of each of the differences . the first - phase insulin response was defined as the sum of insulin levels at the 1st and 3rd min of the intravenous glucose tolerance test . the interventions for the parenteral and oral insulin trials were recombinant human ultralente insulin and recombinant human insulin crystals , respectively . fasting samples were obtained before oral glucose administration ( 1.75 g / kg ; maximum 75 g carbohydrate ) and then at 30 , 60 , 90 , and 120 min . if ogtts were in the diabetic range , participants were asked to return for confirmation with another ogtt ( unless contraindicated ) . methodologies for assessing autoantibody positivity in dpt-1 have been described ( 9 ) . these included measurements of icas by indirect immunofluorescence and insulin autoantibodies by competitive fluid - phase radioassay . the interassay coefficient of variation for the c - peptide assay was 6.9% in a reference pool with relatively high values and 7.8% in a reference pool with relatively low values . fasting c - peptide values in the undetectable range ( < 0.2 ng / ml ) glucose tolerance abnormalities were defined as follows : impaired fasting glucose = fasting glucose value 100125 mg / dl ; indeterminate = 30- , 60- , and/or 90-min glucose value 200 mg / dl ; and impaired glucose tolerance = 2-h glucose value 140199 mg / dl . the thresholds for diabetes were a fasting glucose value 126 mg / dl and/or a 2-h glucose value 200 mg / dl . the sum of c - peptide levels after 30 min was calculated by subtracting the 30-min values from the 60- , 90- , and 120-min values and adding the sum of each of the differences . the first - phase insulin response was defined as the sum of insulin levels at the 1st and 3rd min of the intravenous glucose tolerance test . a total of 504 dpt-1 participants < 15 years of age were included in the overall study cohort ( n = 504 ; 9.2 3.1 years ; 59% male ) . of these individuals , data were analyzed for sequential glucose and c - peptide levels in 36 ( 9.0 3.1 years ; 58% male ) who progressed to type 1 diabetes ( progressors ) and 80 ( 9.0 3.3 years ; 65% male ) who did not progress to type 1 diabetes ( nonprogressors ) . the progressors had ogtts performed every 6 3 months for at least 2 years before diagnosis , whereas the nonprogressors had ogtts performed every 6 3 months for at least 2 years before the last visit . the progressors and nonprogressors all had normal glucose tolerance at baseline . glucose and c - peptide curves from ogtts performed at baseline and at 2.0 and 0.5 years before diagnosis in the progressors , or at baseline and at 2.0 and 0.5 years before the last visit in the nonprogressors , are shown in fig . 1a ) increased significantly from baseline to 0.5 years at all ogtt time points in both the progressors and nonprogressors , but to a greater extent in the progressors . the increase in the auc glucose from baseline to 0.5 years was highly significant for both ( p < 0.001 ) . a : glucose curves from ogtts at baseline and 2.0 and 0.5 years before diagnosis in the progressors , and at baseline and corresponding times before the last visit in the nonprogressors . in both the progressors and nonprogressors , glucose levels increased substantially at all ogtt time points ( auc glucose : p < 0.001 from baseline to 0.5 years in both groups ) . ( mean values are shown for the times before diagnosis or the last visit . ) b : c - peptide curves from ogtts at baseline and 2.0 and 0.5 years before diagnosis in the progressors , and at baseline and corresponding times before the last visit in the nonprogressors . in the nonprogressors , c - peptide levels increased at all ogtt time points ( auc c - peptide : p < 0.001 from baseline to 0.5 years ) . in the progressors , although the fasting and 120-min c - peptide levels were higher at 0.5 years than at baseline , there was no significant overall change ( auc c - peptide : p = 0.936 from baseline to 0.5 years ) . ( mean values are shown for the times before diagnosis or the last visit . ) 1b ) increased significantly at each ogtt time point from baseline to 0.5 years in the nonprogressors ( p < 0.001 for the auc c - peptide from baseline to 0.5 years ) . however , the change in c - peptide levels from baseline to 0.5 years in the progressors varied according to the ogtt time point . fasting and 120-min c - peptide levels increased from baseline to 0.5 years ( p < 0.01 and p < 0.05 , respectively ) in the progressors , but there was no significant change at the other ogtt time points nor in the auc c - peptide ( p = 0.936 ) . we compared the 300 min c - peptide difference at baseline and every 6 months for 2 years before diagnosis in the progressors with the 300 min c - peptide difference at baseline and at corresponding times before the last visit in the nonprogressors . ( among the 504 participants at baseline , there was a positive correlation between the 300 min c - peptide difference and the first - phase insulin response [ r = 0.50 , p < 0.001 ] ) . 2a ) was similar at baseline ; however , by 2.0 years , that difference was lower in the progressors ( p < 0.01 ) . among the progressors , the 300 min c - peptide difference declined from baseline to 0.5 years before diagnosis ( p < 0.01 ) . a : the difference ( mean se ) in c - peptide levels from 0 to 30 min ( the 300 min c - peptide difference ) according to the times before diagnosis ( progressors ) or the times before the last visit ( nonprogressors ) . the 300 min c - peptide difference was consistently lower in the progressors than in the nonprogressors . ( mean values are shown for the times before diagnosis or the last visit . ) b : the difference ( mean se ) in c - peptide levels from 60 to 90 min ( the 9060 min c - peptide difference ) according to the times before diagnosis ( progressors ) or the times before the last visit ( nonprogressors ) . at every time before diagnosis , the 9060 min c - peptide difference was positive in the progressors , whereas at every time before the last visit , it was negative in the nonprogressors . ( mean values are shown for the times before diagnosis or the last visit . ) whereas the 9060 min c - peptide difference ( fig . 2b ) was negative ( i.e. , the 90-min value was less than the 60-min value ) at all times before the last visit in the nonprogressors , it was positive at all times before diagnosis in the progressors . this contrast was not only significant at 2.0 ( p < 0.01 ) , 1.0 ( p < 0.01 ) , and 0.5 years ( p < 0.001 ) , but also at baseline ( p < 0.05 ) . as an overall measure of later c - peptide responsiveness to oral glucose , we used the sum of each of the c - peptide differences of the 30-min value subtracted from the values at 60 , 90 , and 120 min ( c - peptide sum after 30 min ) . figure 3 shows that those values were significantly higher in the progressors than in the nonprogressors at corresponding time points from 2.0 years to 1.0 year . values were also higher , but not significantly so , at baseline and at 0.5 years . among the progressors , the c - peptide sum after 30 min increased significantly from baseline to all subsequent time points ( p < 0.05 from baseline to 2.0 years and to 0.5 years ; p < 0.01 from baseline to 1.5 years and to 1.0 year ) . the c - peptide sum after 30 min ( mean se ) according to the times before diagnosis ( progressors ) or the times before the last visit ( nonprogressors ) . ( mean values are shown for the times before diagnosis or the last visit . ) consistent with the above findings , the timing of the peak c - peptide was delayed in the progressors . the percentage of individuals with the peak c - peptide occurring at 120 min was significantly higher in the progressors at 2.0 years ( 39 vs. 21% , p < 0.05 ) . by 0.5 years , the peak c - peptide occurred at 120 min in 56% of the progressors compared with 18% of the nonprogressors ( p < 0.001 ) . actual peak c - peptide levels did not differ between the progressors and nonprogressors until 0.5 years ( p < 0.01 ) . among the full cohort of 504 at baseline , the occurrence of the peak c - peptide at 120 min and the 9060 min c - peptide difference ( above versus below 0 ) were both highly predictive of type 1 diabetes with and without age as a covariate ( p < 0.001 ) in proportional hazards models ; the peak c - peptide level was somewhat predictive ( p = 0.028 ) , but not with age in the model . figure 4 shows c - peptide changes in the progressors who had ogtts 0.5 years before diagnosis and at diagnosis ( n = 66 ) , and in the nonprogressors who had ogtts 0.5 years before the last visit and at the last visit ( n = 192 ) . the c - peptide sum after 30 min declined somewhat at diagnosis , but not below levels in the nonprogressors at the last visit ( fig . the peak c - peptide occurred at 120 min in 52% of the progressors at diagnosis compared with 23% of the nonprogressors at their last visit ( p < 0.001 ) . a : the 300 min c - peptide difference ( mean se ) in the progressors who had ogtts 0.5 years before diagnosis and at diagnosis and in the nonprogressors who had ogtts 0.5 years before the last visit and at the last visit . b : the c - peptide sum after 30 min ( mean se ) in the progressors who had ogtts 0.5 years before diagnosis and at diagnosis and in the nonprogressors who had ogtts 0.5 years before the last visit and at the last visit . the c - peptide sum after 30 min declined , but did not fall below that in the nonprogressors . the data suggest that the early c - peptide response to oral glucose can be decreased for at least 2 years before the diagnosis of type 1 diabetes and especially as diagnosis approaches . although the early c - peptide response to the glucose challenge declines , c - peptide levels increase at later time points . this was evident in the interval from 60 to 90 min . the c - peptide increase in that interval in the progressors contrasted with the decline in the nonprogressors . it appears that the increase in c - peptide levels from 60 to 90 min can occur even 3 years before diagnosis . this prolonged increase in c - peptide levels after the glucose challenge is also manifested by a delayed peak c - peptide at least 2 years before diagnosis . it is possible that the continuing increase in c - peptide levels after 30 min in progressors occurs as a result of the deficient early c - peptide response . however , the later c - peptide response still does not prevent glucose levels from rising , as is evident in fig . the decrease in the c - peptide sum after 30 min at diagnosis suggests that the later response is also failing by that time . although we have previously examined changes in c - peptide and glucose indexes with progression to type 1 diabetes ( 4 ) , data pertaining to the timing of the c - peptide response before the diagnosis of type 1 diabetes has not been reported , nor has the prediction of type 1 diabetes by the timing of the c - peptide response been reported . a decreased early insulin response was a risk factor for progression to type 2 diabetes in pima indians . however , in contrast to our findings for type 1 diabetes , a decreased ( rather than increased ) later insulin response was predictive of type 2 diabetes in pima indians with impaired glucose tolerance ( 10 ) . the decreased early c - peptide response together with the increased later c - peptide response to oral glucose that we observed in the pre - diabetic state of type 1 diabetes is similar to the abnormal insulin responses to oral glucose in patients already diagnosed with type 2 diabetes ( 11,12 ) . this suggests that there could be some commonality between the disorders in the progression of metabolic abnormalities . the analysis was limited to children , since the nonprogressors were appreciably older than the progressors in the full dpt-1 cohort . by restricting the analysis to younger individuals , differences in progression due to an age effect were minimized . also , among individuals who developed type 1 diabetes , pathogenetic heterogeneity related to age was lessened . because a number of the nonprogressors would probably ultimately develop type 1 diabetes , and thus were not metabolically normal , differences between progressors and a normal reference group could be even more substantial . it is of interest that there was such a marked increment in c - peptide over time in the nonprogressors . the pattern of change suggests increasing insulin resistance over time . the findings in this report help to explain why such measures as peak c - peptide and auc c - peptide values provide relatively little information with regard to the prediction of type 1 diabetes and to its natural history . those indexes change little with progression because the deficient early c - peptide response to the oral glucose challenge is somewhat balanced by a continuing compensatory response until close to diagnosis . thus , the peak c - peptide and auc c - peptide indexes fail to detect the substantial changes in the -cells that are occurring years before diagnosis . it is evident that ogtts can yield appreciably more prediction and natural history information when they are partitioned according to the time after the glucose challenge .	objectivewe studied the c - peptide response to oral glucose with progression to type 1 diabetes in diabetes prevention trial type 1 ( dpt-1 ) participants.research design and methodsamong 504 dpt-1 participants < 15 years of age , longitudinal analyses were performed in 36 progressors and 80 nonprogressors . progressors had oral glucose tolerance tests ( ogtts ) at baseline and every 6 months from 2.0 to 0.5 years before diagnosis ; nonprogressors had ogtts over similar intervals before their last visit . sixty - six progressors and 192 nonprogressors were also studied proximal to and at diagnosis.resultsthe 300 min c - peptide difference from ogtts performed 2.0 years before diagnosis in progressors was lower than the 300 min c - peptide difference from ogtts performed 2.0 years before the last visit in nonprogressors ( p < 0.01 ) and remained lower over time . the 9060 min c - peptide difference was positive at every ogtt before diagnosis in progressors , whereas it was negative at every ogtt before the last visit in nonprogressors ( p < 0.01 at 2.0 years ) . the percentage whose peak c - peptide occurred at 120 min was higher in progressors at 2.0 years ( p < 0.05 ) ; this persisted over time ( p < 0.001 at 0.5 years ) . however , the peak c - peptide levels were only significantly lower at 0.5 years in progressors ( p < 0.01 ) . the timing of the peak c - peptide predicted type 1 diabetes ( p < 0.001 ) ; peak c - peptide levels were less predictive ( p < 0.05).conclusionsa decreased early c - peptide response to oral glucose and an increased later response occur at least 2 years before the diagnosis of type 1 diabetes .
You are an expert at summarizing long articles. Proceed to summarize the following text: the success of endodontic therapy relies on the three - dimensional sealing of the canal after removing bacteria from the root canal to prevent microleakage and penetration of microorganisms . it is reported that the most common reason for endodontic failure is the inability to achieve a three - dimensional seal . strindberg and allen showed that long - term endodontic failure is due to lack of complete canal seal . the ideal features of a root canal filling material include tissue compatibility , antibacterial properties , radiopacity , easy handling , insolubility in tissue fluids , three - dimensional seal of the canal and no coronal discoloration . gutta - percha , the most common root - filling material does not adhere to the canal walls and shrinks after cooling down , resulting in gaps which provide an access route for bacteria . there are various types of sealers ; zinc oxide - based sealers , resin - based sealers , silicon sealers and mineral trioxide aggregate ( mta)-based sealers . reported that epoxy resin - based sealers , such as ah26 , could adhere to dentinal walls after removal of the smear layer . resin - based sealers have other advantages , including setting in the presence of moisture , insolubility in tissue fluids , good setting time and antibacterial effects . mta cement has a great sealing ability and has been used widely in endodontic treatments for sealing perforation areas , internal and external root resorptions , creating an artificial barrier for non - vital open - apex teeth , root - end filling and pulp capping . one of the mta - based sealers introduced is fillapex ( angelus , londrina , pr , brazil ) , which is a double - paste sealer ( base and catalyst ) . the manufacturer claims that it has good sealing ability and biocompatibility ; it can stimulate the generation of mineralized tissue and has a favorable setting time . camilleri et al . compared the sealing ability of fillapex and pulp canal sealer using fluid filtration technique and reported that the sealing ability of these sealers was comparable . oliveira et al . compared bacterial microleakage resistance of two mta - based sealers ( mta sealer and endo cpm sealer ) with six popular sealers ( ah plus , sealer 26 , epiphany se , sealapex , activ gp and endofill ) . their results showed that ah plus and sealapex had the highest bacterial microleakage resistance whilst activ gp and mta - based sealers showed the greatest microleakage . fillapex sealer has been introduced recently but only a limited number of studies have evaluated the bacterial microleakage of this mta - based sealer . the aim of this study was to evaluate the apical microleakage of fillapex sealer compared with ah26 sealer after filling of root canals prepared with hand instrumentation . sixty single - rooted extracted human teeth with mature and straight roots were included in this experimental study . the teeth were examined clinically and radiographically and those with vertical or transverse fractures , severe curvatures , internal or external resorption and pulpal calcification were excluded . all the remaining calculi and tissue remnants were removed using an ultrasonic devise ( juya electronic co , tehran , iran ) . then the teeth were kept in 5.25% sodium hypochlorite ( golrang , pakshoo co. tehran , iran ) for 30 min for further disinfection and dissolving of the organic tissues . the teeth were decoronated from the cementoenamel junction using a diamond disc ( d and z , berlin , germany ) for achieving the average root length of 15 mm . the working length was established using a k - file # 10 ( mani , utsunomiya , japan ) . the apical portion of the canal was prepared using the step - back technique up to k - file # 30 . canal shaping was performed up to k - file # 60 in the coronal portion . finally , the smear layer was removed using 17% ethylenediaminetetraacetic acid ( edta ) ( merck , darmstadt , germany ) for 1 min . the first group consisted of 25 teeth obturated with gutta - percha ( mani , utsunomiya , japan ) and ah26 sealer ( dentsply , konstanz , germany ) . the second group also contained 25 teeth , which were obturated with gutta - percha ( mani , utsunomiya , japan ) and fillapex sealer ( angelus , londrina , pr , brazil ) . obturation was performed using a finger spreader ( mani , utsunomiya , japan ) by lateral condensation technique . the third group ( positive control ) consisted of five teeth , which were obturated using a single gutta - percha cone without any sealer . the last group ( negative control ) also included five teeth , which were obturated with gutta - percha and ah26 sealer . the teeth were incubated for 48 h at 100% humidity and 37c for complete sealer setting . two layers of nail varnish ( my co. , tehran , iran ) were applied on the root surfaces except for the apical 2 mm . in the negative control group , the contact surface of the teeth and the micropipettes were sealed with two layers of cyanoacrylate glue and one layer of nail varnish . this system was placed inside the hole created on the anti - serum vial lids and then sterilized for 24 h using ethylene oxide gas . afterward , this system was transferred to anti - serum vials containing 10 ml of brain heart infusion ( bhi ) ( merck , darmstadt , germany ) under aseptic conditions . the samples were kept inside an incubator for 3 days to ensure non - contamination . a total volume of 1 ml of the solution containing 10 e. faecalis ( atcc 29212 ) was injected from the upper part of the system and bacterial microleakage was evaluated by observing turbidity in bhi solution . the samples were checked every day for 90 days , bacterial injection was performed every 3 days and the turbidity of the medium was recorded every day . all specimens in positive control group had turbidity of the medium within 24 h , while none in the negative control group became turbid . during the experimental period , the survival analysis showed that the fillapex group had more microleakage ( mean : 77.92 ; standard deviation ( sd ) : 5.48 ) than the ah26 sealer group ( mean : 41.84 ; sd : 7.74 ) . both experimental groups had significant differences with control groups , showing the validity of the experiment ( p < 0.05 ) . gutta - percha is rigid and can not completely fill the inside of the canal ; therefore , sealers are needed to complete the canal seal . all the procedures of this study were done by the same operator to avoid intra - operator discrepancies . only teeth with single straight root canals were included because they can offer a more standardized method for evaluation of apical leakage . the culture medium of positive control group became turbid within the first 24 h , confirming the importance of endodontic sealers when filling root canals . in the present study , evaluation was carried out for 90 days in which the majority of the specimens showed bacterial leakage . within the first 30 days , bacterial leakage was detected in 6% of the specimens filled with ah26 and 28% of the specimens filled with mta fillapex . the first medium turbidity was seen in two specimens of ah26 group at day 4 . oliveira et al . observed bacterial leakage in 33% of the ah plus group 61% of mta sealer group after 1 month . this divergence in bacterial leakage percentages may be due to the number of samples and anatomical complexity of the root canal systems . gomes - filho et al . compared the sealing ability of two mta - based sealers ( fillapex and endo cpm sealer ) with a calcium hydroxide - based sealer ( sealapex ) , using dye penetration technique . the results showed that fillapex and sealapex had significantly lower microleakage than endo cpm sealer . yegin and keser evaluated the sealing ability of mta fillapex , sealapex and ah plus using fluid movement technique . they reported that during the first 24 h fillapex showed the least microleakage , but after 180 days ah plus and sealapex had the least microleakage . ah26 sealer was more resistant against bacterial leakage , which may be related to its flowability and dimensional stability , which led to a reduction in marginal leakage . oliveira et al . also verified that ah plus had good resistance to leakage , which suggests better adaptation of this material to dentine walls . saleh et al . reported that ah26 had less leakage when the smear layer was removed , highlighting the importance of using edta . fillapex is based on mta with additional substances to obtain a consistency suitable to be used as a sealer in root canal treatment . mta can form calcium ions and hydroxyl ions important for stimulation of hard tissue deposition . the presence of mta also suggests a possibility of setting expansion , which might have favored the sealability . most of the dental materials have a tendency to shrink away from their interfacial margins , exposing a gap through which contaminating elements can penetrate . mta setting results in the hydration of anhydrous mineral oxide compounds to produce calcium silicate hydrate and calcium hydroxide phases which produces expansion against its confining margins , enhancing the seal and minimizing leakage . the results of this study showed that ah26 was more resistant to bacterial microleakage compared with mta fillapex . this difference might be attributed to the selected sealers and microleakage evaluation techniques . in the present study , microleakage of fillapex increased over time , which is similar to the results of a study by yegin and keser . fillapex had lower sealing ability compared with ah26 , but resulted in a significantly higher sealing ability than the positive control group , demonstrating that it can improve the root canal seal . various factors affect the sealing ability of sealers , including moisture , removal of the smear layer and penetration of sealer into inaccessible regions , dimensional stability and sealer setting time . the difference between the chemical composition of mta - based materials can lead to differences in sealing ability , which was shown in a study by gomes - filho et al . further studies are necessary , particularly animal studies , to evaluate the sealing ability of mta - based sealers . according to the results of this in vitro study , the sealing ability of gutta - percha and ah26 sealer is higher than that of gutta - percha and mta fillapex .	background : sealing ability is one of the most important features of endodontic sealers . the purpose of this in vitro study was to compare the sealing ability of a resin - based sealer with a mineral trioxide aggregate - based sealer.materials and methods : a total of 60 single - rooted extracted human teeth were randomly divided into two experimental groups ( n = 25 ) and two control groups ( n = 5 ) . after canal preparation and smear layer removal , both groups were obturated with gutta - percha and sealer . resin - based ah26 sealer was used in the first group and fillapex sealer in the second group . two layers of nail varnish were applied on tooth surfaces except for the apical 2 mm . in the negative control group , nail varnish was applied on the entire surface . the teeth were mounted according to lima et al . study and then sterilized by ethylene oxide gas . the samples were evaluated for bacterial microleakage using enterococcus faecalis ( atcc 29212 ) for 90 days . data were analyzed by survival test ( p < 0.05).results : control groups had either immediate leakage or no leakage . the fillapex showed significantly higher amounts of microleakage compared with ah26 sealer ( p < 0.05 ) and both groups exhibited significant differences in comparison with control groups.conclusion:both sealers had bacterial leakage . sealing ability of ah26 was significantly higher than that of fillapex.
You are an expert at summarizing long articles. Proceed to summarize the following text: problem and pathological gambling are recognized as being major societal problems , with millions of individuals suffering from them ( e.g. , see ) . because of this fact , a great deal of effort has been exerted trying to identify who might have such problems . numerous examples of diagnostic screens can be found in the literature , including the south oaks gambling screen ( sogs , ) , the norc dsm - iv screen for problem gamblers , and the canadian problem gambling index [ 4 , 5 ] . the rationale behind these attempts is that if one can determine who might be experiencing problems with gambling , one is in a better position to treat , and potentially prevent , such problems . far less effort has been focused on an equally important issue why people might gamble . having such information would seem important because it seems reasonable to believe that different individuals might gamble for different reasons . it may also be the case that certain contingencies are more closely associated with gambling problems than are others . further , it is quite possible that the reason why someone begins to gamble is different than the reason why the same person continues to gamble . instruments designed to assess the contingencies reinforcing gambling behavior are , therefore , necessary to obtain this information . dixon and johnson were the first to introduce a screening instrument designed for this purpose when they forwarded the gambling functional assessment ( gfa ) . the gfa was patterned off of a similar measure designed to ascertain the contingencies maintaining self - injurious behavior and was proposed to measure four potential maintaining contingencies : gambling for tangible gain , for the sensory experience , for social attention , or as an escape . subsequent psychometric research , however , suggested that the gfa was not measuring four distinct maintaining contingencies . rather , it was only measuring two gambling for positive reinforcement and/or as an escape and was not cleanly parsing those two . because the gfa did not appear to be operating as designed , weatherly et al . revised the gfa ( gfa - r ) with the intention of cleanly measuring gambling maintained by positive reinforcement and escape . the gfa - r contains 16 items , each eight designed to measure gambling maintained by those two contingencies . weatherly et al . reported that the gfa - r had sound psychometric properties and cleanly parsed those two contingencies . likewise , weatherly et al . demonstrated that the gfa - r displayed good temporal reliability and internal consistency . overall , the research to date suggests that the psychometric properties of the gfa - r are superior to the original gfa . in terms of the contingencies maintaining gambling behavior , research using the original gfa uncovered a potentially interesting relationship between gambling problems and endorsing gambling as an escape [ 8 , 11 ] . specifically , miller et al . [ 8 , 11 ] reported that most respondents on the gfa displayed higher scores for gambling for positive reinforcement than for gambling as an escape . however , gambling as an escape , but not gambling for positive reinforcement , was strongly predictive of potential gambling problems as measured by scores on the sogs . subsequent research with the gfa - r has replicated both of these findings [ 10 , 12 ] . it has likewise shown that endorsing gambling as an escape is associated with both executive function and emotional regulation deficits that have been linked to gambling problems . thus , there appears to be a strong relationship between gambling problems and endorsing gambling as an escape . this relationship may not be completely surprising given that gambling as an escape is an official symptom of pathological gambling . gambling as an escape has , in fact , been a central tenant in some theories of pathological gambling ( e.g. , ) . for instance , weatherly and derenne reported a correlation of 0.689 between endorsing gambling as an escape on the gfa - r and scores on the sogs in their sample of 177 participants . one potential criticism of such research is that it has relied on the association between the scores on the gfa - r ( or the gfa ) and the sogs . although the sogs is a widely used screening measure within the research literature , it has been criticized on a number of fronts ( e.g. , see [ 16 , 17 ] ) . a second criticism is that the sogs was developed based on a prior , not the most recent , version of the diagnostic criteria for pathological gambling ( i.e. , ) . the sogs also focuses on the respondent 's gambling history , not necessarily the severity of the issues that face the respondent . in the light of these criticisms , one can not necessarily conclude that a strong relationship between gfa - r escape scores and sogs scores is equivalent to predicting the severity of the respondents ' gambling problems . it is possible that such a relationship might exist , but research to date has not established it beyond using the sogs . attempting to do so was the goal of the present study . in the present study , respondents were recruited to complete three measures : the gfa - r , sogs , and problem gambling severity index ( pgsi ) , which is part of the canadian problem gambling index [ 4 , 5 ] . the pgsi measures both gambling behavior and consequences and has been shown to have sound psychometric properties ( e.g. , ) . furthermore , unlike the sogs , the pgsi was designed for the use with the general population . first , participants would endorse gambling for positive reinforcement on the gfa - r to a greater extent than they would endorse gambling as an escape . second , endorsing gambling as an escape on the gfa - r would be a stronger predictor of sogs scores than would be the endorsing gambling for positive reinforcement . third , endorsing gambling as an escape on the gfa - r would be a stronger predictor of pgsi scores than would be the endorsing gambling for positive reinforcement . finally , it was predicted that sogs scores would be significant predictors of pgsi scores , but gfa - r escape scores would explain a significant amount of the variance in pgsi scores above and beyond that account for by sogs scores . the participants were 249 ( 180 females and 69 males ) students enrolled in psychology courses at the university of north dakota . the mean age of the participants was 19.8 years ( sd = 3.6 years ) , and their self - reported grade point average was 3.4 out of 4.0 ( sd = 0.5 ) . all participants received ( extra ) course credit in their psychology class in return for their participation . participants completed the study online using an experiment management system ( sona systems , ltd , version 2.72 ; tallinn , estonia ) . this system guaranteed that participants could complete the materials only one time even if they were enrolled in multiple psychology courses . the first item presented to all participants was an informed consent document that outlined the study and the participant 's rights . one of them was a brief demographic survey that asked them about their sex , age , grade point average , and race . they also completed the gfa - r , which consists of 16 items that respondents respond to on a scale that ranges from 0 ( never ) to 6 ( always ) . both eight - item subscales ( i.e. , positive reinforcement and escape ) are summed to provide a score for that particular subscale . gfa - r scores have also been shown to have good temporal reliability ( r = 0.80 at four weeks and r = 0.81 at 12 weeks ) . participants also completed the sogs , which consists of 20 items pertaining to the respondent 's gambling history . a score of 3 or 4 suggests possible problem gambling , and a score of 5 or more suggests the probable presence of pathology . original research reported that the sogs had high internal consistency ( = 0.97 ) , and subsequent research has reported that it has fair ( = 0.69 , ) to good ( = 0.81 , ) internal consistency . research has also shown that the sogs has good temporal reliability ( r = 0.89 at four weeks and r = 0.67 at 12 weeks ) . the pgsi consists of 12 items , only nine of which are included when calculating respondents ' scores . all items are answered on a four - point scale that ranges from 0 ( never ) to 3 ( almost always ) . scores from the nine counted items are summed , with scores of 0 indicating no gambling problems , 1 - 2 indicating a low level of gambling problems with few negative consequences , 37 indicating a moderate level of gambling problems with some negative consequences as a result , and 8 or more indicating problem gambling that includes negative consequences . initial research on the pgsi indicated that internal consistency was good ( = 0.84 ) , and subsequent research ( e.g. , ) has replicated that finding . ferris and wynne also reported that the pgsi had good temporal reliability ( r = 0.78 ) . the order of presentation of the demographic form , the gfa - r , the sogs , and the pgsi varied randomly across participants . table 1 presents the descriptive statistics for each gambling measure . because females constituted a majority of the sample and because the prevalence of gambling problems varies as a function of sex ( see ) , furthermore , 47 participants ( 18.9% ) scored 0 on the gfa - r , suggesting that these participants either did not gamble or gambled for reasons not measured by the gfa - r ( scores on the gfa - r , rather than the sogs or pgsi , were used to make this distinction because the gfa - r was the focus of the current study . data from participants scoring 0 on the gfa were included to provide information about the observed relationships when using a sample that may be representative of the general population , some of whom do not gamble ) . thus , table 1 also presents the descriptive statistics for the different groups when the data from these potential nongamblers were excluded . the data in table 1 appear to support the first hypothesis ; that respondents would endorse gambling for positive reinforcement to a greater extent than they would endorse gambling as an escape . when analyzing the data from the entire sample , results from wilcoxon signed rank tests indicated that scores on the gfa - r positive reinforcement subscale were higher than scores on the escape subscale for both females ( z = 10.03 , p < .001 ) and males ( z = 6.95 , p < .001 ) . when the nongamblers ( i.e. , gfa - r = 0 ) were excluded , scores remained significantly higher on the positive reinforcement subscale than on the escape subscale for both females ( z = 10.03 , p < .001 ) and males ( z = 6.95 , p < .001 ) ( the z values are identical for the two sets of analyses because , in the original analysis , nonresponders scored 0 on both subscales resulting in a tie score between the subscales ) . thus , participants endorsed gambling for positive reinforcement to a greater extent than they did gambling as an escape . in these analyses , and all that follow , statistical significance that was considered met at p < .05 . table 2 presents the bivariate correlations that were observed between scores on the different measures for females and males in both the entire sample and for only those respondents who scored above 0 on the gfa - r . the correlations in table 2 would seem to support the second hypothesis in that , in all cases , stronger correlations were observed between gfa - r escape subscales scores and sogs scores than were observed between gfa - r positive reinforcement subscale scores and sogs scores . tests of differences between two nonindependent rs indicated that the correlation between the scores on the gfa - r escape subscale and on the sogs was significantly stronger than the correlation between the gfa - r positive reinforcement subscale and the sogs for both female samples . thus , stronger correlations were observed between endorsing gambling as an escape and sogs scores than between endorsing gambling for positive reinforcement and sogs scores for the female respondents , but not for the male respondents . one could argue that these results are influenced by the fact that both gfa - r escape subscale scores and sogs scores were positively skewed . to assess this possibility , gfa - r escape subscale scores were transformed into a categorical variable , with scores of 0 coded as 0 , scores between 1 and 5 coded as 1 , and scores of 6 or more coded as 2 ( these categories were informed by previous research [ 11 , 13 ] ) . sogs scores were similarly coded into a categorical variable , with scores between 0 and 2 coded as 0 , between 3 and 4 coded as 1 , and 5 or more coded as 2 . simultaneous linear regressions were then conducted with the transformed sogs scores serving as the dependent measure and participants ' gfa - r positive reinforcement subscale scores , which were not skewed , and the transformed gfa - r escape subscale scores serving as the potential predictor variables . simultaneous regressions were conducted because these analyses allow for an assessment of the variance of that accounted for by each predictor variable independent of the other . for the entire sample of female participants , the overall regression model was significant , f(2 , 177 ) = 34.17 , p < .001 , and r = .279 . in this model , the transformed escape subscale scores were a significant predictor of the transformed sogs scores , = .449 , p < .001 , but the positive reinforcement subscales were not , = .132 , p = .076 . when the data from the females who scored 0 on the gfa - r were excluded , the regression model was again significant f(2 , 135 ) = 24.98 , p < .001 , and r = .270 . both the escape , = .434 , p < .001 , and positive reinforcement subscale scores , = .168 , p = .036 , were significant predictors of sogs scores , although the escape scores were the stronger predictor . for the entire sample of male participants , the overall regression model was significant , f(2 , 65 = 8.81 , p < .001 , and r = .213 . in this model , the transformed escape subscale scores were a significant predictor of the transformed sogs scores , = .429 , p = .001 , but the positive reinforcement subscales were not , = .062 , p = .622 . when the data from the males who scored 0 on the gfa - r were excluded , the regression model was again significant f(2 , 60 ) = 7.79 , p = .001 , and r = .206 , and escape subscale scores , = .413 , p = .002 , but not positive reinforcement subscale scores , = .086 , p = .496 , were significant predictors of sogs scores . thus , results from these regression analyses would suggest that , for both females and males , escape scores on the gfa - r were stronger predictors of sogs scores than were the positive reinforcement subscale scores . the correlations in table 2 would also seem to support the third hypothesis in that , in all cases , stronger correlations were observed between gfa - r escape subscales scores and pgsi scores than were observed between gfa - r positive reinforcement subscale scores and pgsi scores . like the correlations with the sogs , however , these differences were not always statistically significant . tests of differences between two nonindependent rs indicated that the correlation between the scores on the gfa - r escape subscale and the pgsi was significantly stronger than the correlation between the scores on the gfa - r positive reinforcement subscale and the pgsi for both female samples . the correlations were not significantly different for either male sample . thus , as with the sogs , whether gfa - r escape subscales scores were significantly more correlated with pgsi scores than were gfa - r positive reinforcement subscale scores varied as a function of sex . as with the sogs scores , pgsi scores were positively skewed ( see table 1 ) . thus , these scores were transformed into a categorical variable , with scores of 0 coded as 0 , of between 1 and 2 coded as 1 , of between 3 and 7 coded as 2 , and of 8 or more coded as 3 ( as suggested by ) . a series of simultaneous linear regressions was then conducted with the transformed pgsi scores serving as the dependent measure and participants ' gfa - r positive reinforcement subscale scores and their transformed gfa - r escape subscale scores serving as the predictor variables . for the entire sample of female participants , the overall regression model was significant , f(2 , 177 ) = 57.04 , p < .001 , r = .392 . in this model , both the transformed escape subscale scores , = .522 , p < .001 , and the positive reinforcement subscales , = .171 , p = .012 , were significant predictors of the transformed pgsi scores , with the escape scores being the stronger predictor . when the data from females scoring 0 on the gfa - r were excluded , the identical results were observed . the regression model was significant f(2 , 135 ) = 35.72 , p < .001 , and r = .346 , and both the escape , = .510 , p < .001 , and positive reinforcement subscale scores , = .159 , p = .036 , were significant predictors of the pgsi scores , with the escape scores being the stronger predictor . for the entire sample of male participants , the overall regression model was significant , f(2 , 65 ) = 11.64 , p < .001 , r = .264 . in this model , both the transformed escape subscale , = .244 , p = .048 , and the positive reinforcement subscales scores , = .350 , p = .005 , were significant predictors of the transformed pgsi scores . in this instance , the positive reinforcement subscale scores were the stronger predictor . when the data from males scoring 0 on the gfa - r were excluded , the regression model was again significant f(2 , 60 ) = 8.49 , p = .001 , and r = .221 . escape subscale scores did not significantly predict pgsi scores , = .240 , p = .058 , but positive reinforcement subscale scores did , = .319 , p = .013 . which subscale was the better predictor , however , varied as a function of sex . for females , gfa - r escape subscale scores were the best predictors of pgsi scores . for males , gfa - r positive reinforcement subscale scores the sogs was designed to identify potential pathological gamblers , whereas the pgsi was designed to test for potential gambling problems in the general population . however , one would suspect that there might be a significant overlap between the two . hypothesis four pertains to the question of whether the gfa - r subscales are related to gambling problems ( as measured by the pgsi ) independent of potential pathology ( as measured by the sogs ) . to answer this question , multiple hierarchical linear regressions were conducted using the transformed pgsi scores as the dependent measure and transformed sogs scores as the initial predictor . the gfa - r positive reinforcement subscale scores and the transformed gfa - r escape subscales scores were then simultaneously entered in the second block as additional predictor variables . when the data from all female participants were subjected to this analysis , the initial model was significant , f(1 , 178 ) = 194.99 , p < .001 , and r = .523 , and sogs scores were a significant predictor of pgsi scores , = .723 , p < .001 . when the gfa - r subscale scores were added to the analysis , the model was again significant , f(3 , 176 ) = 90.11 , p < .001 , and r = .606 . furthermore , the r increase of .083 was statistically significant ( p < .001 ) . both the sogs , = .544 , p < .001 , and the gfa - r escape subscale scores , = .278 , p < .001 , were significant predictors of pgsi scores , but gfa - r positive reinforcement subscale scores were not , = .100 , p = .073 . when this analysis was repeated excluding the female participants who scored 0 on the gfa - r , identical results were observed . the initial model was significant , f(1 , 136 ) = 148.82 , p < .001 , and r = .523 , and sogs scores were a significant predictor of pgsi scores , when the gfa - r subscale scores were added , the model was again significant , f(3 , 134 ) = 62.93 , p < .001 , and r = .585 , and the r increase of .062 was statistically significant ( p < .001 ) . sogs , = .572 , p < .001 , and gfa - r escape subscale scores , = .262 , p < .001 , were significant predictors of pgsi scores , but gfa - r positive reinforcement subscale scores were not , = .063 , p = .301 . when data from all male participants were analyzed , the initial model was significant , f(1 , 66 ) = 32.68 , p < .001 , and r = .331 , and sogs scores were significant predictors of pgsi scores , = .575 , p < .001 . when the gfa - r subscale scores were added , the model was again significant , f(3 , 64 = 16.61 , p < .001 , and r = .438 . the r increase of .107 was statistically significant ( p = .004 ) , and sogs scores were again significant predictors , = .470 , p < .001 . however , gfa - r positive reinforcement subscale scores , = .321 , p = .004 , and not gfa - r escape subscale scores , = .042 , likewise , when data from male participants scoring 0 on the gfa - r were analyzed , the initial model was again significant , f(1 , 61 ) = 29.07 , p < .001 , and r = .323 , and sogs scores were again significant predictors of pgsi scores , = .568 , p < .001 . when the gfa - r subscale scores were added , the model was again significant , f(3 , 59 ) = 13.29 , p < .001 , r = .403 , and the r increase of .081 was statistically significant ( p = .024 ) . sogs , = .480 , p < .001 , and gfa - r positive reinforcement subscale scores , = .278 , p = .014 , were significant predictors of pgsi scores , but gfa - r escape subscale scores were not , = .042 , p = .724 . thus , for both female and male participants , the gfa - r subscales accounted for a significant amount of the variance in pgsi scores above and beyond that accounted for by sogs scores . however , which subscale accounted for that increase varied as a function of sex . for females , it was endorsing gambling as an escape . previous research on the gfa - r has suggested that respondents typically endorse gambling for positive reinforcement to a greater extent that they endorse gambling as an escape , but that endorsing gambling as an escape is more strongly related to potential pathology , as measured by the sogs , than is endorsing gambling for positive reinforcement . results from the present study replicated the former finding ; both female and male participants endorsed gambling for positive reinforcement to a significantly greater extent than they did gambling as an escape . likewise , endorsing gambling as an escape was strongly related to potential pathology , as measured by the sogs , for respondents of both sexes . however , endorsing gambling as an escape was only significantly more correlated with sogs scores than endorsing gambling for positive reinforcement for the female participants . one potential criticism of previous research is that conclusions about the gfa - r have been drawn by comparing gfa - r scores to sogs scores . the present study , therefore , had participants complete a widely used measure designed to study potential gambling problems in the general population ( i.e. , the pgsi ) . as with the comparisons to the sogs , gfa - r subscale scores were strongly correlated with pgsi scores . also as with the sogs comparisons , endorsing gambling as an escape was a significantly more correlated with pgsi scores than was endorsing gambling for positive reinforcement , but only for the female participants . in fact , for males , endorsing gambling for positive reinforcement was more predictive of pgsi scores than was endorsing gambling as an escape . although created to measure somewhat different things in different populations , the sogs and pgsi scores were , not surprisingly , highly correlated . furthermore , sogs scores were significant predictors of pgsi scores . however , the gfa - r subscale scores accounted for a significant amount of the variance in pgsi scores above and beyond that accounted for by sogs scores . which gfa - r subscale accounted for that variance varied by sex . for females , first , although the gfa - r and sogs are strongly correlated , the gfa - r is measuring something beyond what is measured by the sogs . second , there appears to be a significant relationship between the contingencies maintaining gambling behavior and the display of gambling problems in a university sample of participants . one might argue that given the fact that males suffer from pathological gambling significantly more frequently than females ( see ) , the results from the male participants might be most important . before taking that tack , however , it should be noted that the display of gambling problems as measured by either the sogs or pgsi was relatively similar for both female and male participants ( see table 1 ) . finding that the gfa - r was predictive of gambling problems above and beyond scores on the sogs and that the different gfa - r subscales were differentially predictive of gambling problems between the two sexes should indicate two things to researchers and practitioners . first , there is additional information about a person 's gambling behavior that can be gained by measuring the function of the behavior rather than only whether the behavior is problematic . second , different functions of the behavior might be more indicative of gambling problems depending on the sex of the individual . one could also argue that the sex differences reported here are consistent with the broader research . that is , research indicates that men tend to be more sensation seeking than women , which is consistent with the present finding that endorsing gambling for positive reinforcement was a predictor of gambling problems for male respondents . likewise , females tend to suffer from certain disorders , such as eating disorders , more frequently than men , and those disorders have been linked to the contingencies of escape ( e.g. , [ 24 , 25 ] ) . thus , although the present results should be generalized with caution because they require replication , there does appear to be convergent validity for them within the research literature . there are , in fact , numerous reasons to be cautious in generalizing the present results . first , the participants were all university students from the upper midwest of the united states and were relatively young even relative to the legal age to gamble in many states . it is , for instance , possible that university students gamble for different reasons than individuals in the general population . next , the present procedure particularly targeted a largely nonpathological sample using a measure of gambling problems ( i.e. , the pgsi ) that is the best at identifying moderate , rather than severe , gambling problems ( see ) . thus , one can not assume that similar results would be observed if the present procedure was conducted using a clinical sample . in fact , one might argue that a major limitation of the present study was the lack of focus on pathological gamblers . one potential counter to that argument is that identifying predictor variables of gambling problems in individuals who have yet to be diagnosed as pathological allows for the potential development of preventative measures . then again , one can not assume that any of the present participants will eventually become pathological gamblers . one could also criticize the present analyses because p values were not adjusted to accommodate the multiple analyses that were conducted on the same data . however , it should be noted that , had such adjustments been made , the conclusions would not have changed . in the vast majority of cases , the outcomes that were statistically significant were significant at p < .001 and would have remained statistically significant had the threshold been raised to be more conservative . the present results are largely consistent with prior findings that endorsing gambling as an escape might be indicative of the respondent experiencing gambling problems . for both sexes , endorsing gambling as an escape appears to be a strong predictor of the potential presence of problem or pathological gambling as measured by the sogs . however , when using a more general measure of gambling problems ( i.e , the pgsi ) , there appear to be sex differences in whether endorsing gambling for positive reinforcement ( males ) or as an escape ( females ) is more predictive of those problems . this finding is augmented by the fact that scores on the gfa - r subscales accounted for a significant amount of the variance in pgsi scores beyond that accounted for by sogs scores . thus , the present results lead to the conclusions that the function of one 's gambling is potentially predictive of whether one might be prone to experience gambling problems . further , finding that gambling to get something versus to get away from something might be differentially predictive of gambling problems depending on the sex of the individual highlights the need for more theoretical and empirical research in this area . it may be possible that the current results can be incorporated into existing theories of pathological gambling ( e.g. , ) . then again	previous research has reported a strong relationship between endorsing gambling as an escape and problem / pathological gambling as measured by the south oaks gambling screen ( sogs ) . the present study recruited 249 university students to complete the gambling functional assessment - revised ( gfa - r ) , which measures the function of the respondent 's gambling , as well as the sogs and the problem gambling severity index ( pgsi ) , which was designed to identify gambling problems in the general population . endorsing gambling as an escape on the gfa - r was again predictive of sogs scores . the function of one 's gambling was also predictive of the respondents ' pgsi scores , but whether gambling for positive reinforcement or as an escape was the significant predictor differed between male and female respondents . scores on the gfa - r subscales also accounted for a significant amount of variance in pgsi scores above and beyond that accounted for by sogs scores . the present results support the idea that both practitioners and researchers should be interested in the function of an individual 's gambling as well as the presence or the absence of pathology . they also suggest that differences in the function of gambling might also exist between the sexes .
You are an expert at summarizing long articles. Proceed to summarize the following text: in the usa , the prevalence of hiv among prisoners is approximately three times greater than in surrounding communities.1 indeed , 14% of all people living with hiv encounter the criminal justice system annually.2 as such , prisons are an important place to detect hiv , treat it , and serve as a conduit to care after release . the availability of highly active antiretroviral therapy has transformed hiv into a chronically manageable condition , even for prisoners.34 unfortunately for prisoners , including those with hiv , the 3-month period after release is a highly vulnerable time period , resulting in high rates of recidivism to prison,5 relapse to drug use,68 and overdose resulting in death.9 for those with hiv , decreased access to antiretroviral therapy,10 poor virological and immunological treatment outcomes,1112 and high rates of hiv risk behaviors have been confirmed.13 thus , researchers , practitioners , and policy makers have emphasized that the first 3 months after release to the community is a crucial period for newly released prisoners . if successful reintegration is not achieved during this period , it is not likely to occur.1416 these findings create an urgent need to adapt and test evidence - based transitional interventions for this population . opioid agonist therapy ( oat ) , including methadone and buprenorphine / naloxone ( bpn / nlx ) therapy , decrease heroin use , time to relapse , criminal activity and hiv risk behaviors and increase retention in treatment and are cost - effective to society.1718 moreover , 8590% of inmates with histories of opioid dependence relapse to heroin use within one year after release , 67 yet oat is rarely available.1921 for unclear reasons , the criminal justice system has not adopted these effective treatments , particularly for those with hiv who might benefit most.22 we therefore sought to establish acceptability , feasibility , and early hiv and substance abuse treatment outcomes after introducing bpn / nlx at the time of release from prison in hiv - infected patients receiving antiretroviral therapy . bpn / nlx , unlike methadone that is a full opioid mu - agonist , is a partial mu - agonist and was selected for its favorable safety profile , reduced likelihood for overdose and death and few pharmacokinetic drug interactions with antiretroviral medications.23 recruitment all subjects were recruited from within a randomized controlled trial of directly administered antiretroviral therapy ( daart ) among hiv - infected prisoners transitioning to the community within 90 days . those who met dsm - iv criteria for opioid - dependence were assessed for interest in oat with either methadone or bpn / nlx . additional eligibility criteria included : ( 1 ) returning to either new haven or hartford ; ( 2 ) age 18 years ; ( 3 ) a negative urine pregnancy test for women and willingness to use contraception ; and ( 4 ) expressing an interest in bpn / nlx treatment . as part of the ongoing parent study , subjects were randomized 2:1 to receive daart versus self - administered therapy ( sat ) . study procedures within 90 days before community - release , all subjects underwent informed consent , baseline assessments and chart review . assessments included demographic information , mental illness and chemical dependence screening using the mini - international neuropsychiatric interview ( m.i.n.i ) , 24 addiction severity index,2526 and alcohol use disorders identification test.27 alcohol and drug use questions referred to the pre - incarceration period to establish historical diagnoses , as no subject was actively using drugs or alcohol . additional post - release activities included baseline physical exam and weekly assessment of opiate craving ( 10-point likert - scale ) , buprenorphine satisfaction ( 10-point likert - scale ) , urine toxicology screening using the nida-6 ( opiates , cocaine , methadone , benzodiazepines , marijuana , and methamphetamines ) , and separate urine tests for oxycodone and buprenorphine ( redwood biotech , santa rosa , ca ) . baseline and quarterly hiv-1 rna levels ( amplicor 1.5 ; roche ) and cd4 lymphocyte counts ( facs ; quest ) were obtained . buprenorphine induction process bpn / nlx induction was allowed up to 30 days post - release from prison ; however , the day of release was targeted when possible . due to low expected tolerance to opioids , subjects were initially administered 2.0 mg/0.5 mg bpn / nlx and increased by 2 mg/0.5 mg increments of bpn / nlx , as tolerated , to reduce the craving score to 1 , while avoiding opiate agonist side effects . bpn / nlx dose , craving for opiates , opioid withdrawal symptoms , opioid - agonist side effects , and urine drug screening were all collected daily during the induction and weekly thereafter . all subjects received weekly , standardized , and manual - based counseling per protocol28 for 4560 min by a certified substance abuse treatment counselor . buprenorphine administration for those randomized to daart , bpn / nlx was observed daily along with their antiretroviral therapy and other chronically prescribed medications . bpn / nlx dispensing , similar to the sat group , was contingent upon attending weekly counseling sessions . for those in the sat arm , bpn / nlx was contingent upon attending weekly counseling sessions where a voucher was provided to allow the pharmacy to release the bpn / nlx . counselors provided a 7-day prescription voucher after providing a urine specimen and attending weekly counseling sessions . follow - up subjects were evaluated daily by the study clinician during the induction phase and at least monthly thereafter . counselors met with subjects weekly , irrespective of study assignment , and assessed urine toxicology screening , opiate craving , bpn / nlx satisfaction , and adverse side effects . structured interviews and phlebotomy for cd4 lymphocyte count and hiv-1 rna level were conducted at weeks 4 and 12 . analytic strategy outcomes from the first 12 weeks the primary hiv treatment outcome was the proportion with a non - detectable hiv-1 rna level 12 weeks after prison - release since this is the most vulnerable time period . secondary drug treatment outcomes included the percentage of opioid - free urine toxicology results over 12 weeks . missing urine results were adjudicated in the following sequential manner : ( 1 ) self - report at weekly visits ; and ( 2 ) last value carried forward only if a single missing value was noted ; ( 3 ) for subjects who remained in the trial , missing consecutive urine values were considered positive . therefore , the proportion of positive urine tests was calculated as the percent positive out of the number who remained in the trial for each week and included missing value adjudication . craving and satisfaction scores were calculated as the mean for those individuals whose results were reported weekly.institutional review boards at both yale university and the university of connecticut and the research committee at the connecticut department of correction approved the study ; a certificate of confidentiality was also obtained . figure 1 depicts the disposition of the 69 subjects enrolled in the parent study ; 70% ( n = 48 ) met dsm - iv criteria for opioid dependence . of these , 14.5% ( n = 7 ) chose methadone , 37.5% ( n = 18 ) chose no form of oat , and 48% ( n = 23 ) chose to be inducted on bpn / nlx . the 2:1 randomization of the parent study resulted in 16 receiving bpn / nlx as daart and seven self - administering it . all subjects ( n = 23 ) had at least one axis i disorder : 52% had thought disorders ( i.e. schizophrenia and psychosis ) and 78% had mood disorders ( i.e. major depression , and bipolar disorder ) . the majority ( 61% of 23 ) with axis i anxiety disorders ( i.e. post - traumatic stress disorder , general anxiety disorder , obsessive compulsive disorder ) was also prescribed psychiatric medications . table 1baseline characteristic of study participants ( n = 23)characteristicmean age ( years)46.4gender ( m / f ) , n ( % ) 18 ( 78):5 ( 22)race / ethnicity black , n ( % ) 9 ( 39 ) hispanic , n ( % ) 12 ( 52 ) white , n ( % ) 2 ( 9)co - morbid axis i mental disorders23 ( 100 ) thought disorders , n ( % ) 12 ( 52 ) mood disorders , n ( % ) 18(78 ) anxiety disorders , n ( % ) 14(61)prior opioid agonist treatment methadone , n ( % ) 19 ( 82.6 ) bpn / nlx , n ( % ) 5 ( 21.7 ) methadone and bpn / nlx , n ( % ) 5 ( 21.7)randomization ( daart / sat)16:7median months of incarceration ( iq range)7 ( 511)baseline audit score6.78 ( 7.71)mean baseline cd4 + lymphocytes ( cells / ml)344 ( 222)hiv-1 rna < 50 ( % ) 63mean hiv-1 rna ( among vl > 50 , copies / ml)4.11 log10m.i.n.i prison diagnosis and/or psychiatric medications baseline characteristic of study participants ( n = 23 ) m.i.n.i prison diagnosis and/or psychiatric medications for the primary hiv treatment outcome ( see figure 2 ) , the proportion of subjects with a non - detectable hiv-1 rna levels at 12 weeks did not differ from baseline ( 61% vs. 63% , p = 0.91 ) . the mean cd4 lymphocyte count ( 367 vs. 344 , p = 0.89 ) did not differ statistically either . for those subjects whose hiv-1 rna level was detectable , they similarly did not have a change in hiv-1 rna levels ( 4.12 vs. 4.11 log10 copies / ml ) . hiv treatment outcomes . among the 23 subjects initiating bpn / nlx , 91% ( n = 21 ) completed the induction period . two subjects left the study after a single dose of bpn / nlx and were subsequently lost to follow - up to both the bpn / nlx and the parent study . after induction , the mean daily bpn / nlx dose at which subjects were stabilized was 9.5 mg ( range , 2 to 16 mg ) . there were no differences between the mean bpn / nlx dose for those treated and not treated with atazanavir - containing regimens ( 9.20 mg vs. 8.46 mg ; p = 0.82 ) , yet there was a trend toward higher bpn / nlx dosage when co - administered with efavirenz - containing regimens ( 10.33 mg vs. 5.33 mg ; p = 0.10 ) . compared to baseline , mean opioid craving scores decreased from 6 to 1.8 after induction completion ( on average , 3 days ) and remained 2.2 by the end of 12 weeks . the mean satisfaction with bpn / nlx treatment score was high at 9.5 throughout the 12-week period ( see figure 3 ) for the 17 retained subjects . overall , retention was high at 12 weeks74% for all 23 subjects and 81% for the 21 who completed induction . one successfully inducted subject withdrew from bpn / nlx because she required a prescribed narcotic for a pain - related syndrome ; two were reincarcerated after cocaine - relapse ( opiate screens remained negative ) ; and one withdrew due to nausea , a side effect he attributed to bpn / nlx . figure 3.opiate craving and satisfaction with buprenorphine treatment . opiate craving and satisfaction with buprenorphine treatment . figure 4 depicts the urine toxicology results for buprenorphine , other opioids and cocaine over the 12-week period . urine opiate positivity decreased from 29% at baseline to 17% at the end of 12 weeks for the 17 subjects who completed 12 weeks ; it was 20% for the 21 subjects who completed the 3-day induction . similarly urine cocaine positivity ranged from 43% at baseline to 29% at 12 weeks . receiving hiv and bpn / nlx medications as daart vs. sat did not significantly differ for retention between groups ( 72.2% vs 92.9% , p = 0.17 ) , but the study was underpowered to detect a difference . the mean number of days between release from prison and receiving the first induction dose of bpn was 8.5 ( range 0 to 30 days ) . the delay between release and starting buprenorphine resulted in positive urine testing at baseline . comparing the 14 subjects who were inducted early ( within the first 7 days of release ) , versus the nine inducted later ( after 7 days of release ) , there was no statistical difference in the mean retention on treatment ( 11.0 vs. 10.6 weeks , p = 0.79 ) , the proportion completing all 12 weeks ( 84.6% vs. 87.5% , p = 1.00 ) , the percent of negative urine screens for opiates ( 70% vs. 86% , p = 0.47)and cocaine ( 51.0 vs. 70.6% , p = 0.56 ) , and the mean bpn dose at the completion of induction ( 9.8 vs. 8.9 mg , p = 0.31 ) . adverse side effects , including constipation , headache , nausea and drowsiness from bpn / nlx during the 12 weeks of the study were considered mild and easily addressed by the treatment team . the one subject who withdrew from the bpn / nlx treatment for nausea refused treatment with anti - emetics . side effects were not associated with co - administration of atazanavir or efavirenz ( data not shown ) . though small , this pilot feasibility study has important implications for both clinical care and research . first , it is the first study to demonstrate that hiv treatment outcomes persist during transition from prison and there were few adverse consequences . second , it is also the first study to demonstrate the feasibility of bpn / nlx treatment as relapse prevention for opioid - dependent , hiv - infected prisoners transitioning to the community . last , this study demonstrates not only a high preference for bpn / nlx over methadone or no oat among those with opioid dependence , but demonstrates high levels of acceptability and satisfaction . prison - release studies of hiv - infected patients , including those from connecticut , confirm poor virological and immunological outcomes within 12 weeks of release.1112 while case management interventions have successfully linked released prisoners to care , none have confirmed stability in hiv treatment outcomes . recent data from texas suggest that refilling prescriptions is low among released hiv - infected prisoners,10 but this obstacle was not present in this study since all prescriptions were continued for all study subjects . the texas study , however , did not assess the reasons for not filling prescriptions , which might have resulted from relapse to drug use . though these pilot data are not powered sufficiently to determine if bpn / nlx treatment alone led to these successful clinical endpoints , these data remain compelling and suggest that bpn / nlx was an important factor in stabilizing the lives of subjects , resulting in improved adherence to antiretroviral therapy . though insufficiently powered , it is now necessary to recruit larger sample sizes to determine if daart plays a role in retention and outcomes . ultimately , buprenorphine treatment alone without daart needs evaluation through the rigors of a randomized controlled trial to determine its effectiveness . similar to most correctional settings in the northeast where hiv prevalence rates are highest , the majority ( 70% ) of all released hiv - infected prisoners on antiretroviral therapy met dsm - iv criteria for opioid dependence . not only did the majority of those with opioid dependence choose oat over no pharmacological therapy , but also bpn / nlx was preferred over methadone . though numerous explanations may be posited , the greater preference for bpn / nlx may reflect previous negative experiences with or perceptions about methadone among this population;29 this finding is supported by the near universal ( > 80% ) prior experience with methadone treatment ( see table 1 ) . one of the misconceptions by prisoners who are no longer using illicit drugs is that oat would no longer be needed after prolonged periods of forced abstinence . absent from that perspective , however , is the recognition that opioid - relapse is high and approaches 8590%6 even after prolonged imprisonment . there are no other studies of bpn / nlx treatment for hiv - infected released prisoners , but compared with hiv - undifferentiated released prisoners , substance abuse outcomes are similar to other studies . among opioid - dependent prisoners in baltimore with unknown hiv status , the use of methadone resulted in retention , efficacy and urine toxicology results similar to ours three months after release.630 we extended their findings to hiv - infected subjects receiving antiretroviral therapy and demonstrated sustained hiv treatment response , high retention rates ( > 70% ) , low opiate- ( 17% ) and cocaine - positive ( 29% ) urine tests , and no associated adverse consequences between buprenorphine and hiv therapeutics . the retention rate of 74% ( n = 17/23 ) , although potentially affected by the daart intervention itself in this study , is higher than described in smaller studies of bpn / nlx treatment of hiv - infected persons in the usa31 and prisoners in puerto rico.32 this study also confirms findings reported from other small studies that have shown that bpn / nlx can be prescribed safely and effectively in hiv primary care settings;3133 hiv - undifferentiated prisoners in puerto rico;32 and france.53435 nine ( 39% ) of the subjects had relapsed to opioid use within a week after release from prison , accounting for the high proportion of positive urine results at baseline . to avoid early relapse and potential overdose and death , relapse prevention strategies this is particularly true since opioid tolerance is low and risk for overdose and death is high.9 future studies should focus on initiating treatment prior to release . in this study of opioid dependent subjects with prolonged periods of abstinence , bpn / nlx treatment was well tolerated , but required an extended induction period over 7 days . dosing started lower than recommended by existing guidelines for chronic opioid - using patients,36 and increases in dosage was slow and incremental . fortunately , excess opioid - agonist effects were infrequent ( 17% ) during the induction period ( i.e. , drowsiness , nausea , headache , constipation ) , extinguished by the end of the induction period . induction did , however , require longer duration of induction due to the down - regulation of opioid receptors after prolonged periods of abstinence and potentially from known symptomatic pharmacokinetic interactions with ritonavir - boosted atazanavir.37 overall , tolerability remained high despite potential pharmacokinetic drug interactions reported between bpn / nlx and antiretroviral medications.233738 last , this study represents a population of hiv - infected individuals who often have many unmet needs and co - morbidities , and who might not otherwise receive such treatment . the unexpected universal co - occurrence of mental illness ( 100% of subjects with documented axis i disorders ) suggests a particularly challenging population to treat . indeed , collectively all subjects met criteria for triple diagnosis and their management has traditionally been fraught with multiple challenges.3940 left untreated , mental illness and substance use disorders contribute significantly to difficulty with maintaining appointments with medical professionals and often are associated with reincarceration.44142 when effective support is offered , however , they can maintain clinical benefit from treatment.43 bpn / nlx may offer added incentives to correctional settings that might result in increased use of oat . these include decreased : ( 1 ) regulatory procedures and licensing ; ( 2 ) concerns about diversion and overdose ; and ( 3 ) concerns about opioid withdrawal symptoms if a subject is not effectively and immediately linked to community services.22 one of the major barriers to bpn / nlx treatment has been the actual cost of the medication , although it has been found to be similarly cost - effective as methadone.44 this concern , however , may be obviated as the bpn / nlx patent ended in late 2009 , potentially allowing for less expensive generics to emerge . buprenorphine induction and stabilization is a highly acceptable , tolerable and effective treatment to prevent relapse to opiate use in released hiv - infected prisoners . importantly , it appears to be effective at maintaining hiv outcomes and potentially decreasing hiv - associated morbidity and mortality . this study initiated bpn / nlx after release from prison , but future studies , including randomized trials examining bpn / nlx induction before and after release are needed to determine the optimal timing for treatment . more importantly , better understanding of the impact that bpn / nlx treatment may have on recidivism , continuity of hiv care , art adherence and hiv mortality through larger randomized controlled trials are urgently needed .	hiv - infected prisoners fare poorly after release . though rarely available , opioid agonist therapy ( oat ) may be one way to improve hiv and substance abuse treatment outcomes after release . of the 69 hiv - infected prisoners enrolled in a randomized controlled trial of directly administered antiretroviral therapy , 48 ( 70% ) met dsm - iv criteria for opioid dependence . of these , 30 ( 62.5% ) selected oat , either as methadone ( n = 7 , 14.5% ) or buprenorphine / naloxone ( bpn / nlx ; n = 23 , 48.0% ) . twelve - week hiv and substance abuse treatment outcomes are reported as a sub - study for those selecting bpn / nlx . retention was high : 21 ( 91% ) completed bpn / nlx induction and 17 ( 74% ) remained on bpn / nlx after 12 weeks . compared with baseline , the proportion with a non - detectable viral load ( 61% vs 63% log10 copies / ml ) and mean cd4 count ( 367 vs 344 cells / ml ) was unchanged at 12 weeks . opiate - negative urine testing remained 83% for the 21 who completed induction . using means from 10-point likert scales , opioid craving was reduced from 6.0 to 1.8 within 3 days of bpn / nlx induction and satisfaction remained high at 9.5 throughout the 12 weeks . adverse events were few and mild . bpn / nlx therapy was acceptable , safe and effective for both hiv and opioid treatment outcomes among released hiv - infected prisoners . future randomized controlled trials are needed to affirm its benefit in this highly vulnerable population .
You are an expert at summarizing long articles. Proceed to summarize the following text: the incidence of fungal liver abscess is rare compared to pyogenic or amebic liver abscess . routes of exposure of bacteria can occur by biliary tree , portal vein , hepatic artery , direct extension , and cryptogenic abscess . a 65-year - old female patient admitted with a complaint of upper abdominal pain for 15 days , more on the right hypochondrium and epigastric region , associated with a history of nausea and vomiting . she was a known case of diabetes mellitus for 5 years , on oral hypoglycemic drugs . respiratory , cardiovascular , central nervous system , and musculoskeletal system found to be normal . routine blood investigations such as complete blood count and renal and liver function tests were normal . ultrasound abdomen showed 7.15 cm 4.43 cm ill - defined echo texture lesion with internal anechoic area seen in segment vii and v of the liver [ figure 1 ] . we are not sure whether we are dealing with chronic liver abscess or infected hydatid cyst ; hence , we proceeded with contrast computed tomography ( ct ) abdomen and immune assay for igg antibodies against hydatid cyst ( echinococcus ) . contrast ct abdomen showed enlarged liver of 20.8 cm with ill - defined hypoechoic collection with air fluid level of size 13 cm 10 cm 7 cm noted in the right lobe of liver with enhancing walls suggestive of liver abscess [ figures 2 and 3 ] . however , serum enzyme - linked immunosorbent assay ( elisa ) against echinococcus igg - positive ( 0.78 od ) ( reference interval - negative < 0.3 , positive > 0.3 ) . the patient was started on antibiotics and albendazole for hydatid cyst . we are not sure with diagnosis whether synchronous hydatid cyst with pyogenic liver abscess or hydatid cyst becomes infected . gram stain showed many pus cells and fungal filaments branching septate hyphae , suggestive of candida albicans and culture and sensitivity were also suggestive of c. albicans . later , we placed pigtail catheter inside the abscess cavity and the cavity started resolving [ figure 4 ] . ultrasound abdomen showed 7.15 cm 4.43 cm ill - defined echo texture lesion with internal anechoic area seen in segment vii and v of the liver axial computed tomography abdomen showed enlarged liver with hypodense fluid collection contrast computed tomography abdomen showed enlarged liver of 20.8 cm with ill - defined hypo echoic collection with air fluid level of size 13 cm 10 cm 7 cm noted in the right lobe of liver with enhancing walls suggestive of liver abscess ultrasound abdomen with pigtail catheter inside the abscess cavity with resolving abscess hydatid cyst occurs due to infection with echinococcus granulosus . after ingestion of parasite embryo , it releases an oncosphere which penetrate the mucosa of intestine . larvae of echinococcus reach portal venous system through intestine and spread to various organs such as liver , lung , spleen , brain , muscle , and other parts . ultrasound abdomen mostly diagnoses hydatid cyst , but when the diagnosis is uncertain , we need ct abdomen or magnetic resonance imaging ( mri ) abdomen to fetch diagnosis . when imaging fails to identify the lesion or when the lesion is doubtful , we need serological examination , which is confirmatory . following serological test used for the diagnosis of hydatid cyst . they are complement fixation test , indirect hemagglutination test , latex agglutination test , elisa , indirect immunofluorescence antibody test , immunoelectrophoresis , and immunoblotting . sensitivity and specificity of elisa vary from organ to organ , in which hydatid cyst is involved . hydatid cyst liver carries higher sensitivity ( 90% ) compared to hydatid cyst of the lung ( 75% ) . elisa for igg carries higher sensitivity ( 90% ) compared to elisa for igm ( 85% ) or ige ( 80% ) antibodies . ultrasound abdomen shows calcification of the cyst wall with double echogenic shadow due to pericyst . mri abdomen shows mixed low signals on t1-weighted images whereas mixed high signals on t2-weighted images with septations . mostly , hydatid cysts with immunocompromised status are more prone to develop liver abscess following hydatid cyst . the occurrence of abscess in hydatid cyst creates diagnostic difficulty when seropositive for hydatid cyst , but imaging fails to identify hydatid cyst or serology positive for hydatid cyst , but aspirate shows negative for hydatid . in our case , there was a diagnostic difficulty because imaging failed to identify typical hydatid cyst , but seropositive for hydatid cyst . with extensive search on pubmed , this is the first case reported with hydatid cyst with fungal liver abscess created diagnostic difficulty . the previous description of fungal abscess mostly associated with liver malignancies or hematological malignancies . fungal liver abscess following hydatid cyst was treated with pair and medical management such as albendazole with antifungal agents . this case created diagnostic difficulty whether we are dealing with liver abscess or hydatid cyst . sometimes , it may be possible that concurrent occurrence of hydatid cyst with liver abscess or hydatid cyst became infected and mimic like liver abscess . immunocompromised states with hydatid may be more prone for fungal liver abscess compared to pyogenic liver abscess . this kind of cases can be managed with initially pair followed by pigtail for proper resolution .	liver abscess is mostly either pyogenic or amebic . fungal and mycobacterial liver abscesses are rare and mostly associated with immunosuppression . the occurrence of fungal liver abscess with hydatid cyst was never reported previously . this case created diagnostic difficulty , whether we are dealing with liver abscess or hydatid cyst . sometimes , it may be possible that concurrent occurrence of hydatid cyst with liver abscess or hydatid cyst become infected and mimic like liver abscess .
You are an expert at summarizing long articles. Proceed to summarize the following text: therapeutics based on rna interference hold great promise for the treatment of numerous diseases that are not currently druggable . in recent years , both viral and nonviral methods for delivery and expression of interfering rna molecules have been explored . examples of nonviral delivery vectors include peptides , proteins , aptamers , cationic polymers , lipoplexes , and liposomes . some of these agents are not suitable for systemic in vivo delivery due to factors such as instability in serum , undesirable pharmacokinetics , or the expense or feasibility of scaling - up to obtain therapeutic quantities . in addition to meeting these criteria , an effective delivery agent requires targeting to disease - relevant tissue to avoid potentially toxic side effects of either the agent or the associated interfering rna . one approach for delivering rna to specific cells is to use small proteins or polypeptides that contain a receptor - binding or cell - homing domain to direct the complex to the surface of the desired cell population . these vectors also require rna - binding capability ; a property sometimes conferred using cationic peptide sequences that electrostatically interact with negatively charged rna backbones . as an alternative to cationic peptides , there are many examples of both prokaryotic and eukaryotic proteins that contain canonical double - stranded rna ( dsrna)-binding motifs . these proteins can bind to rna duplexes both specifically ( i.e. , they do not bind duplexed dna ) and with high affinity . a well - studied example of these is protein kinase r ( pkr ; also known as dsrna - activated protein kinase dai ) , which contains two dsrna - binding motifs . in isolation , both motifs can bind dsrna , with affinities of kd = 3.8 10 mol / l and kd = 2 10 mol / l for the n - terminal motif 1 and c - teminal motif 2 , respectively . together , the motifs act cooperatively to enhance the affinity of dsrna binding by 100-fold ( kd = 4 10 pkr drbd motif 1 was fused to a protein transduction domain ( ptd ) composed of three repeats of the cell - penetrating peptide ( cpp ) tat . this fusion protein , termed ptd - drbd , and now commercially available as transductin , facilitated robust gene silencing in cultured cells and effectively reduced luciferase reporter gene expression in the nasal and tracheal passages of a transgenic mouse . while ptd - drbd is effective at small interfering rna ( sirna ) delivery in vitro or when directly administered to target tissues in vivo , it is not suitable for intravenous systemic delivery ; tat motifs interact with serum molecules such as glycosaminoglycans , which can block its internalizing activity . further , because tat is a general cell - binding peptide and cpp , it does not target specific cells or tissue types . we reasoned that targeting drbd with cell - homing peptides or receptor ligands in place of tat would provide a strategy for cell type - specific delivery of sirnas . in the present study , we evaluated drbds derived from pkr as a general sirna delivery vector when fused with either cell - penetrating domains or cell - homing peptides . we found that ptd - drbd , which contains the cationic cell - binding domain tat and a single drbd , can non - specifically interact with sirna , but that stable and specific binding of sirna by fusion proteins lacking tat requires two drbds . unexpectedly , while fusion proteins containing two drbds can mediate internalization of sirna directed by both cpp and cell - homing peptides , gene silencing is attenuated by the inclusion of the larger rna - binding domain . in addition , regardless of the number of drbd motifs , we found that the concentration of protein required for maximal gene silencing exceeds the amount required for sirna binding . these studies demonstrate that the structural properties of drbd , and possibly other protein delivery vectors , can significantly affect the accessibility of sirna to the rna - induced silencing complex and , ultimately , the efficacy of gene silencing . we first tested whether drbd could complex sirna when fused to a peptide with affinity for a specific cell type . for this , we generated a protein , pps - drbd , in which the three tandem copies of the tat peptide in ptd - drbd were replaced with the brain vascular targeting peptide dspahps ( pps ) , a motif that our lab previously identified by in vivo biopanning of mouse brain endothelium ( figure 1 ) . for comparison we also generated drbd alone , which lacks a cell - binding domain ( figure 1 ) . the ability of each protein to bind sirna was measured by electrophoretic mobility shift assay ( figure 2 ) . both pps - drbd and drbd failed to induce a marked sirna mobility shift , even at a 40-fold molar excess of protein relative to sirna ( figure 2a , b ) . to address the possibility that the purification method caused a loss of rna - binding activity , we also tested drbd purified under denaturing conditions followed by refolding ; this protein preparation yielded a comparable result ( supplementary figure s1 ) . in contrast , incubating ptd - drbd with sirna resulted in a mobility shift in a dose - dependent manner ( figure 2c ) . we hypothesized that the ptd domain might be mediating nonspecific interaction with sirna . to test this , we performed an electrophoretic mobility shift assay using dsdna of the same sequence as the sirna ( figure 2c ) . ptd - drbd induced a dsdna shift at a 4:1 molar ratio , though , in contrast to sirna , the dsdna complex failed to migrate into the gel , indicating possible aggregation ( figure 2c ) . to test if the polybasic tat motifs within the ptd domain facilitate binding , we incubated synthetic peptides with sirna and measured electrophoretic mobility . sirna migration was shifted when incubated with tat , but not control peptides , including the polybasic tlh and b2 peptides ( figure 2d ) . these data indicate that drbd alone or fused to the pps - targeting domain is not sufficient to stably bind sirna . however , the tat motifs in ptd - drbd enable nonspecific nucleic acid binding . it was previously reported that proteins containing the complete pkr rna - binding motif ( 2 drbd ) can complex with small dsrna ( ~2025 base pairs ) and sirna . given this , we next tested whether the pps domain ( pps-2 drbd ) fused to 2 drbd could interact with sirna . both 2 drbd alone and pps-2 drbd yielded near complete sirna binding at a 2:1 molar ratio ( figure 3a , b ) . an additional mobility shift was observed at 4:1 molar ratios , which is consistent with previous studies indicating that the length of a small dsrna is sufficient to allow binding of two molecules of 2 drbd in a concentration - dependent manner . increasing the concentration of protein did not result in a further mobility shift or aggregation as evidenced by the same band migration pattern at ratios as high at 16:1 ( figure 3c ) . unlike ptd - drbd , ptd fused to 2 drbd also did not bind to dsdna , and it displayed a sirna - binding profile nearly identical to 2 drbd ( figure 3d ) . in a previous report , ptd - drbd was complexed with sirna at relatively high molar ratios ( 20 - 60:1 ) for in vitro gene silencing . at these concentrations , complexes formed with ptd-2 drbd migrate into the gel but those with ptd - drbd are retained in the gel well , indicative of aggregation ( figure 3e ) . interestingly , at ratios of 40 and 60:1 , the complex undergoes a third mobility shift compared to that at 4:1 ( figure 3d versus figure 3e ) . this suggests that at high concentrations , a third protein molecule may be able to associate with the sirna . collectively , these results indicate that 2 drbd fused to either ptd or cell - targeting peptides can specifically and stably bind sirna , but not dsdna . having established that our 2 drbd fusion proteins could bind to sirna , we next evaluated their ability to deliver sirna and mediate gene silencing in vitro . we hypothesized that the greater specificity and affinity of the sirna interaction with ptd-2 drbd would confer more potent knockdown relative to ptd - drbd . to test this , we measured knockdown of the housekeeping gene hypoxanthine - guanine phosphoribosyltransferase ( hprt ) in hela cells treated with sirna complexes formed with each protein ( figure 4 ) . pps-2 drbd served as a negative control as pps peptide does not bind to hela cells ( supplementary figure s2 ) . as previously mentioned , at least a 2060-fold molar excess of ptd - drbd is typically complexed with sirna for in vitro gene silencing , despite our observation that much less is required for sirna binding . in order to directly compare silencing efficacy of ptd - drbd to ptd-2 drbd , we chose to form complexes with a 60-fold excess of protein over sirna . after 24 hours , hprt message was reduced ~82% in cells treated with ptd - drbd , and no knockdown was observed from treatment with pps-2 drbd ( figure 4 ) . unexpectedly , treatment with ptd-2 drbd only resulted in ~19% knockdown ( figure 4 ) . to test if the size of the transduction domain might somehow be inhibitory when fused to 2 drbd , we also tested a truncated form of ptd that only contained a single tat motif ( 1 tat-2 drbd ) . however , this protein did not mediate any knockdown of hprt ( figure 4 ) . to determine whether a cpp other than tat might yield knockdown , we tested 2 drbd fused to the cpp , penetratin ; however this construct also failed to mediate knockdown ( supplementary figure s3 ) . together , these findings suggest that 2 drbd , but not drbd , inhibits robust gene silencing in vitro . a possible explanation for lack of gene silencing is that the interaction between the ptd and the larger rna - binding domain prevents efficient binding and/or internalization into cells . to address this , we incubated hela cells with complexes containing protein and fluorescently labeled sirna ( figure 5 ) . after incubation , cells were acid washed to remove extracellular complexes and sirna localization was monitored by fluorescence microscopy . cells treated with ptd - drbd showed punctate , vesicular signal , localized near the nucleus ( figure 5a ) . treatment with ptd-2 drbd resulted in a similar pattern but the puncta were larger and there was a higher overall level of internal cell fluorescence ( figure 5a , b ) . treatment using pps-2 drbd or sirna alone did not result in appreciable intracellular sirna localization ( figure 5a ) . since sirna needs to escape into the cytoplasm to interact with the silencing machinery , we also evaluated whether the vesicular localization of sirna changed over time when treated with the different proteins . after 24 hours of incubation , sirna delivered with both ptd - drbd and ptd-2 drbd remained primarily punctate with perinuclear staining ( figure 5c ) . these results show that sirna complexed to both ptd - drbd and ptd-2 drbd can bind to and be internalized into cells . we hypothesized that minimal gene silencing resulted from limited dissociation of the sirna from the high affinity rna - binding domain and/or that the sirna that did dissociate remained trapped in endocytic compartments . to test these possibilities , we treated cells with sirna complexes in the presence or absence of chloroquine , an endosomolytic agent used to enhance gene delivery ( figure 6a ) . in contrast to treatment without chloroquine , hprt message was reduced 92% in cells treated with either ptd - drbd or ptd-2 drbd ( figure 6a ) . hprt levels also decreased after treatment with 1 tat-2 drbd , though the silencing was more modest ( 59% ) ( figure 6a ) . a comparable level of knockdown ( ~60% ) was achieved with penetratin-2 drbd , indicating that sirna delivery can be achieved with a distinct cpp when fused to 2 drbd ( supplementary figure s3 ) . minimal knockdown ( 12% ) was observed using pps-2 drbd ( figure 6a ) . as our initial hypothesis was that ptd-2 drbd 's higher affinity for sirna would enhance silencing activity ( i.e. , less protein is required to deliver the same amount of sirna ) , we tested whether lower concentrations of protein ( 0.4 versus 3.0 mol / l in figure 6a ) could mediate knockdown ( figure 6b ) . in the absence of chloroquine , no protein constructs , including ptd - drbd , reduced hprt mrna levels ( figure 6b ) . however , in the presence of chloroquine , cells treated with ptd-2 drbd but not ptd - drbd had significantly lower levels of hprt message ( 63 versus 4% , respectively ) ( figure 6b ) . together , these results suggest that lack of silencing with ptd-2 drbd in the absence of chloroquine is due to restricted endosomal escape of sirna rather than a lack of dissociation from the rna - binding domain . further , on a molar basis , ptd-2 drbd has a greater ability to deliver sirna into cells as compared with ptd - drbd . it is possible that targeting 2 drbd fusion proteins to specific receptors , rather than using nonspecific cpps , may result in an altered trafficking pathway that yields greater localization of cargo to the cytoplasm versus the endosome . to test this and to evaluate sirna delivery with a receptor - targeted fusion protein , we generated a 2 drbd fusion protein containing three repeats of the b2 peptide sequence ( ghkvkrpkg ) in place of the tat ptd domains ( figure 1a ) . we previously identified the b2 peptide by phage display panning against recombinant transferrin receptor ( tfr ) and showed that adenovirus capsids engineered to present b2 display enhanced transduction of cells in a tfr - mediated manner . coincidentally , the b2 peptide also shares a high degree of similarity with the mature prion protein ( prp ) n - terminal polybasic domain ( kkrpkp ) , which has been shown to have membrane interacting and cell - internalizing properties ( figure 7b ) . we first tested delivery of sirna and hprt knockdown in hela cells , which express tfr and have been used to study transferrin trafficking . interestingly , in the absence of chloroquine , treatment with b2 - 2 drbd sirna complexes resulted in a 30% reduction in hprt message , which is 12% greater knockdown than that obtained with ptd-2 drbd ( figure 7a versus figure 4 ) . in the presence of chloroquine , hprt knockdown was increased to 65% ( figure 7a ) . to test whether sirna delivery and knockdown occurs through the tfr , we competed binding by pre - incubating cells with transferrin or bovine serum albumin control protein before adding the sirna complex . while there was a decrease in hprt knockdown with transferrin pre - treatment relative to the control ( 67 versus 49% ) , this difference was variable and not statistically significant ( figure 7a ) . these results indicate that b2 - 2 drbd can mediate gene silencing but that internalization might be occurring through multiple pathways which include binding tfr and a cell - penetrating mechanism involving the prp - like internalization motif present within the b2 sequence . we first tested whether drbd could complex sirna when fused to a peptide with affinity for a specific cell type . for this , we generated a protein , pps - drbd , in which the three tandem copies of the tat peptide in ptd - drbd were replaced with the brain vascular targeting peptide dspahps ( pps ) , a motif that our lab previously identified by in vivo biopanning of mouse brain endothelium ( figure 1 ) . for comparison we also generated drbd alone , which lacks a cell - binding domain ( figure 1 ) . the ability of each protein to bind sirna was measured by electrophoretic mobility shift assay ( figure 2 ) . both pps - drbd and drbd failed to induce a marked sirna mobility shift , even at a 40-fold molar excess of protein relative to sirna ( figure 2a , b ) . to address the possibility that the purification method caused a loss of rna - binding activity , we also tested drbd purified under denaturing conditions followed by refolding ; this protein preparation yielded a comparable result ( supplementary figure s1 ) . in contrast , incubating ptd - drbd with sirna resulted in a mobility shift in a dose - dependent manner ( figure 2c ) . we hypothesized that the ptd domain might be mediating nonspecific interaction with sirna . to test this , we performed an electrophoretic mobility shift assay using dsdna of the same sequence as the sirna ( figure 2c ) . ptd - drbd induced a dsdna shift at a 4:1 molar ratio , though , in contrast to sirna , the dsdna complex failed to migrate into the gel , indicating possible aggregation ( figure 2c ) . to test if the polybasic tat motifs within the ptd domain facilitate binding , we incubated synthetic peptides with sirna and measured electrophoretic mobility . sirna migration was shifted when incubated with tat , but not control peptides , including the polybasic tlh and b2 peptides ( figure 2d ) . these data indicate that drbd alone or fused to the pps - targeting domain is not sufficient to stably bind sirna . however , the tat motifs in ptd - drbd enable nonspecific nucleic acid binding . it was previously reported that proteins containing the complete pkr rna - binding motif ( 2 drbd ) can complex with small dsrna ( ~2025 base pairs ) and sirna . given this , we next tested whether the pps domain ( pps-2 drbd ) fused to 2 drbd could interact with sirna . both 2 drbd alone and pps-2 drbd yielded near complete sirna binding at a 2:1 molar ratio ( figure 3a , b ) . an additional mobility shift was observed at 4:1 molar ratios , which is consistent with previous studies indicating that the length of a small dsrna is sufficient to allow binding of two molecules of 2 drbd in a concentration - dependent manner . increasing the concentration of protein did not result in a further mobility shift or aggregation as evidenced by the same band migration pattern at ratios as high at 16:1 ( figure 3c ) . unlike ptd - drbd , ptd fused to 2 drbd also did not bind to dsdna , and it displayed a sirna - binding profile nearly identical to 2 drbd ( figure 3d ) . in a previous report , ptd - drbd was complexed with sirna at relatively high molar ratios ( 20 - 60:1 ) for in vitro gene silencing . at these concentrations , complexes formed with ptd-2 drbd migrate into the gel but those with ptd - drbd are retained in the gel well , indicative of aggregation ( figure 3e ) . interestingly , at ratios of 40 and 60:1 , the complex undergoes a third mobility shift compared to that at 4:1 ( figure 3d versus figure 3e ) . this suggests that at high concentrations , a third protein molecule may be able to associate with the sirna . collectively , these results indicate that 2 drbd fused to either ptd or cell - targeting peptides can specifically and stably bind sirna , but not dsdna . having established that our 2 drbd fusion proteins could bind to sirna , we next evaluated their ability to deliver sirna and mediate gene silencing in vitro . we hypothesized that the greater specificity and affinity of the sirna interaction with ptd-2 drbd would confer more potent knockdown relative to ptd - drbd . to test this , we measured knockdown of the housekeeping gene hypoxanthine - guanine phosphoribosyltransferase ( hprt ) in hela cells treated with sirna complexes formed with each protein ( figure 4 ) . pps-2 drbd served as a negative control as pps peptide does not bind to hela cells ( supplementary figure s2 ) . as previously mentioned , at least a 2060-fold molar excess of ptd - drbd is typically complexed with sirna for in vitro gene silencing , despite our observation that much less is required for sirna binding . in order to directly compare silencing efficacy of ptd - drbd to ptd-2 drbd , we chose to form complexes with a 60-fold excess of protein over sirna . after 24 hours , hprt message was reduced ~82% in cells treated with ptd - drbd , and no knockdown was observed from treatment with pps-2 drbd ( figure 4 ) . unexpectedly , treatment with ptd-2 drbd only resulted in ~19% knockdown ( figure 4 ) . to test if the size of the transduction domain might somehow be inhibitory when fused to 2 drbd , we also tested a truncated form of ptd that only contained a single tat motif ( 1 tat-2 drbd ) . however , this protein did not mediate any knockdown of hprt ( figure 4 ) . to determine whether a cpp other than tat might yield knockdown , we tested 2 drbd fused to the cpp , penetratin ; however this construct also failed to mediate knockdown ( supplementary figure s3 ) . together , these findings suggest that 2 drbd , but not drbd , inhibits robust gene silencing in vitro . a possible explanation for lack of gene silencing is that the interaction between the ptd and the larger rna - binding domain prevents efficient binding and/or internalization into cells . to address this , we incubated hela cells with complexes containing protein and fluorescently labeled sirna ( figure 5 ) . after incubation , cells were acid washed to remove extracellular complexes and sirna localization was monitored by fluorescence microscopy . cells treated with ptd - drbd showed punctate , vesicular signal , localized near the nucleus ( figure 5a ) . treatment with ptd-2 drbd resulted in a similar pattern but the puncta were larger and there was a higher overall level of internal cell fluorescence ( figure 5a , b ) . treatment using pps-2 drbd or sirna alone did not result in appreciable intracellular sirna localization ( figure 5a ) . since sirna needs to escape into the cytoplasm to interact with the silencing machinery , we also evaluated whether the vesicular localization of sirna changed over time when treated with the different proteins . after 24 hours of incubation , sirna delivered with both ptd - drbd and ptd-2 drbd remained primarily punctate with perinuclear staining ( figure 5c ) . these results show that sirna complexed to both ptd - drbd and ptd-2 drbd can bind to and be internalized into cells . we hypothesized that minimal gene silencing resulted from limited dissociation of the sirna from the high affinity rna - binding domain and/or that the sirna that did dissociate remained trapped in endocytic compartments . to test these possibilities , we treated cells with sirna complexes in the presence or absence of chloroquine , an endosomolytic agent used to enhance gene delivery ( figure 6a ) . in contrast to treatment without chloroquine , hprt message was reduced 92% in cells treated with either ptd - drbd or ptd-2 drbd ( figure 6a ) . hprt levels also decreased after treatment with 1 tat-2 drbd , though the silencing was more modest ( 59% ) ( figure 6a ) . a comparable level of knockdown ( ~60% ) was achieved with penetratin-2 drbd , indicating that sirna delivery can be achieved with a distinct cpp when fused to 2 drbd ( supplementary figure s3 ) . minimal knockdown ( 12% ) as our initial hypothesis was that ptd-2 drbd 's higher affinity for sirna would enhance silencing activity ( i.e. , less protein is required to deliver the same amount of sirna ) , we tested whether lower concentrations of protein ( 0.4 versus 3.0 in the absence of chloroquine , no protein constructs , including ptd - drbd , reduced hprt mrna levels ( figure 6b ) . however , in the presence of chloroquine , cells treated with ptd-2 drbd but not ptd - drbd had significantly lower levels of hprt message ( 63 versus 4% , respectively ) ( figure 6b ) . together , these results suggest that lack of silencing with ptd-2 drbd in the absence of chloroquine is due to restricted endosomal escape of sirna rather than a lack of dissociation from the rna - binding domain . further , on a molar basis , ptd-2 drbd has a greater ability to deliver sirna into cells as compared with ptd - drbd . it is possible that targeting 2 drbd fusion proteins to specific receptors , rather than using nonspecific cpps , may result in an altered trafficking pathway that yields greater localization of cargo to the cytoplasm versus the endosome . to test this and to evaluate sirna delivery with a receptor - targeted fusion protein , we generated a 2 drbd fusion protein containing three repeats of the b2 peptide sequence ( ghkvkrpkg ) in place of the tat ptd domains ( figure 1a ) . we previously identified the b2 peptide by phage display panning against recombinant transferrin receptor ( tfr ) and showed that adenovirus capsids engineered to present b2 display enhanced transduction of cells in a tfr - mediated manner . coincidentally , the b2 peptide also shares a high degree of similarity with the mature prion protein ( prp ) n - terminal polybasic domain ( kkrpkp ) , which has been shown to have membrane interacting and cell - internalizing properties ( figure 7b ) . we first tested delivery of sirna and hprt knockdown in hela cells , which express tfr and have been used to study transferrin trafficking . interestingly , in the absence of chloroquine , treatment with b2 - 2 drbd sirna complexes resulted in a 30% reduction in hprt message , which is 12% greater knockdown than that obtained with ptd-2 drbd ( figure 7a versus figure 4 ) . in the presence of chloroquine , hprt knockdown was increased to 65% ( figure 7a ) . to test whether sirna delivery and knockdown occurs through the tfr , we competed binding by pre - incubating cells with transferrin or bovine serum albumin control protein before adding the sirna complex . while there was a decrease in hprt knockdown with transferrin pre - treatment relative to the control ( 67 versus 49% ) , this difference was variable and not statistically significant ( figure 7a ) . these results indicate that b2 - 2 drbd can mediate gene silencing but that internalization might be occurring through multiple pathways which include binding tfr and a cell - penetrating mechanism involving the prp - like internalization motif present within the b2 sequence . in this study , we sought to determine whether pkr - derived drbd could serve as a protein platform for cell type - specific targeted delivery of sirna . for this , we compared knockdown mediated by cell - binding peptides fused to either a single drbd or the complete pkr rna - binding motif that contains two drbds . our evaluation revealed that ptd - drbd , which can mediate robust gene silencing as shown in earlier work , does not stably bind sirna in the absence of ptd . this result is consistent with previous studies of the rna - binding activity of pkr demonstrating that although the n - terminal drbd motif 1 can bind short rnas ( 2025 bp ) , the binding is of low affinity and unstable . this suggests that the tat peptides in ptd - drbd are acting in tandem with the drbd to enable stable sirna binding through electrostatic interaction , which is further supported by the observation that dsdna also binds to ptd - drbd when in molar excess . the requirement for ptd raises the question as to what degree the drbd contributes to the sirna - binding activity of ptd - drbd . it could be that the drbd is not absolutely required and that a polypeptide composed of only the ptd or several tandem repeats of tat may also have sirna complexation properties that could confer silencing activity . in the contrast to ptd - drbd , we showed that the inclusion of both pkr - derived drbds results in stable and dsrna - specific binding , even when the drbds are fused to the tat - containing ptd . this means that 2 drbd can be fused to various cell - binding peptides , including highly positively charged sequences , and still maintain binding of the sirna restricted to the drbd . this property is important as it prevents the cell - binding peptide from interacting with the sirna , which could interfere with its ability to associate with cell surface moieties . both ptd - drbd and ptd-2 drbd can complex with sirna and be internalized , however , ptd-2 drbd complexes are more readily internalized at lower concentrations compared with ptd - drbd , as inferred from a greater degree of gene silencing . this may occur because at lower molar ratios of protein to sirna , the sirna binds to the ptd domain of ptd - drbd and this impedes uptake of the complex into the cell . in contrast , at the same molar ratios , sirna binding to ptd-2 drbd is restricted to the drbds , allowing the unencumbered ptd to mediate more effective cell surface binding and internalization . despite the improved sirna binding and internalization properties of 2 drbd fusion proteins , we found that these complexes are not nearly as robust at mediating gene silencing as ptd - drbd . this was true for both tat and penetratin fusions , which supports the hypothesis that it is not the specific cpp that is restricting silencing but rather a property of the 2 drbd domain . penetratin has been used to mediate rapid internalization of sirna when directly conjugated to modified sirna via a disulfide bond . the resulting molecule is smaller than the complexes tested in our studies , which may explain its greater silencing activity . interestingly , despite the observation that sirna can bind to 2 drbd proteins at a 24 molar excess , maximal silencing requires a greater than eightfold molar excess . it may be that excess , unbound protein facilitates knockdown in a manner that does n't necessarily require direct interaction with the sirna . one possibility is that at high concentrations , free cpp stimulates cargo uptake as reported for tat - dna complexes . alternatively , the high protein concentration may prevent sirna dissociation from drbd proteins after dilution into the transfection media . treatment of cells with chloroquine resulted in silencing comparable with ptd - drbd , which indicates that the 2 drbd complexes are sequestered to endosomal / lysosomal compartments and do not efficiently escape into the cytoplasm . this is consistent with earlier studies showing that sirna complexed with a protein containing the pkr drbds could facilitate gene silencing , but only when admixed with the fusogenic peptide kala . one explanation for this observation is that both ptd - drbd and 2 drbd fusion proteins traffic through the same compartments but that the larger relative size and/or structural features of the 2 drbd fusion protein complex hinder endosomal escape . alternatively , the 2 drbd constructs might enter the cell through a pathway distinct from ptd - drbd . it has been reported that ptd - drbd and other tat fusion proteins utilize a macropinocytic uptake mechanism that promotes cytoplasmic cargo delivery . however , there are differing reports on the exact mechanism of tat uptake and recently it was shown that tat peptide fused to relatively small cargo could directly translocate across the plasma membrane whereas fusion to larger cargo results in endocytic internalization . when considering the relationship between complex size and bioactivity , it is of note that at the molar concentrations of protein used for in vitro gene silencing ( 20 - 60:1 ) , ptd - drbd complexes are aggregated . counter - intuitively , aggregates are more efficient at mediating gene silencing than soluble or lower - order complexes . thus , 2 drbd complexes might preferentially traffic through endocytic compartments that lead to entrapment while ptd - drbd trafficking allows for enhanced cytoplasmic localization of bound sirna . in support of the concept that utilizing different internalization mechanisms may affect gene - silencing efficacy , our transferrin receptor - targeted protein provided enhanced knockdown relative to ptd-2 drbd in the absence of chloroquine . however , because transferrin did not compete with b2 - 2 drbd - mediated gene knockdown , the b2 peptide may be utilizing both the transferrin receptor as well as other cell surface moieties , via interaction with the b2 polybasic motif , for internalization . one possibility is that the net positive charge of the b2 domain facilitates nonspecific electrostatic interaction with negative charges on the cell surface ( e.g. , from glycosaminoglycans ) . in support of this , internalization of the prp cell - penetrating motif is inhibited by exogenous heparin and chondroitin sulfate . this property may , in part , explain the incomplete inhibition of silencing by transferrin receptor competition . it would be interesting to evaluate whether the b2 peptide , in addition to binding tfr , is a novel cpp . overall , our study demonstrates that protein - based sirna delivery vectors utilizing canonical drbds display several properties that could be used for cell type - specific targeting . however , the effect of the drbd structure on the bioavailability of sirna delivered with the 2 drbd proteins evaluated in this study poses a significant limitation to their utility . it would be interesting to test whether further vector engineering could overcome these shortcomings . for example , a membrane - destabilizing fusogenic / endosomolytic peptide could be introduced into the 2 drbd fusion protein . alternatively , chemical modification of the proteins with endosome - disrupting groups might enhance cytoplasmic cargo delivery . future studies will show whether these , or other strategies , will yield more potent 2 drbd - based vectors for delivery of sirna targeted to specific cell types . hela cells were cultured in complete medium ( dulbecco 's modified eagle 's medium containing 10% fetal bovine serum ) at 37 c , 5% co2 . unlike the other constructs , penetratin-2 drbd contains a flexible linker previously used to generate a penetratin - mcherry protein for cellular delivery . oligonucleotides ( idt , coralville , ia ) were annealed , extended , and pcr amplified . digested pcr products were ligated into pet11d expression vector ( stratagene , santa clara , ca ) backbone generated by digesting with ncoi and bamhi restriction enzymes ( new england biolabs , ipswich , ma ) . the sequence of each expression plasmid was confirmed by sequencing performed at the university of iowa dna core facility . all proteins were expressed and purified except for ptd - drbd , which was obtained from idt ( commercially sold as transductin ) . pet11d expression plasmids were transformed into bl21 star e. coli ( life technologies , carlsbad , ca ) . a 25 ml starter culture grown using a freshly transformed colony was inoculated into 500 ml of lb containing 100 g / ml ampicillin and grown at 37 c , 220 rpm shaking , until the od600 reached between 0.6 and 0.8 . protein expression was induced with 1 mmol / l iptg and cultures were grown an additional 4 hours at 37 c , 220 rpm shaking . cultures were centrifuged at 5,000 rpm at 4 c for 15 minutes to pellet bacteria . protein constructs containing 2 drbd required denaturing purification and on - column refolding in order to remove bacterial rna bound to the protein . for purification under denaturing conditions , the pellet was resuspended in 5 ml of buffer a ( 50 mmol / l sodium phosphate , ph 7.4 ) containing complete mini protease inhibitors ( roche , basel , switzerland ) ; 30k units of rlysozyme ( novagen , madison , wi ) , 150 units of benzonase ( sigma - aldrich , st . louis , mo ) , and mgcl2 to a final concentration of 2 mmol / l and the suspension was incubated on ice for 20 minutes . ten milliliter of 1.1 buffer b ( 50 mmol / l sodium phosphate , 555.5 mmol / l nacl , 22.2 mmol / l imidazole , 6.66 mol / l guanidine hcl , 2.2 mmol / l -mercaptoethanol ; ph 7.4 ) was added to the suspension that was then sonicated on ice seven times for 30 seconds with the amplitude set at 50 using a model ge50 ultrasonic processor ( sonics & materials , newton , ct ) ; 35 ml of buffer b was added for a final volume of 50 ml and the lysate was allowed to incubate on ice for 25 minutes to solubilize proteins . the lysate was clarified by centrifugation for 40 minutes at 16,000 g , 4 c . the supernatant was then filtered through a 0.4 m pvdf filter ( millipore , billerica , ma ) . using a biorad biologic lp system ( bio - rad , hercules , ca ) , denatured proteins were bound to a hitrap imac column ( ge healthcare , waukesha , wi ) charged with cobalt chloride . the column was washed with 10 ml of buffer b diluted to 1 with buffer a. bound protein was refolded on - column by applying a 0100% linear gradient over 80 minutes at 0.5 ml / minute flow rate starting with 100% buffer b/0% buffer c ( 50 mmol / l sodium phosphate , 500 mmol / l nacl , 20 mmol / l imidazole ; ph 7.4 ) and ending with 0% bufferb/100% buffer c. purified , refolded proteins were eluted with buffer d ( 50 mmol / l sodium phosphate , 300 mmol / l nacl , 150 mmol / l imidazole ; ph 7.4 ) and concentrated using amicon ultra 10k mwco centrifugal filters ( millipore ) . concentrated eluates were buffer exchanged into phosphate - buffered saline + 10% glycerol using zeba desalting columns ( thermo fisher scientific , waltham , ma ) . for non - denaturing purification of the drbd and pps - drbd proteins used in figure 2a , b , benzonase- and lysozyme - treated lysates were clarified by centrifugation and then combined with 1 buffer b containing 750 mmol / l nacl . extracts were applied to the imac cobalt column and washed with buffer b containing 1 mol / l nacl . after elution , extracts were treated again with benzonase to remove co - purifying bacterial nucleic acid . electrophoretic mobility shift assays . the sirna used was ambion silencer rest sirna ( catalog no . dsdna was prepared using dna oligonucleotides ( idt ) of the same sequence as the sirna . dna oligonucleotides were annealed by heating to 95 c for 4 minutes in annealing buffer ( 100 mmol / l potassium acetate , 2 mmol / l magnesium acetate , 30 mmol / l hepes , ph 7.4 ) and then allowed to cool to room temperature . in all gel shifts except for in figure 3e , 8 pmol of sirna or dsdna were combined with varying amounts of each protein in a final volume of 8 l , yielding concentrations of 1 mol / l sirna or dsdna . in figure 3e , 1 pmol of sirna was complexed with proteins in order to achieve the higher molar protein ratios in a small volume . mixtures were incubated at room temperature for 15 minutes and then combined with 2 l of 5 tbe loading buffer ( life technologies ) . the entire reaction volumes were electrophoresed on a 0.5 tbe , 6% acrylamide gel at 90 v for 45 minutes in 0.5 tbe running buffer . the gel was stained with 0.5 g / ml ethidium bromide ( bio - rad ) and visualized using a versadoc 5000 mp ( bio - rad ) . for gel shifts with synthetic peptides , n - terminally biotinylated tat ( rkkrrqrrr ) , pps ( dspahps ) , tlh ( gwtlhnk ) , or b2 ( ghkvkrpkg ) peptides ( american peptide company , sunnyvale , ca ) were incubated at a final concentration of either 20 or 40 mol / l with 1 mol / l sirna and analyzed as described above . hela cells were plated in 48-well dishes at 23 10 cells / well in regular media ( dulbecco 's modified eagle 's medium with 10% fetal bovine serum ) 1416 hours before sirna treatment . complexes were formed by incubating 6 pmol of hprt s1 dicer - substrate sirna ( idt ) or universal negative control ( nc1 ) dicer - substrate sirna ( idt ) with either 48 or 360 pmol of protein on ice for 30 minutes . dulbecco 's modified eagle 's medium containing 10% q - serum ( q - media ) prepared as previously described was added to complexes to yield a final concentrations of 50 nmol / l sirna and 0.4 or 3 mol / l protein . media and sirna / protein mixtures were added to cells that were previously washed one time with q - media and then preincubated in q - media for 30 minutes at 37 c . complexes were incubated with cells for 4 hours and then removed and replaced with fresh complete medium . for chloroquine experiments , chloroquine was added during both the sirna incubation step and during the 24-hour incubation period . for transferrin receptor blocking experiments , hela cells were pretreated for 30 minutes with 5 mol / l holo - transferrin ( sigma - aldrich ) or bovine serum albumin ( new england biolabs ) in q - media before knockdown . trizol reagent ( life technologies ) was used to extract and purify total rna from treated cells . cdna was synthesized from rna using the multiscribe high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's protocol . cycling was performed on a 7900ht fast real time pcr system ( applied biosystems ) . gapdh was used to calculate the ct for hprt in hela cells , knockdown with hprt sirna was calculated as the average relative quantity from three independent experiments and variation is represented as the sd of the three relative quantities . the variation of hprt in cells treatment with nc1 sirna was calculated as the population sd of the ct s for each treatment condition in the three independent experiments . hela cells were incubated with complexes containing 50 nmol / l scrambled sequence cy3-sirna ( idt ) and 2 mol / l protein prepared as described above for hprt gene - silencing experiments . after 3 hours of incubation , the medium containing complexes was removed and the cell nuclei were stained for 10 minutes at 37 c with hoechst 3342 dye ( life technologies ) diluted into complete media . cells were then washed three times with complete media followed by washing with low ph acid buffer ( 0.2 mol / l glycine , ph 2.8 ) . after the acid wash , cells were washed one additional time with dulbecco 's pbs ( life technologies ) before examination by fluorescence microscopy . the brightness and contrast of the primary images were adjusted equivalently using image j software ( nih , bethesda , md ) . the fluorescence intensity ( integrated density ) was measured for 16 cells per analyzed image . the integrated density was calculated and corrected by subtracting background signal from regions proximal to the measured cells . mean fluorescence intensity and sd was calculated from two independent experiments . peptide cell binding and recombinant protein characterization . electrophoretic mobility shift assay of sirna binding to drbd prepared under denaturing purification conditions . silencing of hprt mediated by penetratin-2 drbd fusion protein in the presence or absence of chloroquine . silencing of hprt mediated by penetratin-2 drbd fusion protein in the presence or absence of chloroquine .	delivery of small interfering rna ( sirna ) targeted to specific cell types is a significant challenge for the development of rna interference - based therapeutics . recently , ptd - drbd , a double - stranded rna binding domain ( drbd ) fused to the tat protein transduction domain ( ptd ) , was shown to be effective at delivering sirna in a non - cell type - specific manner . here , we evaluated the potential of drbd as a general protein platform for targeted small interfering rna ( sirna ) delivery . we found that a single drbd was insufficient to stably complex sirna when fused to targeting peptides other than ptd , which facilitated nonspecific nucleic acid binding . in contrast to ptd - drbd , fusion proteins containing two drbds ( 2 drbd ) yielded specific and stable sirna binding . these proteins could mediate the cellular uptake of sirna in vitro , though compared with ptd - drbd gene silencing was attenuated by endosomal entrapment . our findings suggest that unlike a single drbd , 2 drbd inhibits sirna escape into the cytoplasm and/or induces an internalization pathway distinct from that of ptd - drbd . collectively , these data indicate that while 2 drbd retains sirna - binding activity when fused to different cell surface - interacting peptides , the utility of 2 drbd for cell - specific rna interference is limited without further protein engineering to enhance the bioavailability of the delivered sirnas .
You are an expert at summarizing long articles. Proceed to summarize the following text: the nhis is an ongoing survey of the health status , health care access , and behaviors of the u.s . civilian noninstitutionalized population conducted by the national center for health statistics ( nchs ) ( 11 ) . the nhis uses multistage probability sampling to select approximately 41,000 households and 107,000 individuals each year . the annual response rate of nhis between 1997 and 2004 ranged from 87 to 92% . here , we used data from 242,383 ( approximately 30,000 per year ) adults aged 18 years and older ( one randomly selected from each family to receive additional nhis questions ) from the survey years 19972004 whose data were linked to the national death index , a computer database of all deaths in the u.s . approximately 89% of all participants data ( range of 8693% across survey years ) were considered adequate for accurate linkage ( 12 ) . interviewers assessed diabetes status by asking participants if a doctor or other health professional had ever told them that they had diabetes or sugar diabetes and the number of years since diagnosis . in addition , respondents were queried for age , race / ethnicity , sex , education , family income , history of cvd , and self - reported height and weight , which were used to compute bmi ( kg / m ) . income was used to compute the poverty index ratio , an index of income assessed in relation to need , with a score of 1 representing the official federal poverty threshold , a score of < 1 indicating a relative level of poverty , and a score of > 1 representing income as a multiple of the poverty level . mortality , including causes of death , was determined from the national death index , which was linked with the nhis ( 12 ) . the participants vital status was available from the time of the survey 19972004 through 31 december 2006 ( up to 10 years ) . underlying causes of death were classified according to the codes of the international classification of diseases , ninth revision including those coded 390 to 448 classified as cvd deaths ( icd-10 codes 100178 ) . our primary objective examined whether death rates in later samples of persons with diabetes were different from those of earlier samples . secondary objectives examined whether changes in mortality over time differed between the diabetic and nondiabetic cohorts and between various age , sex , race , and socioeconomic subgroups of the diabetic population . we grouped the survey participants into four consecutive 2-year samples ( 19971998 , 19992000 , 20012002 , and 20032004 ) and calculated death rates ( deaths per person - years ) for a maximum of 3 years of follow - up . participants data were censored at the time ( quarter year ) of death or after 3 years if still alive . we used tests and anova to determine whether demographic and health characteristics of the population changed across surveys . we compared survival distributions between the samples according to diabetes status and key covariates using life table analysis and log rank tests . we then used cox proportional hazards analysis to compare death rates between earlier and later samples among the diabetic population , nondiabetic population , and among various subgroups of the diabetic population . we also compared death rates between individuals with and without diabetes . to examine the potential bias that would exist if later cohorts are diagnosed earlier in the natural history of diabetes , we excluded those with less than 2 years of diabetes duration from the most recent sample . survey weights were applied to make study estimates representative of the u.s . civilian noninstitutionalized adult population with diabetes . in addition , survey weights were adjusted to account for potential nonresponse bias associated with age , sex , or race / ethnicity . results were considered significant if p < 0.05 , and analyses were conducted with sudaan version 10.0.1 to account for the complex nhis design ( 13 ) . interviewers assessed diabetes status by asking participants if a doctor or other health professional had ever told them that they had diabetes or sugar diabetes and the number of years since diagnosis . in addition , respondents were queried for age , race / ethnicity , sex , education , family income , history of cvd , and self - reported height and weight , which were used to compute bmi ( kg / m ) . income was used to compute the poverty index ratio , an index of income assessed in relation to need , with a score of 1 representing the official federal poverty threshold , a score of < 1 indicating a relative level of poverty , and a score of > 1 representing income as a multiple of the poverty level . mortality , including causes of death , was determined from the national death index , which was linked with the nhis ( 12 ) . the participants vital status was available from the time of the survey 19972004 through 31 december 2006 ( up to 10 years ) . underlying causes of death were classified according to the codes of the international classification of diseases , ninth revision including those coded 390 to 448 classified as cvd deaths ( icd-10 codes 100178 ) . our primary objective examined whether death rates in later samples of persons with diabetes were different from those of earlier samples . secondary objectives examined whether changes in mortality over time differed between the diabetic and nondiabetic cohorts and between various age , sex , race , and socioeconomic subgroups of the diabetic population . we grouped the survey participants into four consecutive 2-year samples ( 19971998 , 19992000 , 20012002 , and 20032004 ) and calculated death rates ( deaths per person - years ) for a maximum of 3 years of follow - up . participants data were censored at the time ( quarter year ) of death or after 3 years if still alive . we used tests and anova to determine whether demographic and health characteristics of the population changed across surveys . we compared survival distributions between the samples according to diabetes status and key covariates using life table analysis and log rank tests . we then used cox proportional hazards analysis to compare death rates between earlier and later samples among the diabetic population , nondiabetic population , and among various subgroups of the diabetic population . we also compared death rates between individuals with and without diabetes . to examine the potential bias that would exist if later cohorts are diagnosed earlier in the natural history of diabetes , we excluded those with less than 2 years of diabetes duration from the most recent sample . survey weights were applied to make study estimates representative of the u.s . civilian noninstitutionalized adult population with diabetes . in addition , survey weights were adjusted to account for potential nonresponse bias associated with age , sex , or race / ethnicity . results were considered significant if p < 0.05 , and analyses were conducted with sudaan version 10.0.1 to account for the complex nhis design ( 13 ) . population according to diabetes status across the four consecutive 2-year nhis samples . among the population with diabetes , there are consistent increases over time in the levels of education , income , and obesity and a decrease in the proportion of smokers , sedentary behavior , and difficulty walking . there were no significant changes in age , race / ethnicity , history of cvd , or diabetes duration . demographic trends over time were similar for the nondiabetic population , except that there was also a slight increase in age and the number of hispanics . characteristics of the u.s . adults with and without diabetes according to 2-year study cohorts , 19972004 among the population with diagnosed diabetes , 3-year cvd death rates declined by 4.0 deaths per 1,000 person - years from the 19971998 sample ( 9.5 per 1,000 person - years ) to the 20032004 sample ( 5.6 per 1,000 person - years ) ( p for trend < 0.01 ) ( table 2 ) . in multivariate analyses adjusting for age , sex , race / ethnicity , and diabetes duration , diabetic adults in the most recent sample ( 20032004 ) had 40% lower cvd ( hazard rate ratio [ hrr ] , 0.60 [ 95% ci 0.460.77 ] ) mortality and 23% lower all - cause mortality ( 0.77 [ 0.650.90 ] ) than people in the earliest sample ( 19971998 ) ( p < 0.05 for each ) . there were no significant changes in the rates of cancer mortality among persons either with or without diabetes . in sensitivity analyses excluding those with less than 2 years of diabetes duration from the most recent sample , all - cause and cvd mortality rates ( deaths per 1,000 person - years ) according to cohort and diabetes status cvd death rates declined among the nondiabetic population as well , but the magnitude of decline was weaker ( from 3.7 to 3.3 deaths per 1,000 ; hrr 0.89 [ 95% ci 0.78 to 1.03 ] , p for trend = 0.07 ) than that observed for diabetic adults and there was not a significant decline in all - cause mortality ( p = 0.36 ) . as a result , the excess cvd death rate associated with diabetes ( i.e. , compared with those without diabetes ) declined from 5.8 to 2.3 deaths per 1,000 , and the all - cause death rate difference between people with and without diabetes declined from 10.8 to 6.1 deaths per 1,000 . all - cause and cvd death rates in the diabetic population declined among both men and women ( table 3 ) . however , there was a slightly greater magnitude of decline among men ( 5.2 deaths per 1,000 decline for men vs. 3.5 per 1,000 for women ; p = 0.02 for interaction term ) . these trends for both men and women again paralleled less dramatic reductions in the nondiabetic population , resulting in reductions in the excess cvd mortality from 7.5 to 2.5 deaths per 1,000 for diabetic men and from an excess 4.8 to 1.8 deaths per 1,000 women . all - cause and cvd mortality rates according to cohort and diabetes status and sex stratified analyses indicated that reductions in all - cause and cvd mortality were observed relatively uniformly across the population ( table 4 ) . hrrs of decline were similar across groups , but absolute rates of decline tended to be greater in that subgroup with high initial rates , such as older adults and people with long - duration diabetes . of note , however , was the high relative reduction in cvd and all - cause mortality among young adults with diabetes ( age 1844 years ) . rates of decline ( deaths per 1,000 person - years ) and multivariate hrrs for all - cause mortality associated with cohort status among specific subgroups of the diabetic population this analysis of nationally representative samples of adults with and without diabetes reveals impressive reductions in cvd and all - cause mortality between 1997 and 2006 . the rates of improvement among those with diabetes have exceeded those of the nondiabetic population , resulting in more than a 50% reduction of the excess death rates that have been repeatedly attributed to diabetes . although excess mortality risk remains high about 2 deaths per 1,000 due to cvd and about 6 all - cause deaths this excess risk is now considerably lower than previous reports and consistent with improvements in several risk factors , complications , and indicators of medical care and representative of gradual , ongoing improvement in health for people with diagnosed diabetes ( 35,14 ) . improvements were observed approximately equally in women and men , which contrasts with earlier analyses . finland have suggested that the declines in mortality were stronger among men than women ( 9,10,15 ) . although no explanation for that finding was confirmed , differences in risk factor management and pathophysiology between diabetic men and women were both cited as possible factors ( 16 ) . the present analyses suggest , however , that previous sex - related differences in trends may have dissipated over time . the rate of decline in death was as great among women as men , and this finding was maintained after adjusting for differences in demographic factors , comorbidity , and time since diagnosis of disease . our findings of improved life expectancy support recent regional studies in the u.s . , including north dakota , framingham , and minnesota , as well as population - based studies in ontario , denmark , scotland , norway , and finland ( 68,17,18 ) . these trends parallel other improvements in levels of risk factors and rates of complications among the overall u.s . in addition , steady improvements in quality and organization of care , self - management behaviors , and medical treatments , including pharmacological treatment of hyperlipidemia and hypertension , could each have contributed to reductions in death rates ( 21 ) . incidence of lower extremity amputation , end - stage renal disease , and cvd hospitalization have each declined steadily ( 22 ) . reductions in mortality are likely to be influenced by multiple factors , however , and thus may lag behind declines in specific risk factors . the possibility of such lags in secular trends is a reminder that , since improvements in smoking prevalence and blood pressure levels may have slowed during the past decade ( 23 ) , a flattening of death rates in the near future is still conceivable and should be evaluated . although the nhis provides the largest nationally representative cohort data with diagnosed diabetes , our findings are limited by reliance on self - report to define diabetes ; at least 20% of cases with diabetes are undiagnosed ( 13 ) . because the fraction of diabetes cases that remain undiagnosed may be decreasing , later cohorts of diabetes could be enriched with people who had their diabetes detected earlier , possibly contributing to lower mortality . in addition , individuals with diabetes who were unaware of their condition would have been misclassified into the nondiabetic group . because this group is likely to have an increased death rate relative to people without diabetes , the excess mortality risk associated with diabetes is likely to be an underestimate . to examine this , we conducted sensitivity analyses in which we excluded those with less than 2 years of diabetes duration from the most recent sample . this had little difference in our findings , indicating that the reductions in death rates across the samples appears not to be explained by earlier diagnosis . there was also little difference in the prevalence of clinically significant cvd or functional impairment ( i.e. , difficulty walking ) across samples . nevertheless , to rule out a bias due to earlier detection in later samples , studies will need to compare death rates from cohorts that include individuals with undiagnosed diabetes , for which few studies exist . however , our analyses have the advantage of having a larger cohort of people with diabetes ( about 2,000 cases per year ) , along with a national probability sampling methodology , making it the most representative contemporary examination of death rates among the u.s . although our analyses indicate encouraging reduction in mortality and , indirectly , continued success in diabetes care , these findings have ironic implications for the future u.s . death rates , along with earlier detection of undiagnosed diabetes and incidence of new diabetes cases , are the principal determinants of future diabetes prevalence and the disease and economic burden that follows . recently published models indicate thatdeclining mortality among people with diabetes can lead to a substantial increase in prevalence ( 24,25 ) . studies from national registries in denmark , sweden , and provincial registries in canada portray a similar picture , wherein the death rate has declined sufficiently so that prevalence is likely to continue to increase even if incidence trends flatten ( 18,26 ) . thus , the excess mortality associated with diabetes , though declining , will be spread among a considerably greater proportion of the population . ultimately , this means that the need for vigilant efforts to prevent vascular and neuropathic complication and early mortality associated with diabetes along with efforts to reduce diabetes incidence will continue to be major demands into the future .	objectiveto determine whether all - cause and cardiovascular disease ( cvd ) death rates declined between 1997 and 2006 , a period of continued advances in treatment approaches and risk factor control , among u.s . adults with and without diabetes.research design and methodswe compared 3-year death rates of four consecutive nationally representative samples ( 19971998 , 19992000 , 20012002 , and 20032004 ) of u.s . adults aged 18 years and older using data from the national health interview surveys linked to national death index.resultsamong diabetic adults , the cvd death rate declined by 40% ( 95% ci 2354 ) and all - cause mortality declined by 23% ( 1035 ) between the earliest and latest samples . there was no difference in the rates of decline in mortality between diabetic men and women . the excess cvd mortality rate associated with diabetes ( i.e. , compared with nondiabetic adults ) decreased by 60% ( from 5.8 to 2.3 cvd deaths per 1,000 ) while the excess all - cause mortality rate declined by 44% ( from 10.8 to 6.1 deaths per 1,000).conclusionsdeath rates among both u.s . men and women with diabetes declined substantially between 1997 and 2006 , reducing the absolute difference between adults with and without diabetes . these encouraging findings , however , suggest that diabetes prevalence is likely to rise in the future if diabetes incidence is not curtailed .
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Proceed to summarize the following text: according to the world health organization ( who ) , the assessing of health and before - after therapy outcomes are not sufficient for indicating the severity of disease , rather evaluation of quality of life is essential as a complementary assessment ( 13 ) . a common method in order to evaluate quality of life is questionnaires , which the developed english ones are usually translated and adapted to be applicable according to the other cultures and languages ( 4 , 5 ) . in a more specific view to health , it is true also regards to the speech disorders that despite of numerous objective evaluations to assess speech behavior in adults ( 6,7 ) or children ( 8,9 , 10 ) , however the importance of subjective questions to evaluate the speech and in a more details voice disorders , using patient - centered measures have been asserted in some studies ( 1113 ) . although there are several tests to do it but one of the most conventional is voice handicap index [ vhi ] ( 14 ) . ( 12 ) that consists of three domains including functional , physical , and emotional aspects of voice disorders and is administered by patients themselves regardless to the type of their voice disorder in a five - point likert - type scale manner for each item ( from 0 = never to 4 = always ) . patients with severe voice disorder would gain higher total vhi scores , which the highest score can be 120 . it is a valid and reliable instrument for assessing self - perception of patient s voice handicap ( 12 ) , which has been translated and validated into many languages ( 1323 ) . it is remarkable that despite of lack of the strong relationship between objective voice evaluations ( e.g. acoustic and aerodynamic ) and vhi scores ( 2426 ) , it is widely applied as a useful instrument to evaluate patient s self - perception from their severity of voice disorder , its effect on their life and also therapy outcomes ( 27 ) . although many studies have indicated that objective measurements are of valuable importance in diagnosis voice disorders however those are not applicable to evaluate patients self - assessment of the range of their disability ( 28 ) . the experienced disability resulted from voice disorder is affected by several factors including occupation , psychological mood , social interactions , and individual s routine functions ( 29 ) . there are several instruments to assess subjective self - assessment of voice disorders such as the voice symptom scale ( voiss ) ( 30 ) , the voice related quality of life measure ( v - rqol ) ( 31 ) , the vocal performance questionnaire ( vpq ) ( 32 ) , and the voice handicap index ( vhi ) ( 12 ) . although all of these questionnaires were valuable but vhi was more accepted and has been used widely in evaluation of outcome of treatments at clinics and in research . in addition , the agency of healthcare research and quality in 2012 announced the vhi as a valid and reliable diagnostic instrument ( 33 ) . nowadays translation and adaptation has been done on the vhi into many languages including german ( 34 ) , chinese ( 35 ) , portuguese ( 36 ) , and polish ( 37 ) . all of them used vhi for patients with different voice disorders but there are only two studies compared patients group with control group ( 29 , 36 ) . jacobson s study indicated high test - retest reliability , internal consistency , and correlation between vhi scores and patient s self - judgment of severity of their disorder ( 12 ) . by getting information about validity and reliability of the vhi versions in other languages and showing high scores for them , in addition to research goals , some studies supported the applicability of vhi in clinic and found high sensitivity to show patient s perceived changes related to their voice after variety of interventions including surgery , medical , and voice therapy ( 3840 ) . one of the advantages of the vhi is its attention to the main dimensions of the patient s qol voice related experiences including the functional domain ( the range of the patient s ability to communicate in different conditions via their voice ) , the physical domain ( whatever the patient percept during vocalization ) , and the emotional domain [ attends to the emotional aspects of the voice problem ] ( 41 ) . in order to define item - total correlations of vhi , in a study by applying pearson s correlation those with lower than 0.35 coefficiency were deleted or reformulated ( 42 ) . the first is regard to its popularity of usage in clinics for patients with voice disorder . the second one is the highest frequency of vhi applicability as an instrument to evaluate voice related qol compared to the others in voice studies ( 43 ) . in addition , confirmation of the vhi s validity and reliability by agency of healthcare research and quality increased its value and importance ( 44 ) . after the acknowledgment at 2002 , the vhi was accepted to be translated and adapted as a tool to assess the voice problems impacts on patient s participation in other cultures and languages and also its psychometric values ( reliability and validity ) were defined for every languages including ; german ( 4549 ) , portuguese ( 50 ) , lithuanian ( 51 ) , polish ( 52,53 ) , french ( 54 ) , taiwanese ( 55,56 ) , hebrew ( 57 ) , swedish ( 58 ) , italian , dutch , and flemish dutch ( 59 ) . in addition , recently equalization was done on eight european translations ( 59 ) . reliability refers to the equality of values produced from an instrument under the same conditions . there are different prevalent methods to define reliability of an instrument consist of parallel - test reliability , test retest reliability , split half reliability , and internal consistency ( 60 ) . validity implies that what is intended to be measured is really what the diagnostic tool evaluates . in order to investigate validity two applied approaches are content validity and construct validity ( 61 ) . content equivalency of the translated items is not expected by only direct translation an instrument from a language to another ( 62 , 63 ) . it has been accepted that in order to validate and possibility of applying a tool in a cross - cultural study the back - translation is necessary ( 64 , 65 , and 66 ) . the brislin s back - translation model is used as the most widely preferred method among all translation models . although researchers because of some problems in performing brislin s back - translation model use the modified rather the classic one and apply it in a team method or use a combination of the other translation approaches ( 67 ) . however , these researchers may eliminate their problems in translation temporarily by not using the classic brislin s back - translation model completely but unfortunately , usually it results in decreased validity and content equivalency of the translated version . therefore , the fact emphasizes the importance of considering appropriate methods , which guaranties valid translated instrument especially in cross - cultural studies before conducting it . brislin has suggested some rules to enhance content equivalency between translated and original instrument in cross - cultural studies such as avoiding from copying the content and length of the original version in direct translation ( 68 ) . brislin in order to gain this object has also recommended four methods consist of back translation procedure , committee technique , pretest method , and bilingual approach ( 62 ) . generally , in the back translation procedure the instrument firstly is translated from original language to the target language and then is performed reversely by another independent translator and finally the two original and target language translated versions are compared and edited several times by another translators until all of them agree about the equivalency of the two versions ( 62 ) . given the fact that the back translation technique is performed by several independent bilingual translators ( 69 ) therefore , the problem of finding high numbers of bilingual translators would be a weakness of this model . the committee procedure is administered by a team of bilingual expert translators ( more than three people ) and it can be more useful when there is the problem of availability to the bilingual translators . the pretest method is another translation procedure that is applied as a pilot study to determine the potential possible problems that may occur during the main study in bigger size sample ( 62 ) . the fourth method , the bilingual model , is a participant - centered technique , which the tool is administered by bilingual participants in the both original and object languages and then the causes of the differences between completed instruments in two languages are discussed by the researches ( 62 ) . because of some reported limitations from studies used brislin s classic back - translation model to elevate their translated instrument validity and equivalency , jones et al . proposed a combined translation technique ( 65 ) . in this approach a combination of back translation , committee , and bilingual models are used in a several steps method until reach to a translated version , which is reliable and equivalent to the original one . although there may be some preferences for this combination technique over brislin s classic back - translation model but it has also some limitations that should be mentioned ( 65 ) . by getting information about validity and reliability of the vhi versions in other languages and showing high scores for them , in addition , it would encourage the other authors in other languages to choose the best method in order to translate vhi to their own languages . for example , the studies are more valuable that have performed combined translation technique , and included both validity and reliability on their translated questionnaire and applying it for patient group will increase its reliability and validity . therefore regards to the importance of vhi in evaluation of voice - disordered patients and its widespread usage across languages and the lack of a systematic review on it , so performing this paper is unavoidable . defined study criteria for eligibility were articles published from 1997 [ the year of vhi development by jacobson ( 12 ) ] to 2014 in english language which included translation , reliability , and validity reports of vhi-30 version . collection data was done by searching electronic resources including ; cochrane , googlescholar medline ( via pubmed gate ) , sciencedirect , web of science , and their reference lists . in some cases , which the full text was not available , and also in order to find more related studies , by contact with the correspondent author s article the paper was gotten . all the databases were investigated by key word of voice handicap index by title limitation and time of publication ( from 1997 to 2014 ) albeit without applying any other limitation , format of article , language , and so on . all the researched articles were gathered in step 1 , then in step 2 all the papers were investigated by title , nonrelated ones were deleted , and the included articles transferred to next stage , screening by abstract , which has its own several steps . therefore in next steps , papers written in non - english languages , those about other versions of vhi rather than vhi-30 , articles without having translation or validity and reliability processing report and repeated ones were excluded in third , fourth , fifth , and sixth steps respectively , this process was done for every databases ( fig . 1 ) . process of reviewing diagram in order to assessment of methodology of selected studies and assessing the risk of bias of individual studies , a 12 item diagnostic test checklist in critical appraisal skills programme or ( casp)(http://creativecommons.org / licenses / bync - sa/3.0/ ) was used to judge about the methodological quality of the studies . every checklist was performed by each author independently , and judged studies to be excluded according to the checklist were again proposed at a panel of 3 authors , therefore if two of them decided to delete or accept the paper , it was done . all the including and excluding process by the title , abstract , and full text was done by 3 authors . after this stage , according to the mentioned 6 steps , the variables for which data were sought included translation procedure , content validity , construct validity , internal consistency , and test - retest reliability . according to the first searches throughout the mentioned electronic resources in method section , generally 351 articles were found . after screening by title and omitting the digressed ones follow by that , in applying the exclusion criteria at screening by abstract stage , writing language of 9 papers were not english , and 23 ones did not precede on vhi-30 therefore were excluded . as well as , 37 articles because of lack of translation or validity and reliability method were deleted until after lay away repeated ones finally 16 paper identified to be included in the last step of review by full article . after getting help from the diagnostic test checklist of casp , from the 16 remained papers ; one study was deleted because it could not compliance the questions from 9 to 12 for all three author s assessment , because the study was designed on swedish vhi version adapted for singers . as well as three other studies due to the above questions were deleted at the panel ( fig . , translation process and the status of reliability and validity have been explained in the 11 different languages ( 2 different studies in arabic ) in the next section below . hebrew ( ofer amir , odelia ashkenazi , tali leibovitzh , orit michael , yael tavor , and michael wolf , 2006 ) : the original english was translated to hebrew by 4 lay native hebrew who were highly proficient in writing and speaking english . the four hebrew versions were , then , translated back to english by four native speakers of english , who are also lay persons , highly proficient in written and spoken hebrew . these four retranslated english versions were compared with the original vhi , individually for each item of the questionnaire . following this procedure , a final hebrew version was obtained , which was assembled by the items that translated most accurately from english to hebrew and then back to english . the final hebrew version was then presented , along with the original version , to four english hebrew bilingual judges . these judges confirmed that the final hebrew version is comparable with the original english version ( 21).spanish(faustino nez - batalla , paz corte - santos , blanca searis - gonzlez , jos l. llorente - pends , carmen grriz - gil , and carlos surez - nietoa , 2007 ) : after the first translation , the different items were discussed among the authors of this study and other professionals dealing with voice disorders . as a result the modifications were introduced on the basis of linguistic and cultural differences ( 42).arabic : ( khalid h. malki , tamer a. mesallam , mohamed farahat , manal bukhari , thomas murry , 2010 ) : the original english version of the vhi was translated into arabic by two arabic bilingual experienced phoniatricians items on the questionnaire were translated into arabic , back - translated into english , and compared with the original items by a qualified professional translator familiar with american english and arabic . the arabic version of the vhi was then pilot - tested with ten consented saudi subjects with voice disorders . subsequently , the vhi was amended according to their suggestions after reviewing the pilot data ( 70).arabic ( ahmad f. saleem 1 & yaser s. natour , 2010 ) : the english version of the vhi was translated into msa by the authors this version was then reviewed by a bilingual professional linguistic translator ( english/ arabic ) , who provided valuable suggestions for the sentence structure to make it easier for the layperson to understand . this revised version was then presented to a number of arabic speakers with no voice problems , who confirmed that all items were well understood . following this , the revised version emerged as the final version of the vhi , and its wording was maintained ( 71).greek ( meropi e. helidoni , thomas murry , joanna moschandreas , kchristos lionis , athanasia printza , and george a. velegrakis , 2011 ) : it was done according to the minimal translation criteria which its forward translation performed by two greek slp , who were proficient in the english and greek also a greek / english bilingual professional translator did the backward translation ( 14).italian ( antonio schindler , francesco ottaviani , francesco mozzanica , cinzia bachmann , elena favero , ilenia schettino , and giovanni ruoppolo , 2010 ) : forward and back translation and comparison with the original items has been done but its process was not reported ( 72).mandarin chinese : ( wen xu , demin han , hongyan li , rong hu , and li zhang , 2010 ) : the original english version of the vhi was translated into mandarin chinese independently by four translators . the initial translations in mandarin chinese were reviewed and modified by clinical voice experts . in a preliminary investigation , the reviewed versions of the mandarin chinese vhi were given to 30 dysphonic patients and 30 control subjects ( they were not included in 1886 cases discussed in this article ) . after this preliminary trial , the items were further evaluated and modified according to chinese cultural and language habits to develop the revised mandarin chinese versions of the vhi . the revised mandarin chinese versions were translated back into english independently by qualified professional translators . the retranslated english versions were reviewed by professor thomas murry , and the final retranslated english version confirmed by professor barbara jacobson . the revised mandarin chinese version that corresponded to the final retranslated english version was chose to be the final mandarin chinese version of the vhi for presentation in this article ( 73).bizilian portuguese ( mara behlau , luciana de moraes alves dos santos , and gisele oliveira , 2011 ) : forward and backward translations were done by 3 bilingual english - portuguese people separately , also a committee of five voice specialists revised the final protocol . to evaluate cultural and linguistic equivalency , the option not applicable was introduced to each item of the questionnaire , which was then administered to 10 patients . none of the questions were shown to be invalid ( 74).hindi ( datta r , sethi a , singh sh , nilakantan a , venkatesh md , 2011 ) : the first translation from english to hindi was done by one of the coauthors and then was discussed in a committee ( authors ) and finally back translation ( hindi to english ) performed by a school teacher ( 75).norwegian ( karlsen t , grieg arh , heimdal jh , aarstad jh , 2012 ) : the original english vhi was translated into norwegian by a slp and a physician follow by that it was investigated in a panel of five slps for elevating its accuracy of translation . after doing back translation into english by an independent person and another evaluating the equivalency the final norwegian version tested in a normal voice group as a pilot study ( 76).croatian ( bonetti a , bonetti l , 2013 ) : the primary croatian vhi version was prepared by 3 experts from its original english version and after applying croatian cultural and linguistic habits and back translation ( croatian - english ) the final vhi croatian version was ready to use ( 77).persian ( moradi n , pourshahbaz a , soltani m , javadipour s , hashemi h , soltaninejad n , 2013 ) : forward translation performed by two english translators and a slp who were native persian language . then the translation was discussed in a panel including 5 experts . after preparing the refined persian translated one , the backward translation was done by a bilingual clinical expert and was found understandable and equivalent to original english vhi-30 by another translator . at last the final persian vhi was presented to a group of voice disordered people as pilot in order to defining the non - understandable words ( 78 ) . hebrew ( ofer amir , odelia ashkenazi , tali leibovitzh , orit michael , yael tavor , and michael wolf , 2006 ) : the original english was translated to hebrew by 4 lay native hebrew who were highly proficient in writing and speaking english . the four hebrew versions were , then , translated back to english by four native speakers of english , who are also lay persons , highly proficient in written and spoken hebrew . these four retranslated english versions were compared with the original vhi , individually for each item of the questionnaire . following this procedure , a final hebrew version was obtained , which was assembled by the items that translated most accurately from english to hebrew and then back to english . the final hebrew version was then presented , along with the original version , to four english hebrew bilingual judges . these judges confirmed that the final hebrew version is comparable with the original english version ( 21 ) . spanish(faustino nez - batalla , paz corte - santos , blanca searis - gonzlez , jos l. llorente - pends , carmen grriz - gil , and carlos surez - nietoa , 2007 ) : after the first translation , the different items were discussed among the authors of this study and other professionals dealing with voice disorders . as a result the modifications were introduced on the basis of linguistic and cultural differences ( 42 ) . arabic : ( khalid h. malki , tamer a. mesallam , mohamed farahat , manal bukhari , thomas murry , 2010 ) : the original english version of the vhi was translated into arabic by two arabic bilingual experienced phoniatricians items on the questionnaire were translated into arabic , back - translated into english , and compared with the original items by a qualified professional translator familiar with american english and arabic . the arabic version of the vhi was then pilot - tested with ten consented saudi subjects with voice disorders . subsequently , the vhi was amended according to their suggestions after reviewing the pilot data ( 70 ) . arabic ( ahmad f. saleem 1 & yaser s. natour , 2010 ) : the english version of the vhi was translated into msa by the authors this version was then reviewed by a bilingual professional linguistic translator ( english/ arabic ) , who provided valuable suggestions for the sentence structure to make it easier for the layperson to understand . this revised version was then presented to a number of arabic speakers with no voice problems , who confirmed that all items were well understood . following this , the revised version emerged as the final version of the vhi , and its wording was maintained ( 71 ) . greek ( meropi e. helidoni , thomas murry , joanna moschandreas , kchristos lionis , athanasia printza , and george a. velegrakis , 2011 ) : it was done according to the minimal translation criteria which its forward translation performed by two greek slp , who were proficient in the english and greek also a greek / english bilingual professional translator did the backward translation ( 14 ) . italian ( antonio schindler , francesco ottaviani , francesco mozzanica , cinzia bachmann , elena favero , ilenia schettino , and giovanni ruoppolo , 2010 ) : forward and back translation and comparison with the original items has been done but its process was not reported ( 72 ) . mandarin chinese : ( wen xu , demin han , hongyan li , rong hu , and li zhang , 2010 ) : the original english version of the vhi was translated into mandarin chinese independently by four translators . the initial translations in mandarin chinese were reviewed and modified by clinical voice experts . in a preliminary investigation , the reviewed versions of the mandarin chinese vhi were given to 30 dysphonic patients and 30 control subjects ( they were not included in 1886 cases discussed in this article ) . after this preliminary trial , the items were further evaluated and modified according to chinese cultural and language habits to develop the revised mandarin chinese versions of the vhi . the revised mandarin chinese versions were translated back into english independently by qualified professional translators . the retranslated english versions were reviewed by professor thomas murry , and the final retranslated english version confirmed by professor barbara jacobson . the revised mandarin chinese version that corresponded to the final retranslated english version was chose to be the final mandarin chinese version of the vhi for presentation in this article ( 73 ) . bizilian portuguese ( mara behlau , luciana de moraes alves dos santos , and gisele oliveira , 2011 ) : forward and backward translations were done by 3 bilingual english - portuguese people separately , also a committee of five voice specialists revised the final protocol . to evaluate cultural and linguistic equivalency , the option not applicable was introduced to each item of the questionnaire , which was then administered to 10 patients . hindi ( datta r , sethi a , singh sh , nilakantan a , venkatesh md , 2011 ) : the first translation from english to hindi was done by one of the coauthors and then was discussed in a committee ( authors ) and finally back translation ( hindi to english ) performed by a school teacher ( 75 ) . norwegian ( karlsen t , grieg arh , heimdal jh , aarstad jh , 2012 ) : the original english vhi was translated into norwegian by a slp and a physician follow by that it was investigated in a panel of five slps for elevating its accuracy of translation . after doing back translation into english by an independent person and another evaluating the equivalency the final norwegian version tested in a normal voice group as a pilot study ( 76 ) . croatian ( bonetti a , bonetti l , 2013 ) : the primary croatian vhi version was prepared by 3 experts from its original english version and after applying croatian cultural and linguistic habits and back translation ( croatian - english ) the final vhi croatian version was ready to use ( 77 ) . persian ( moradi n , pourshahbaz a , soltani m , javadipour s , hashemi h , soltaninejad n , 2013 ) : forward translation performed by two english translators and a slp who were native persian language . then the translation was discussed in a panel including 5 experts . after preparing the refined persian translated one , the backward translation was done by a bilingual clinical expert and was found understandable and equivalent to original english vhi-30 by another translator . at last the final persian vhi was presented to a group of voice disordered people as pilot in order to defining the non - understandable words ( 78 ) . regarding to the translation procedures used to convert the english version of vhi-30 to other languages , generally it was according to brislin s classic back - translation model ( 1970 ) method which described before but only in some languages [ e.g. hebrew , portuguese , mandarin chinese , arabic ( 70 ) , and spanish ] the model was applied completely and in order to evaluate cultural and linguistic equivalency of the translated vhi , a panel including voice specialists judged the questionnaire items to enhance the validity of the instrument . in addition , some studies asserted that because the vhi is intended for use by lay persons , the use of professional terminology was not considered to be desirable . therefore , they decided to use translators who are not professionally knowledgeable in the field of speech and voice . the studies include portuguese ( only for forward translation ) , hebrew ( for both forward and back translation ) , and also in arabic study ( 71 ) speech pathologist translated it but lay bilingual english - arabic confirmed and edited them . another thing that in some vhi language versions such as mandarin chinese and hebrew was considered was performing the translation by several people separately and independently which this decreased the bias of affecting the translator s ideas on each other and also increasing the reliability of the translation . in addition , across the all of the investigated studies , only in mandarin chinese vhi version , the final retranslated english version sent to the original english vhi developer ( jacobson ) to confirm it so this may be positive point for mandarin vhi version . attending to the table 1 two essentially issues for a test to be applicable are remarkable which includes reliability and validity : table 1:validity and reliability of translated versions of vhi-30 across languages.languagecontent validityconstruct validityinternal consistency ( cronbach s alpha coefficient)test - retest reliabilitygreek - spearman s rank correlation coefficient = 0.55/ ( p < 0.0001)0.95icc= 0.96brazilian / portuguesedone-0.88wilcoxon = 0.100 ( p < 0.001)italian--0.93pearson correlation test= 0.97mandarinepearson correlation coefficients 0.86principal - component analysis > 10.95pearson correlation coefficient : r = 0.991(50 ) , p < 0.01hebrewdonespearman correlation coefficients = 0.760.97arabic/ ( ahmad f)-pearson : r = 0.52 , p = 0.010.955r = 0.89arabic/ ( khalid h)done-0.97r = 0.89 , p = 0.001also by spearman ( correlation between items):r 0.88spanishdoner=0.76 ; p < 0.0010.93r=0.822 ; p < 0.001persiandoneclinical validity:/ ( p = 0.000)0.87r=0.96hindidoner=0.44 ; p < 0.0010.95r=0.95 ; p < 0.001clinical validity : p < 0.001norwegiandonevariance ranged between 40 and 60% ( allp<0.001)0.93not donecroatiandoneclinical validity : p = 0.0000.940.92 validity and reliability of translated versions of vhi-30 across languages . there are two different types of reliability , which has done in the mentioned vhi versions , which consists of test - retest and internal consistency . except to hebrew and norwegian versions ( did not report ) all of them reported high test - retest reliability ( external reliability ) which this would be an important strength for vhi versions in different languages especially because this high reliability is across all of the 3 domains of vhi test ( table 2 ) . evaluation of different vhi-30 versions between languages t : translation , bt : back translation , ( ) : not reported , ( + ) : reported in case of internal consistency all of them used cronbach s alpha coefficient , which strong results were concluded except to greek version ( was not reported ) , therefore all 3 domains of vhi test despite their different language versions were homogeny and consistent . regarding to content validity in most of languages it was done rather than the greek , italian , and arabic ( 71 ) versions , which were not reported , but unfortunately , only a few of them described its process precisely . therefore , we can not judge about their procedure to perform it . in order to determine construct validity , participants completed general questions , regarding their satisfaction with their voice then the correlation between the responses and vhi total scores were calculated by different statistics methods but it was not done in some studies such as portuguese , italian , and arabic ( 70 ) . surprisingly despite the fact that different self - rating questionnaires were applied in the languages but all of them showed moderate correlation with total vhi score , this finding is in concordance with jacobson et al . ( 12 ) , and supporting his prognosis which high score of self - rating of voice handicap may concurrent with high scores in three domains of vhi . regarding to this review , vhi seems to be a valid and reliable instrument to evaluate the impacts of voice disorder on patient s life despite of its translated language version . in addition , vhi can be applied for varieties of voice disorders as a base rate and effects of different interventions such as voice therapy , surgery , and so on . also , even by some adaptations for special groups such as singers . there were limitations in getting full text of some studies searched from the mentioned databases therefore ; we were forced to get them from its correspondent author that took a lot of time from us . as well as in this paper it was tried to cover important electronic databases , however access to some of the prime ones was not possible so we preferred to ignore them . therefore , it may create the risk of not including all of the existing studies on vhi . although there may be bias of losing studies without having the voice handicap index phrase or vhi in their title , although the studies consist of vhi translation , reliability , and validity process . there are two different types of reliability , which has done in the mentioned vhi versions , which consists of test - retest and internal consistency . except to hebrew and norwegian versions ( did not report ) all of them reported high test - retest reliability ( external reliability ) which this would be an important strength for vhi versions in different languages especially because this high reliability is across all of the 3 domains of vhi test ( table 2 ) . evaluation of different vhi-30 versions between languages t : translation , bt : back translation , ( ) : not reported , ( + ) : reported in case of internal consistency all of them used cronbach s alpha coefficient , which strong results were concluded except to greek version ( was not reported ) , therefore all 3 domains of vhi test despite their different language versions were homogeny and consistent . regarding to content validity in most of languages it was done rather than the greek , italian , and arabic ( 71 ) versions , which were not reported , but unfortunately , only a few of them described its process precisely . therefore , we can not judge about their procedure to perform it . in order to determine construct validity , participants completed general questions , regarding their satisfaction with their voice then the correlation between the responses and vhi total scores were calculated by different statistics methods but it was not done in some studies such as portuguese , italian , and arabic ( 70 ) . surprisingly despite the fact that different self - rating questionnaires were applied in the languages but all of them showed moderate correlation with total vhi score , this finding is in concordance with jacobson et al . ( 12 ) , and supporting his prognosis which high score of self - rating of voice handicap may concurrent with high scores in three domains of vhi . regarding to this review , vhi seems to be a valid and reliable instrument to evaluate the impacts of voice disorder on patient s life despite of its translated language version . in addition , vhi can be applied for varieties of voice disorders as a base rate and effects of different interventions such as voice therapy , surgery , and so on . also , even by some adaptations for special groups such as singers . there were limitations in getting full text of some studies searched from the mentioned databases therefore ; we were forced to get them from its correspondent author that took a lot of time from us . as well as in this paper it was tried to cover important electronic databases , however access to some of the prime ones was not possible so we preferred to ignore them . therefore , it may create the risk of not including all of the existing studies on vhi . although there may be bias of losing studies without having the voice handicap index phrase or vhi in their title , although the studies consist of vhi translation , reliability , and validity process . according to this manuscript , vhi-30 is a valuable and reliable instrument across different languages . therefore , it can be considered as an effortful evaluating tool to be translated in other languages , to present more services to voice disordered patients . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .	background : in this systematic review , the aim is to investigate different vhi-30 versions between languages regarding their validity , reliability and their translation process.methods:articles were extracted systematically from some of the prime databases including cochrane , googlescholar , medline ( via pubmed gate ) , sciencedirect , web of science , and their reference lists by voice handicap index keyword with only title limitation and time of publication ( from 1997 to 2014 ) . however the other limitations ( e.g. excluding non - english , other versions of vhi ones , and so on ) applied manually after studying the papers . in order to appraise the methodology of the papers , three authors did it by 12-item diagnostic test checklist in critical appraisal skills programme or ( casp ) site . after applying all of the screenings , the papers that had the study eligibility criteria such as ; translation , validity , and reliability processes , included in this review.results:the remained non - repeated articles were 12 from different languages . all of them reported validity , reliability and translation method , which presented in details in this review.conclusion:mainly the preferred method for translation in the gathered papers was brislin s classic back - translation model ( 1970 ) , although the procedure was not performed completely but it was more prominent than other translation procedures . high test - retest reliability , internal consistency and moderate construct validity between different languages in regards to all 3 vhi-30 domains confirm the applicability of translated vhi-30 version across languages .
You are an expert at summarizing long articles. Proceed to summarize the following text: desmoplastic small round cell tumor ( dsrct ) is a malignant neoplasm that was first described in 1987 but was only clinically recognized in 1989 . it predominantly affects young males , and it typically presents as a large intraabdominal mass with smaller peritoneal implants . histologically , it is formed by nests of small round cells within a desmoplastic stroma . immunohistochemically , it shows a polyphenotypic differentiation with positivity for epithelial , mesenchymal , myogenic , and neural markers . our aim is to describe the unusual findings of dsrct on a postchemotherapy setting and to create awareness of such changes the clinical history is that of a 13-year - old male who complained of dysuria and was found to have a 16 cm pelvic mass , adhered to the bladder and two liver nodules . he received 1-year of chemotherapy , which included : ifosfamide , etoposide , vincristine , cyclophosphamide , doxorubicin , and vincristine ) . six months after , the liver mass decreased in size and he was started on total abdominal radiotherapy ( until complete 4500 cgys ) . he received a total of 11 cycles of chemotherapy and 44 cycles of radiotherapy and was found to have a good response to treatment with persistence of the pelvic mass . he was taken to surgery 14 months after primary diagnosis and was found to have irresectable mass that surrounded bladder and ureters . one month after surgery he presented to the er with nausea , vomiting , and abdominal pain . physical examination revealed a distended abdomen that was painful upon palpation . he was admitted to our institution and received palliative care ( hydromorphone and midazolam ) for a 2 month duration before he died . cytospins [ figure 1a b ] and cell block [ figure 1c ] were hypercellular , with numerous large , single and pleomorphic cells with an epithelioid appearance , eccentrically located and convoluted nuclei . hypercellular cytospin showing single , large , neoplastic cells with eccentric nuclei , prominent nucleoli , abundant cytoplasm , mitosis and apoptosis ( a to e , 400 ) ( a : h and e ) . ( c ) cell block with numerous neoplastic cells with marked apoptosis in a hemorrhagic background ( h and e ) . ( e ) positive desmin ( ihc ) immunohistochemical studies performed on the cell block showed positivity of the malignant cells with vimentin , epithelial membrane antigen [ ema , figure 1d ] , desmin [ cytoplasmic , figure 1e ] and cytokeratin ae1/ae3 . the tumor cells were negative for calretinin , d2 - 40 and ck 5 - 6 . the polyphenotypic immunoprofile , in conjunction with the clinical history , confirmed the persistence of dsrct . desmoplastic small round cell tumor is a rare tumor with aggressive behavior , morphologically the tumor overlaps with other lesions of small cell morphology such as ewing 's sarcoma , rhabdomyosarcoma , and small cell mesothelioma . these tumors are characterized by reciprocal translocation t(11;22)(p13;q12 ) that can be confirmed by ews - wt1 gene fusion transcript by fluorescent in - situ hybridization . in our institution , we did not have the molecular confirmation , but the primary diagnosis was done pretreatment and showed the classic morphology and immunophenotype with divergent differentiation ( positive for vimentin , ema , desmin and ae1/ae3 and negative for calretinin , d2 - 40 and ck 5 - 6 ) . the therapeutic paracentesis showed a hypercellular specimen composed of large single and discohesive cells , with an abundant eosinophilic cytoplasm , perinuclear hoff and frequent mitosis and apoptosis . due to the lack of groups , stroma and the epithelioid appearance of cells with absence of small cells with high n : c ratio immunocytochemical studies were done on the cell block to rule out reactive mesothelial origin . there are very few reports that describe the cytological findings of dsrct tumor after chemotherapy . most of the reports describe the usual cytological features of this tumor , including hypercellular aggregates of oval , round or fusiform cells of medium size with a high n : c ratio . the nuclei can show molding , membrane irregularities , granular chromatin , inconspicuous nucleoli , very few mitotic figures and some fragments of metachromatic stroma . the presence of stroma is crucial to the differential diagnosis ; however , it is always necessary to perform immunohistochemical confirmation . dsrct classically is positive for epithelial , mesenchymal and neural markers and although mesothelium share some markers , studies have demonstrated negativity for d2 - 40 and ck5 - 6 . in general , postchemotherapy changes in mesothelial cells have been described as atypia characterized by irregular nuclei , hyperchromasia and prominent nucleoli the findings of dsrct in effusions are the same described for fine needle aspiration specimens [ table 1 ] . to our knowledge , only two of these papers were in patients who received chemotherapy and described the loss of cohesion with the hypercellularity , prominent cells size and nucleoli , no mitotic figures and abundant stroma . summary of cytologic findings for dsrct on effusions herein , we report another case of dsrct status postchemotherapy , where we found a hypercellular specimen of large single and discohesive cells , as previously described , but in addition to what has been reported , we did not see any presence of stroma , the cells had abundant eosinophilic cytoplasm and there were frequent mitosis and apoptosis . the immunocytochemical studies performed on the cell block along with the previous clinical history helped to confirm the neoplastic nature of the effusion . we believe it is important to report unusual morphologic findings in such uncommon tumor due to therapy in order to be able to diagnose recurrence .	desmoplastic small round cell tumor ( dsrct ) is a malignant neoplasm that most often presents in male adolescents as an abdominal mass . cytological features have been previously described , but only two reports noted post chemotherapy changes on effusions . we report a case of a 15-year - old male with dsrct status postchemotherapy that presented with ascitis . unusual morphology was seen : numerous malignant large and single cells with prominent nucleoli and abundant cytoplasm in a background without the stroma , occasional mitosis , and the abundant apoptosis . cell block immunocytochemistry was confirmatory . awareness of the postchemotherapy changes in this tumor will allow us to diagnose recurrence .
You are an expert at summarizing long articles. Proceed to summarize the following text: patients with acute central retinal artery occlusion ( crao ) typically relate a history of painless visual loss occurring over several seconds followed by classic signs of retinal infarction in the fundus . spontaneous recanalization frequently occurs in many patients making crao a challenging diagnosis.1 in such cases , electroretinogram ( erg ) can be a helpful ancillary test for crao diagnosis . as the retinal artery supplies the middle and inner layers of the retina , the physiological consequence of crao is a predominant reduction of the scotopic maximal erg b - wave with preservation of the a - wave.2 a negative full - field erg usually describes international standard for clinical electrophysiology of vision standard maximal response in which the amplitude of b - wave is smaller than the minimally attenuated a - wave.35 this reduced b - wave is usually associated with a number of congenital and acquired conditions such as x - linked juvenile retinoschisis,6,7 congenital stationary night blindness,8,9 crao,10,11 birdshot chorioretinopathy,1214 paraneoplastic and autoimmune retinopathies,1517 and retinal toxicity.18,19 we report an atypical presentation of crao in which a unilateral negative erg was valuable in diagnosis and the initiation of systemic work - up . a 94-year - old ophthalmologist noted central vision deterioration in the right eye over the past two years . medical history was significant for an 18-year history of systemic hypertension , for which the patient was being treated with beta - blocker ( atenolol ) and diuretic ( hydrochlorotiazide ) drugs . screening procedures for malignancy including a careful medical history , a chest x - ray , and liver enzymes were negative . there was neither history of retinotoxic medication use , nor family history of eye disease . ultrasound examination of the carotid arteries was performed and showed tiny atherosclerotic plaque without hemodynamically compromised lesion . two years before his presentation , his ophthalmologist recorded 20/60 vision in the right eye and 20/20 in the left eye . visual acuity at presentation was 20/80 in the right eye and 20/20 in the left eye . the patient s central vision in the right eye progressively deteriorated but stabilized over six months , resulting in acuity of 20/100 . anterior segment examination was unremarkable , except for mild superficial punctate keratitis , a few guttata , more prominently on the left cornea , and pseudophakia . the funduscopic examination revealed hard drusen on the macular area in both eyes , and atrophic retinal pigment epithelium ( rpe ) changes with pigmentary clumps on the macular area of left eye . minor retinal artery narrowing was observed in both eyes without optic atrophy ( figure 1a and 1b ) . fundus fluorescein angiography ( fa ) was unremarkable in the right eye and revealed transmission defect in the posterior pole compatible with rpe abnormalities in the left eye ( figure 1c and 1d ) . fa choroidal phase was 6 seconds in the right eye and 5 seconds in the left eye ; fa arteriovenous phase was 14 seconds in the right eye and 11 seconds in the left eye . spectral domain optical coherence tomography ( sd - oct ) demonstrated reduced thickness of the inner retina in both eyes but more prominently in the right eye ( figure 2 ) . the thickness of the inner retina ( from retinal nerve fiber layer to inner nuclear layer ) was measured 600 m , 1,000 m , and 2,000 m nasally and temporally from the fovea . we also measured the inner retina thickness at the same locations described previously in 10 normal age - matched control eyes . the measurements were performed using the 1,000 m caliper available in the oct - slo spectralis , heidelberg retina angiograph 2 ( heidelberg engineering , dossenheim , germany ) . our measurements showed that the inner retina thicknesses were decreased at 600 m , 1,000 m , and 2,000 m nasally and temporally from the fovea in the patient s right eye in comparison to the left eye and normal control eyes ( figure 3 ) . goldmann perimetry demonstrated central scotoma in the right eye and no abnormalities in the left eye . full - field erg in the right eye was attenuated in both scotopic and photopic responses and revealed a b - wave smaller than the a - wave amplitude , ie , electronegative maximal response . in the left eye , minimal scotopic and photopic attenuation was observed ( figure 4 ) . western blot analysis of 1:500 dilutions of the patient s serum did not show any reactivity against retinal protein extract . our patient presented with a history of systemic hypertension treated with antihypertensive medications and vision loss of unknown origin with minimal abnormalities in the clinical and imaging studies . based on his history , imaging examinations , and on the erg result , crao and autoimmune retinopathies were considered as differential diagnosis . although autoimmune retinopathies , including cancer - associated retinopathy and melanoma - associated retinopathy , can cause electronegative erg , they are bilateral . indeed , autoimmune retinopathy patients tend to have antiretinal antibody activity often with different antibodies found on immunoblots.2022 the findings of generalized vascular narrowing , even though minimal , lack any sign of acute ischemia and the unilateral electronegative maximal erg in the right eye were consistent with inner retinal dysfunction due to an old central arteriolar occlusion in the right eye . a reduction of the b - wave with a relative preservation of the a - wave of the scotopic maximal erg is a characteristic of eyes with crao.2,5 in addition , the reduced inner retina thickness more prominent in the right eye than in the left eye demonstrable by sd - oct measurements strengthens the diagnosis of crao since crao is classically associated with a reduced thickness in the inner retinal layers.23,24 although the classic retinal findings of crao have been described , a high degree of suspicion is needed to diagnose crao in some cases because of transitory abnormalities in the fundus appearance , fa findings for crao , the insensitivity of carotid doppler imaging and echocardiography for associated lesions , and the subtlety of residual findings after crao . in such cases , mainly represented by the transient type , the diagnosis of crao is intricate and an electronegative erg can support the diagnosis of artery occlusion.1,2 crao is reported to be the most common cause of a unilateral negative erg.25 we report an atypical case of crao without its characteristic angiographic findings , except mild retinal artery narrowing detected on his fundus and imaging exams , in which erg was essential for diagnosis . our case , therefore , illustrates that erg testing is helpful for the work - up of individuals with suspected retinal vascular disorders .	a unilateral electronegative electroretinogram ( erg ) was seen in a 94-year - old man with presumed central retinal artery occlusion . goldmann perimetry revealed central scotoma in the right eye and no abnormalities in the left eye . full - field erg in the right eye described a reduction of the b - wave with a relative preservation of the a - wave which is characteristic of electronegative erg . hence , our case illustrates that erg testing is essential for the work - up of individuals with suspected retinal vascular disorders .
You are an expert at summarizing long articles. Proceed to summarize the following text: after several conferences and workshops , each study group selected the tumors and determined the provisional behavior codes . the study group for hematopathology selected in situ lesions of malignant lymphomas , and histiocytic lesions for discussion . in situ lesions included mantle cell lymphoma in situ and follicular lymphoma in situ , and histiocytic lesions included follicular dendritic cell tumors , interdigitating dendritic cell tumors , and langerhans cell histiocytosis . the bone and soft tissue pathology study group initially selected diseases that do not have an icd - o3 code among the bone and soft tissue tumors included in the who classification , including:6 fibromatosis colli , juvenile hyaline fibromatosis , inclusion body fibromatosis , superficial fibromatosis , lipofibromatosis , hyalinizing spindle cell tumor , neoplasm with perivascular epithelioid cell differentiation ( pecoma ) , and clear cell myomelanocytic tumor among the soft tissue tumors , and aneurysmal bone cyst , fibrous dysplasia , osteofibrous dysplasia , and erdheim - chester disease among bone tumors . pecoma were further divided into pecoma , not otherwise specified ( pecoma nos ) , angiomyolipoma ( aml ) , lymphangioleiomyomatosis ( lam ) , clear cell " sugar " tumor of the lung ( ccst ) , and clear cell myomelanocytic tumor of the falciform ligament / ligamentum teres ( ccmmt ) . hyalinizing spindle cell tumor was also excluded because it is a variant of low grade fibromyxoid sarcoma , and thus should be regarded as a malignant disease . under the heading of aml , we excluded renal aml , because its biologic behavior is well known to be benign even though sarcomatous change is rarely reported.7 aneurysmal bone cyst , fibrous dysplasia / osteofibrous dysplasia , and erdheim - chester disease were initially included in the discussion , but finally excluded due to well - known benignancy or rarity . the study group for hematopathology determined the codes presented both in the preexisting icd - o3 and the who classification 2008 for the selected tumors so that other pathologists could compare the results , because icd - o3 does not provide a behavior code for follicular lymphoma in situ and mantle cell lymphoma in situ , but does provide behavior code -/1 for the histiocytic and dendritic cell neoplasms . this is in contrast to the who classification 2008 , which considers them as malignant tumors.5 in the field of bone and soft tissue tumors , three pathologists classified these tumors on the basis of previous reports and made provisional behavior codes . they prepared materials for the charged parts , and the presentation , the discussion , and the convergence of opinions were conducted during the workshop held within the bone and soft tissue study group . because reports regarding these diseases were insufficient in korea , we referred to several foreign literature databases and who classifications to determine provisional behavior codes . the cancer registration committee held additional discussions regarding provisional behavior codes provided by the study group for hematopathology and the bone and soft tissue pathology study group . the final lists of tumors were determined and the provisional biologic behavior codes for the corresponding diseases were established . the questionnaire was made considering these decisions and distributed to the members of ksp during the academic congress of pathology and cytopathology . after several conferences and workshops , each study group selected the tumors and determined the provisional behavior codes . the study group for hematopathology selected in situ lesions of malignant lymphomas , and histiocytic lesions for discussion . in situ lesions included mantle cell lymphoma in situ and follicular lymphoma in situ , and histiocytic lesions included follicular dendritic cell tumors , interdigitating dendritic cell tumors , and langerhans cell histiocytosis . the bone and soft tissue pathology study group initially selected diseases that do not have an icd - o3 code among the bone and soft tissue tumors included in the who classification , including:6 fibromatosis colli , juvenile hyaline fibromatosis , inclusion body fibromatosis , superficial fibromatosis , lipofibromatosis , hyalinizing spindle cell tumor , neoplasm with perivascular epithelioid cell differentiation ( pecoma ) , and clear cell myomelanocytic tumor among the soft tissue tumors , and aneurysmal bone cyst , fibrous dysplasia , osteofibrous dysplasia , and erdheim - chester disease among bone tumors . pecoma were further divided into pecoma , not otherwise specified ( pecoma nos ) , angiomyolipoma ( aml ) , lymphangioleiomyomatosis ( lam ) , clear cell " sugar " tumor of the lung ( ccst ) , and clear cell myomelanocytic tumor of the falciform ligament / ligamentum teres ( ccmmt ) . hyalinizing spindle cell tumor was also excluded because it is a variant of low grade fibromyxoid sarcoma , and thus should be regarded as a malignant disease . under the heading of aml , we excluded renal aml , because its biologic behavior is well known to be benign even though sarcomatous change is rarely reported.7 aneurysmal bone cyst , fibrous dysplasia / osteofibrous dysplasia , and erdheim - chester disease were initially included in the discussion , but finally excluded due to well - known benignancy or rarity . the study group for hematopathology determined the codes presented both in the preexisting icd - o3 and the who classification 2008 for the selected tumors so that other pathologists could compare the results , because icd - o3 does not provide a behavior code for follicular lymphoma in situ and mantle cell lymphoma in situ , but does provide behavior code -/1 for the histiocytic and dendritic cell neoplasms . this is in contrast to the who classification 2008 , which considers them as malignant tumors.5 in the field of bone and soft tissue tumors , three pathologists classified these tumors on the basis of previous reports and made provisional behavior codes . they prepared materials for the charged parts , and the presentation , the discussion , and the convergence of opinions were conducted during the workshop held within the bone and soft tissue study group . because reports regarding these diseases were insufficient in korea , we referred to several foreign literature databases and who classifications to determine provisional behavior codes . the cancer registration committee held additional discussions regarding provisional behavior codes provided by the study group for hematopathology and the bone and soft tissue pathology study group . the final lists of tumors were determined and the provisional biologic behavior codes for the corresponding diseases were established . the questionnaire was made considering these decisions and distributed to the members of ksp during the academic congress of pathology and cytopathology . the study group for hematopathology suggested the behavior code -/3 ( malignant ) for mantle cell lymphoma in situ and follicular lymphoma in situ . for the histiocytic and dendritic cell neoplasms , the majority of the study group members agreed on the malignant behavior ( table 2 ) . for the bone and soft tissue tumors , the tumors were categorized as fibromatosis or pecoma in order to avoid the complexities of the classification . among the fibromatosis category , fibromatosis colli , juvenile hyaline fibromatosis , and inclusion body fibromatosis were described as benign , and given the behavior code -/0 . lipofibromatosis was regarded as a borderline tumor and given behavior code -/1 . among the pecoma category , ccmmt was suggested to be a benign disease and the behavior code -/0 was applied . aml , lam , and ccst were given behavior code -/1 , and pecoma nos was given behavior code -/3 ( table 3 ) . a total of 274 members of the ksp provided their opinions by completing the questionnaire . of the responders , the following percentages indicated a behavior code of -/3 ( malignant ) for the respective tumor types : 75.5% ( 207/274 ) for follicular lymphoma in situ , 77% ( 211/274 ) for mantle cell lymphoma in situ , 88.0% ( 241/274 ) for follicular dendritic cell tumor , 81.0% ( 222/274 ) for interdigitating dendritic cell tumor , and 74.8% ( 205/274 ) for langerhans the remaining members listed behavior code -/1 ( uncertain malignant potential or borderline tumor ) for these tumor types , with some members providing code -/2 ( carcinoma in situ ) and a few others suggesting that we postpone coding ( table 4 ) . regarding responses to the questionnaire on soft tissue tumors , 288 members of the ksp provided opinions . for the fibromatosis category , most responders gave behavior code -/0 ( benign tumor ) for fibromatosis colli , juvenile hyaline fibromatosis , and inclusion body fibromatosis , and -/1 ( uncertain malignant potential or borderline tumor ) for lipofibromatosis as the provision of the bone and soft tissue study group . for the pecoma category , most responders assigned pecoma nos to -/3 ( malignant neoplasm ) , aml , lam , and ccst to -/1 , and ccmmt to -/0 ( table 5 ) . the study group for hematopathology suggested the behavior code -/3 ( malignant ) for mantle cell lymphoma in situ and follicular lymphoma in situ . for the histiocytic and dendritic cell neoplasms , the majority of the study group members agreed on the malignant behavior ( table 2 ) . for the bone and soft tissue tumors , the tumors were categorized as fibromatosis or pecoma in order to avoid the complexities of the classification . among the fibromatosis category , fibromatosis colli , juvenile hyaline fibromatosis , and inclusion body fibromatosis were described as benign , and given the behavior code -/0 . lipofibromatosis was regarded as a borderline tumor and given behavior code -/1 . among the pecoma category , ccmmt was suggested to be a benign disease and the behavior code -/0 was applied . aml , lam , and ccst were given behavior code -/1 , and pecoma nos was given behavior code -/3 ( table 3 ) . a total of 274 members of the ksp provided their opinions by completing the questionnaire . of the responders , the following percentages indicated a behavior code of -/3 ( malignant ) for the respective tumor types : 75.5% ( 207/274 ) for follicular lymphoma in situ , 77% ( 211/274 ) for mantle cell lymphoma in situ , 88.0% ( 241/274 ) for follicular dendritic cell tumor , 81.0% ( 222/274 ) for interdigitating dendritic cell tumor , and 74.8% ( 205/274 ) for langerhans the remaining members listed behavior code -/1 ( uncertain malignant potential or borderline tumor ) for these tumor types , with some members providing code -/2 ( carcinoma in situ ) and a few others suggesting that we postpone coding ( table 4 ) . regarding responses to the questionnaire on soft tissue tumors , 288 members of the ksp provided opinions . for the fibromatosis category , most responders gave behavior code -/0 ( benign tumor ) for fibromatosis colli , juvenile hyaline fibromatosis , and inclusion body fibromatosis , and -/1 ( uncertain malignant potential or borderline tumor ) for lipofibromatosis as the provision of the bone and soft tissue study group . for the pecoma category , most responders assigned pecoma nos to -/3 ( malignant neoplasm ) , aml , lam , and ccst to -/1 , and ccmmt to -/0 ( table 5 ) . the study group for hematopathology focused on in situ lesions of malignant lymphomas and histiocytic lesions . mantle cell lymphoma in situ and follicular lymphoma in situ are defined morphologically when tumor cells are restricted to the mantle zone and intrafollicular area , respectively . however when the cells of these tumors circulate in the body , they should be classified as malignant diseases.5,8 - 10 because the term " in situ " can cause confusion not only to pathologists , but also to clinicians , the study group for hematopathology decided to include these lesions in order to clearly indicate that " in situ lesions " are malignant . there were some disputes in the study group regarding histiocytic neoplasms . in the who classification 2008 , histiocytic and dendritic cell neoplasms are classified as malignant disease with behavior code /3 of icd - o3.5 even before this edition , it has been suggested that langerhans cell histiocytosis should be considered a malignant tumor because of its risk of multisystem involvement and refractoriness to treatment,11 although it is treated as a tumor of uncertain malignant potential.4 dendritic cell neoplasms also have morphological obscurity between borderline and malignant tumors , but they were eventually defined as malignant disease without distinction , which was in contrast to the previous edition of the who classification . therefore the study group decided to include these histiocytic and dendritic cell neoplasms in this study because it may be meaningful to compare behavior codes between icd - o3 and the who classification 2008 . the study group selected mantle cell lymphoma in situ , follicular lymphoma in situ , follicular dendritic cell tumor , interdigitating dendritic cell tumor , and langerhans cell histiocytosis for this study . in the who classification 2001 , in situ lesion of follicular lymphoma was not mentioned.4 it was only introduced in the who classification 2008 under the name intrafollicular neoplasia " in situ " follicular lymphoma , and defined morphologically as architecturally normal appearing lymph nodes . other lymphoid tissues harboring one or more follicles that overexpress bcl-2 in centrocytes and centroblasts with or without a monomorphic cytologic appearance was described to be suggestive of follicular lymphoma.5 the meaning of this tumor has not been completely established yet , but it may represent the tissue counterpart of circulating clonal b - cells possessing bcl-2 rearrangement,5,9 or the earliest evidence of true follicular lymphoma that subsequently progress to overt follicular lymphoma.5,10,12 mantle cell lymphoma " in situ " is even mentioned in the who classification 2001 , but the term " in situ " is only found in the who classification 2008.4,5 by the definition , this tumor is confined to the inner mantle zone of lymphoid tissue.5,8 histiocytic and dendritic cell tumors are classified as borderline tumors and are distinguished from their malignant counterpart sarcoma in the who classification 2001 . they became evenly classified as malignant tumors without distinction of tumor or sarcoma in the who classification 2008.4,5 follicular dendritic cell tumor is a rare malignant tumor originating from follicular dendritic cells . it can arise in any organ , but most commonly occurs in the cervical lymph node . they show an indolent clinical course but have frequent local recurrence and distant metastasis.5,13 interdigitating dendritic cell tumor is also a very rare disease , and usually occurs as a solitary lesion in the lymph node , although extranodal lesions have been reported.5,14 langerhans cell histiocytosis usually occurs as solitary lesion in children . typical of multisystem lesions , it is refractory to treatment and associated with high mortality.5,15 the study group for hematopathology did not propose different behavior codes , but rather decided to provide both codes that are presented in the preexisting icd - o3 and the who classification 2008 . the biologic codes presented in the who classification 2001 are the same as the codes of icd - o3 . the questionnaire form produced by the study group for hematopathology was presented to the members of the ksp at academic congresses . the results of the questionnaire indicated that , members of the ksp generally agreed on the biological behavior of follicular lymphoma in situ , mantle cell lymphoma in situ , follicular dendritic cell tumor , interdigitating dendritic cell tumor , and langerhans cell histiocytosis as malignant disease , and therefore , the study group for hematopathology and the cancer registration committee could propose behavior code -/3 for these tumors . the study group also suggested that the diagnostic names of langerhans cell histiocytosis , unifocal langerhans cell histiocytosis , and multifocal langerhans cell histiocytosis should all be recapitulated under the name of langerhans cell histiocytosis with biologic behavior code -/3 . howerver , the final acceptance of these provisional codes may require additional studies that include long - term follow - up data , greater accumulation of knowledge , and agreement among clinicians . among the bone and soft tissue tumors included in the who classification,6 the study group selected tumors that do not have an icd - o3 code ; however , the biologic behaviors of these tumors are well known among pathologists even though icd - o3 codes were not granted . of these , the following diseases were selected : fibromatosis colli , juvenile hyaline fibromatosis , inclusion body fibromatosis , superficial fibromatosis , lipofibromatosis , hyalinizing spindle cell tumor , pecoma , and clear cell myomelanocytic tumor . among the tumors of bone and soft tissue presented in the who classification , many tumors have behavior code -/1 , such as , desmoid - type fibromatosis , inflammatory myofibroblastic tumor , and myopericytoma . these tumors have some characteristics in common , such as local recurrence , somewhat poor clinical outcome , and infrequent distant metastasis.6 therefore , these characteristics were considered in this study when determining the biologic behavior of the diseases . fibromatosis colli is a benign , site - specific lesion that occurs in the distal sternocleidomastoid muscle of infants.6 it occurs in 4% of live births , and the majority of affected infants are diagnosed before 6 months of age . it is managed in a non - surgical manner , but surgical intervention may be required in some patient.16,17 we considered this lesion to be a benign disease and therefore suggest a behavior code of -/0 . juvenile hyaline fibromatosis is an extremely rare and apparently non - neoplastic disorder that typically presents in infancy , and is characterized by the accumulation of extracellular " hyaline material " within skin , somatic soft tissues , and the skeleton , resulting in tumor - like masses.6 the hyaline material is produced by an aberrant population of fibroblasts . this tumor has a progressive nature and forms superficial and deep nodules with resulting deformity and dysfunction . it most frequently occurs in skin of the face and neck , gum , periarticular soft tissues , and several bone tissues . surgery is the treatment of choice , but the recurrence rate is high.18,19 there are reports that squamous cell carcinoma can arise from juvenile hyaline fibromatosis.20,21 we considered this disease to be locally invasive with malignant potential , and therefore behavior code -/1 may be appropriate . however , definitive evidence of malignancy or metastasis was not found , and therefore we suggest biologic code -/0 . inclusion body fibromatosis is a rare benign proliferation of fibroblastic and myofibroblastic cells that typically occurs on the digits of young children . it is named for the intracytoplasmic inclusions that are detected in a minority of the lesional cells.6 treatment is surgical excision , but the local recurrence rate is high.22,23 thus , based on this characteristic , the biologic behavior may be /1 , but because definitive evidence of malignancy or metastasis was not found , we also suggest behavior code -/0 for this disease as well . lipofibromatosis is a rare pediatric tumor and a histologically distinctive fibrofatty tumour of childhood , which was previously designated as infantile fibromatosis of non - desmoid type with a predilection for distal extremities . the tumor has a high rate of non - destructive local recurrence , but exhibits no metastatic potential.24,25 because of its local recurrence , we suggest biologic behavior code -/1 . pecoma is a neoplasm of perivascular epithelioid cells , and includes several diseases within the pecoma family.6 however , their biologic behaviors have not been established because of the rarity of the disease and uncertain criteria for malignancy . the subset of pecoma tumors with malignant behavior do show a high mitotic index , necrosis , marked cytologic atypia , and an infiltrative growth pattern.26 aml in this study represented non - renal aml . aml usually occurs in the kidney , but is also found in the liver , lung , uterus , skin and oral cavity in rare situations.27 one report has indicated that lam has a poor prognosis due to respiratory failure.28 ccst is considered benign , but tumors larger than 2 cm that are symptomatic and focally necrotic should be regarded as potentially malignant neoplasms.29 ccmmt has predilection for children and young adults , and it is known as a relatively benign disease.30 as presented above , aml , lam , and ccst could be considered as behavior code -/1 , and ccmmt as -/0 . pecoma nos , which does not show aml , lam , ccst , or ccmmt characteristics , is considered a subset of pecoma with malignant behavior,26 and therefore we suggest behavior code -/3 for this tumor type . we could not find studies that were similar to this study in literature databases from around the world , so comparison between our data and others was not possible . however , new disease entities and a gradual increase of our understanding of rare diseases have resulted in a need for the continuous reconsideration of the biologic behavior of theses tumors . in korea , the incidence of tumors of lymphoid tissue is increasing , and therefore the results of this study should be considered in the future as a comparator for newly reported studies . tumors of the bone and soft tissue undergo continual changes in the concept , classification , nomination , and biologic behavior . bone and soft tissue tumors have a relatively low incidence , and therefore experience regarding these tumors is limited , especially for the diseases selected in this study . although the majority of diseases entities in the study were benign or had uncertain biologic behavior and were not pertinent to the purpose of cancer registration , we think believe it is of great value to discuss and provide the behavior code for these rare disease entities . therefore , this study has provided behavior codes for these selected tumors , but as future studies provide a more detailed understanding of these tumors , a further refinement may be necessary . in addition , tumor types not included in our questionnaire will be assessed in the future .	backgroundunderstanding the biologic behavior of a tumor is a prerequisite for tumor registration code assignment . the aim of this report was to propose appropriate behavior codes of the international classification of disease oncology 3 ( icd - o3 ) to rare , yet pathologically interesting hematopoietic and soft tissue tumors.methodsthe study group for hematopathology , the bone and soft tissue pathology study group , and the cancer registration committee prepared the questionnaire containing provisional behavior codes of selected diseases.resultsin situ lesions of mantle cell and follicular lymphomas , dendritic cell tumors , and neoplasms with perivascular epithelioid cell differentiation ( pecoma ) , not otherwise specified were classified as malignant ( -/3 ) . the fibromatosis group , with the exception of lipofibromatosis , was proposed as benign ( -/0 ) . lipofibromatosis and several diseases that belong to the pecoma group were proposed as uncertain malignant potential ( -/1 ) . for the hematologic and soft tissue tumors , 274 and 288 members of the korean society of pathologists , respectively , provided opinions through questionnaire , and most responders showed agreement with the provisional behavior code proposed.conclusionsthe determination of behavior codes for the rare diseases described in this study , especially those of the pecoma group or malignant lymphoma , could be viewed as impractical and premature , but this study provides the basis for future research on this topic .
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Proceed to summarize the following text: incidence of gastric cancer in korea is very high , and , despite early detection , it continues to be a major health problem . through achievement of remarkable advances in cancer treatment , one problem , however , is that because patients are living longer , they may be at increased risk of developing second primary cancer . the incidence of metachronous dual primary gastric cancer has increased with the concomitant increase in the prevalence of gastric cancer . patients with metachronous cancer are defined as those with an interval of six months or more after diagnosis of gastric cancer . some studies of double primary cancer in patients with gastric cancer have reported on prognosis or characteristics of metachronous cancer [ 3 - 5 ] . however , the general pattern of metachronous gastric cancer still needs to be clarified . due to the high incidence of gastric cancer in korea , although numerous reports on metachronous cancers have been issued , most have been simple case reports , as relatively few studies on the incidence and characteristics of metachronous cancers that develop during the postsurgical follow - up period have been conducted . studies evaluating the characteristics of synchronous and metachronous cancer are limited , and the incidence of metachronous cancer varies to the extent that the clinical results differ in each study . further characterization of metachronous gastric cancer could provide valuable information for use in early diagnosis and treatment of these diseases . the purpose of this study was to investigate the prognosis and clinical features of metachronous gastric cancer . between october 1996 and november 2007 , 5,778 patients underwent surgical treatment or chemotherapy for gastric cancer at dongsan medical center ; however , these patients did not undergo endoscopic resection . patients who had been diagnosed with new primary cancer developing from other origins were selected from the original group . among the 5,778 patients who underwent treatment for gastric cancer , the metachronous cancer patients described above could be defined as those with an interval of six months or more after diagnosis of gastric cancer . the histological diagnosis was established by gastrointestinal endoscopic biopsies and operative specimens . as part of the standard preoperative workup for gastric cancer , the staging workup included a complete physical examination , a complete blood count and biochemical profile , a gastrointestinal contrast study , and a chest and abdominal computed tomography scan . second primary cancer was confirmed by histologic diagnosis , however , hepatocellular carcinoma , pancreatic cancer , and biliary cancer proved difficult to diagnose using this procedure in cases of poor performance . in such cases , in addition , the age of patients at onset of metachronous cancer , or , the tumor , node and metastasis ( tnm ) stage , according to the american joint committee on cancer ( ajcc ) 6th edition , and the presence of factors that could mediate effects on the survival rate were examined retrospectively . evaluation of the interval from the first gastric surgery to development of second primary cancer , clinical characteristics , and survival rate after the second surgery was based on medical records . the kaplan - meier method was used for generation of overall survival curves , and the log - rank test was used for comparison of between - group differences . univariate and multivariate analyses with the cox proportional hazard model were used for analysis of prognostic factors . the survival curve for patients with metachronous double primary cancer was calculated from the date of diagnosis of the second cancer . a p - value of < 0.05 was considered to indicate statistical significance . the spss ver . the kaplan - meier method was used for generation of overall survival curves , and the log - rank test was used for comparison of between - group differences . univariate and multivariate analyses with the cox proportional hazard model were used for analysis of prognostic factors . the survival curve for patients with metachronous double primary cancer was calculated from the date of diagnosis of the second cancer . a p - value of < 0.05 was considered to indicate statistical significance . the spss ver . 18.0 ( spss inc . , among 5,778 patients with gastric cancer , 209 patients ( 5.0% ) were diagnosed with synchronous double primary cancer and 214 patients ( 3.7% ) were diagnosed with metachronous double primary cancer . looking at the age distribution between the groups , the proportion was higher in older patients diagnosed with metachronous double primary cancer ( p<0.001 ) ( table 1 ) . the most common group included patients aged 60 to 70 , and the most common metachronous double primary cancer was colorectal cancer , followed by lung , liver ( excluding cholangiocarcinoma ) , ovarian , and cervical cancer . the mean age was 61.810.62 years , and the oldest patient , who had lung cancer , was 68.5 years old , and the youngest patient , with breast cancer , was 51.7 years old . in comparison of sex distribution of patients with metachronous double primary cancer , double primary cancer occurred more frequently in male patients , with the exception of breast , ovary , and cervical cancer ( p<0.001 ) ( table 3 ) . the observed / expected ( o / e ) ratio was significantly higher in male colorectal ( 1.25 ) , male biliary ( 1.60 ) , ovarian ( 8.72 ) , and cervical cancer ( 3.33 ) with primary gastric cancer , compared with double primary cancer . table 4 shows the yearly diagnostic period for metachronous double primary cancer , and does not indicate any notable difference in incidence from year to year . the most common period of occurrence was 1 - 2 years ( n=60 , 40.8% ) . bladder cancer and esophageal cancer were diagnosed earlier , compared to other cancers ( table 5 ) . table 6 shows development of metachronous double primary cancer over a period of fiveyears , after diagnosis of gastric cancer . development of breast cancer ( 54.5% ) , prostate cancer ( 50.0% ) , and laryngeal cancer ( 40.0% ) occurred much more frequently after five years from diagnosis of gastric cancer . the median survival period for metachronous double primary cancer after diagnosis of second primary cancer was 19.83.33 months ( table 7 ) . the four year survival rate for patients with double primary gastric cancer was 4% , however , the rate without second primary cancer was 61.1% ( fig . the median survival period for patients with breast , laryngeal , cervical , and colorectal cancers was longer than that of other cancers , however , no survival difference was observed according to gastric cancer stage ( table 8) . the first study of synchronous and metachronous cancer in patients with gastric cancer was reported by yoshino et al . . results of the study , conducted in japan , showed an incidence of synchronous and metachronous cancer in patients with gastric cancer of 2.0% . other studies have since reported incidence rates of 1.1 - 4.7% [ 3 - 10 ] . in this study , the incidence was 3.7% , which appears to be overestimated , as it included patients who had not been diagnosed with pathologic confirmation due to their physical condition and/or had difficulty in receiving a pathologic diagnosis . in this study , the most common double primary cancer was colorectal cancer , followed by lung , liver ( excluding cholangiocarcinoma ) , ovary , and cervical cancer . comparing this result with the general cancer incidence in korea , the incidence of colorectal cancer is higher than expected . that is , although colorectal cancer is the third most common cancer in the general population , it is the most common double primary cancer . this result has been observed not only in korea , but also in japan , china , and australia . in the general population , colorectal cancer is the fifth most common cancer in china and the third most common cancer in australia ; however , it is the most common double primary cancer in both countries . therefore , performance of regular colonoscopies on patients who have been diagnosed with gastric cancer is important , particularly for those who are over sixty . the median age for secondary gastric cancer was 63.0 years ; the number of males was 137 , the number of females was 59 . hepatocellular carcinoma ( 30 in 2,481 cases , 1.21% ) , esophageal cancer ( 24 in 518 cases , 4.63% ) , and lung cancer ( 20 in 2,814 cases , 0.71% ) were reported as common types of primary cancers . a comparison of clinicopathological characteristics among patients with other primary cancers and patients without them was conducted by kato et al . and lee et al . statistical differences in age , sex , and gastric cancer staging were observed in these previous studies . that is , the mean age , sex ratio , and proportion of early gastric cancer was higher in patients with other primary cancers than in those without . this study reported similar results , except with regard to gastric cancer staging ; no significant difference was observed in gastric cancer staging , compared to the statistics . the incidences of most cancers are higher in males than in females and tend to increase with age . patients with metachronous cancer of ovary or breast were younger than those with other cancers , which means that female patients with metachronous double primary cancer tend to be younger than male patients . in this study , the o / e ratio was found to be significantly higher in colorectal , biliary , ovarian , and cervical cancer with primary gastric cancer . in order to enhance detection and survival rate , conduct of a large scale prospective study is necessary for investigation of the efficacy of colonoscopies , abdomen and pelvic computed tomographys , and pap smears . previous studies have reported that the majority of double primary malignancies develop within a period of three years , and that surveillance gastroscopy is commonly recommended for a period of three years after surgery for treatment of colorectal cancer . however , there was no exact surveillance colonoscopy recommendation period or surveillance tool for detection of colon cancer after gastric cancer surgery . a higher percentage of metachronous cancers was confirmed for both colorectal and lung cancer . ikeda et al . reported that metachronous lung cancer showed a high incidence of advanced - stage disease and was a major cause of death in patients with metachronous double primary cancers . therefore , close attention should be paid and regular checkups should be performed on patients who have undergone surgery for treatment of gastric cancer . patients who have not been diagnosed with secondary cancers within five years after gastric cancer surgery are usually regarded as having been cured . in this study , metachronous double primary cancer was found to occur after 10 years ; therefore , performance of regular checkups for a longer period of time on patients who have undergone surgery for treatment of gastric cancer is important . survival rates of patients with a metachronous or synchronous cancer were analyzed by maehara et al . , the 10-year survival rate was 69.3% for gastric cancer patients without a double primary cancer , 40.1% for patients with synchronous double primary cancer , and 75.2% for patients with metachronous double primary cancer . in this study , the median survival time for patients with metachronous double primary cancer after diagnosis of second primary cancer was 19.8 months , and 55.2 months after diagnosis of gastric cancer . survival rates for patients withpancreatic cancer and hepato - biliary cancer were very low , indicating a strong relationship of survival rate with the type of double primary cancer . other prognostic factors should be evaluated in order to demonstrate the difference in median survival rate of patients with metachronous double primary cancer . patients with gastric cancer develop double primary cancers either synchronously or metachronously , and , in the present study , second primary cancer was the main cause of death for these patients . the o / e ratio showed a significant increase in colorectal , male biliary , ovarian , and cervical cancer with primary gastric cancer , and second primary cancer was the main cause of death for these patients . a follow - up examination for metachronous double primary cancer	purposethe pattern of double primary cancers after treatment for gastric cancer is important for a patient 's survival.materials and methodswe analyzed the clinicopathologic data of 214 gastric cancer patients from october 1996 to november 2007 with regard to metachronous second primary cancers.resultsout of 5,778 patients with gastric cancer , metachronous second primary cancers occurred in 214 patients . the median age was 61.8 years , the number of male and female patients was 140 ( 65.4% ) , 74 ( 34.6% ) , respectively . the median time to the occurrence of second cancers after diagnosis of the first was 39.2 months ( standard deviation , 31.2 months ) . the most common cancer was colorectal cancer , which occurred in 44 patients ( 20.6% ) , and lung cancer in 33 patients ( 15.4% ) , hepatocellular carcinoma in 26 patients ( 12.1% ) , ovarian cancer in 15 patients ( 7.0% ) , cervical cancer in 12 patients ( 7.0% ) , breast cancer in 11 patients ( 5.1% ) , and esophageal cancer in 11 patients ( 5.1% ) . the observed / expected ( o / e ) ratio showed a significant increase in colorectal ( 1.25 ) , male biliary ( 1.60 ) , ovarian ( 8.72 ) , and cervical cancer ( 3.33 ) with primary gastric cancer . after five years from diagnosis of gastric cancer , secondary cancer occurred in 50 patients ( 23.4% ) , and breast cancer , prostate cancer , laryngeal cancer , lung cancer , and hepatocellular carcinoma were the most frequent.conclusionthe o / e ratio showed a significant increase in colorectal , male biliary , ovarian , and cervical cancer with primary gastric cancer , and second primary cancer as the main cause of death for these patients . a follow - up examination for metachronous double primary cancer is needed in order to improve the survival time in patients with gastric cancer .
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Proceed to summarize the following text: an 18-year - old woman patient was admitted to hospital with complaints of abdominal distension , and the patient had experienced intermittent mild abdominal pain since birth . the abdominal girth had slowly increased in diameter over the previous 18 years , and the patient 's general condition of health was poor , presenting symptoms including fatigue , fever , nausea , vomiting , decreased appetite and weight loss . clinically , the diagnosis of a giant hydronephrose ( gh ) was made , and upon physical examination , an extremely distended abdomen was revealed , with a palpable mass in the all - flank region ( fig . the blood laboratory analyses revealed a concentration of blood urea nitrogen of 30.1 mg / dl , and a level of creatinine of 1.56 mg / dl . the level of hemoglobin was 12 g / dl , and the hematocrit was determined to be 45.7% , whereas tumor markers were all revealed to be in normal range , the results of the urine analysis revealed a red blood cell count of 32/high power field ( hpf ) a white blood cell count of 6/hpf and a yeast cell count of 5/hpf . blood sugar , phosphate , liver function , serum calcium , serum amylase and electrolyte analyses all yielded results within the normal limits . a diagnostic aspiration performed from the area of abdominal swelling revealed the presence of urine . ultrasonography revealed the presence of a massive hypoechoic lesion occupying almost the entire abdomen , which exerted pressure on the bowel on the left - hand side . a cystic lesion revealed multiple septations , which were likely to have arisen from the right kidney . a computerized tomography ( ct ) scan revealed a large abdominal mass of water density in the all - abdomen area , which comprised a huge cystic mass ( 41.530.530.5 cm ; fig . c ) that , in the right kidney , occupied almost the entire abdominal cavity . this mass had displaced the small intestine , colon , pancreas , spleen and the left kidney . after admission , the cystic mass was punctured , and a total of ~24 l urinal fluid was drained by urethral catheterization over a period of 8 days ( 3 liters each day ) . there was an improvement in the general condition of the patient : the distended abdomen decreased in size ( fig . a right nephrectomy was performed : when an incision was made in the right lumbar region , the cyst presented as a retroperitoneal soft cystic dilatation mass , lacking in definition , measuring almost 302015 cm ( fig . 3 ) , which was able to adhere to adjacent organs , including the liver and the pelvic cavity , lifting the cecum and the ascending colon . the ureter was normal , and ureteropelvic junction obstruction ( upjo ) was established as the cause of gh . gh is a rare urological condition , which occurs in patients of all ages , defined as the presence of > 1 liter of fluid in the collecting system ( 1 ) . symptomatic nephrolithiasis and hydronephrosis more frequently present as clinical conditions , and gh is a rare clinical phenomenon . although numerous cases of gh have been reported in the literature , only a few of them contained > 2 liters of fluid ( table i ) . in the present case report , the hydronephrotic kidney contained 24 l urine , and ghs seldom fill the entire abdomenal space , as had occurred in our patient . the most common cause of gh , as detailed in the literature , is a condition caused by upjo , although stone disease , trauma , renal ectopy and ureteral tumors have also been reported ( 2 ) . in the present study , the clinical symptoms of gh are non - specific , and may present with vague symptoms , including an increased abdominal girth due to the presence of a mass in the flank . gh is a slowly progressive disease , and a huge abdominal mass or distended abdomen may cause pain , and be symptomatic of hematuria , recurrent urinary tract infection or other symptoms or complications described in the literature , including nausea , fatigue or dyspepsia , urinary tract infection , loss of weight , renal insufficiency , gross hematuria resulting from trauma in the area , the compression of surrounding structures , or even rupture of the kidney ( 3 ) . ultrasonography and ct scans have facilitated the diagnosis of hydronephrosis , and a case of gh may be defined as the presence of hydronephrosis occupying a hemiabdomen , which meets , or extends beyond , the midline , and which extends at least five or six vertebral bodies in length ( 4 ) . however , in a number of cases , a differential diagnosis between gh and other cystic formations proves to be difficult . therefore , as demonstrated in the present case report , an accurate diagnosis of gh in individual cases remains challenging . contrast - enhanced ct of the abdomen and the pelvis provides the gold standard for an accurate diagnosis of gh , and other useful diagnostic imaging techniques include abdominal radiography and intravenous urography . the ideal treatment for gh is nephrectomy , and this is the procedure of choice ; other treatment options in a functional kidney include percutaneous nephrostomy , reduction pyeloplasty with nephropexy calycoureterostomy , and calycocystostomy ( 5 ) . in spite of the widespread use of prenatal ultrasound and the development of new diagnostic techniques , a puncture / drainage procedure may be performed in cases where the condition of the patient does not allow other treatments to be performed , or where hemodynamic changes may occur following a sudden abdominal decompression ( 6 ) . in the present case study , the patient underwent a right nephrectomy and was discharged on postoperative day 8 . in conclusion , gh is a rare condition , which is associated with the occurrence of cystic abdominal masses , and the puncture / drainage procedure or nephrectomy provides the most suitable method for surgical intervention .	the hydronephrotic kidney , resulting from a ureteropelvic junction obstruction ( upjo ) , presents commonly as a clinical condition , with the presence of usually no more than 12 liters in the collecting system , but a very small number of cases of giant hydronephroses ( ghs ) has been reported in adults . a gh is defined as the adult renal pelvis containing > 1 liter of urine , or at least 1.5% of the body weight . in the majority of cases , the range of the hydronephrotic kidney remains restricted to the renal area . however , the patient described in the present case report had a range for the hydronephrotic kidney which occupied almost the entire abdominal cavity ( ~24 l ) , and cases such as these are rarely presented ; therefore the aim of the present case study was to document a clear case of gh resulting from upjo , also accompanied by a review of the current literature .
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Proceed to summarize the following text: measurement of the outcome is critical for any decision - making and results of evaluations in all medical circumstances . the japan orthopaedic association ( joa ) developed and published a specific instrument to measure outcomes for patients with low back problems in 1986.1 it was called the joa score rating system for low back pain , with a full score being 29 points . since then , the instrument has been widely utilized to evaluate the functional results of many types of intervention for patients with such problems . it has been referred to not only in articles by japanese investigators2 but also in those by non- japanese - speaking investigators.3,4 one of the major criticisms of this specific instrument , however , is that it is not a patient - oriented measurement but a physician - based one . it is now widely accepted that a patient s perspective is essential for making medical decisions and for evaluating the results of interventions.5 based on the current needs for measuring outcome , the joa was urged to revise its original score rating system and to develop a new one . in 2002 , a subcommittee on evaluation of back pain and cervical myelopathy was organized in the clinical outcome committee of joa , and work began on revising the original joa scoring system . as described in the previous literature concerning part 1 , the original joa scoring system was revised and a new scoring system ( the joa back pain evaluation questionnaire joabpeq ) was developed.6 the key points of this revision were to make the original joa score more patient - oriented . for the survey in the part 1 study , we first created a preliminary questionnaire consisting of 60 items . the questionnaire was a self - administered , disease - specific measure that was created with reference to the japanese editions of the short form health survey with 36 questions ( sf-36)7 and the roland - morris disability questionnaire ( rdq)8 to assess health - related quality of life . from the survey , a total of 25 items were selected for tentative use on a draft of the joabpeq ( table 1 ) . table 1items ( n = 25 ) selected for the draft of the joabpeq evaluated in this studywith regard to your health condition during the last week , please circle the item number of the answer for the following questions that best applies . if your condition varies depending on the day or time , circle the item number when your condition is at its worst.q1 - 1 . to alleviate low back pain because of low back pain , you do not do any routine housework these days.1 ) no2 ) yesq1 - 3 . because of low back pain , you lie down more often than usual.1 ) yes2 ) noq1 - 4 . because of low back pain , you sometimes ask someone to help you when you do something.1 ) yes2 ) noq1 - 5 . because of low back pain , you refrain from bending forward or kneeling down.1 ) yes2 ) noq1 - 6 . because of low back pain , you have diffi culty standing up from a chair.1 ) yes2 ) noq1 - 7 . your lower back aches most of the time.1 ) yes2 ) noq1 - 8 . because of low back pain , turning over in bed is diffi cult.1 ) because of low back pain , you have diffi culty putting on socks or stockings.1 ) yes2 ) noq1 - 10 . because of low back pain , you walk only short distances.1 ) yes2 ) noq1 - 11 . because of low back pain , you stay seated most of the day.1 ) yes2 ) noq1 - 13 . because of low back pain , you become irritated or angry at other persons more often than usual.1 ) yes2 ) noq1 - 14 . because of low back pain , you go up stairs more slowly than usual.1 ) yes2 ) noq2 - 1 . how is your present health condition?1 ) excellent2 ) very good3 ) good4 ) fair5 ) poorq2 - 2 . do you have diffi culty in climbing stairs?1 ) i have great diffi culty.2 ) i have some diffi culty.3 ) i have no diffi culty.q2 - 3 . do you have diffi culty in any one of the following motions : bending forward , kneeling , or stooping?1 ) i have great diffi culty.2 ) i have some diffi culty.3 ) i have no diffi culty.q2 - 4 . do you have diffi culty walking more than 15 minutes?1 ) i have great diffi culty.2 ) i have some diffi culty.3 ) i have no diffi culty.q2 - 5 . have you been unable to do your work or ordinary activities as well as you would like?1 ) i have not been able to do them at all.2 ) i have been unable to do them most of the time.3 ) i have sometimes been unable to do them.4 ) i have been able to do them most of the time.5 ) i have always been able to do them.q2 - 6 . has your work routine been hindered because of the pain?1 ) greatly2 ) moderately3 ) slightly ( somewhat)4 ) little ( minimally)5 ) not at allq2 - 7 . have you been discouraged or depressed?1 ) always2 ) frequently3 ) sometimes4 ) rarely5 ) neverq2 - 8 . do you feel exhausted?1 ) always2 ) frequently3 ) sometimes4 ) rarely5 ) neverq2 - 9 . do you feel happy?1 ) always2 ) almost always3 ) sometimes4 ) rarely5 ) neverq2 - 10 . do you think you are in reasonable health?1 ) yes ( i am healthy.)2 ) fairly ( my health is better than average)3 ) not ( very much)/particularly ( my health is average)4 ) barely ( my health is poor)5 ) not at all ( my health is very poor)q2 - 11 . do you feel your health will get worse?1 ) very much so2 ) a little at a time3 ) sometimes yes and sometimes no4 ) not very much5 ) not at all items ( n = 25 ) selected for the draft of the joabpeq evaluated in this study the purpose of the part 2 study in this project was to evaluate the reliability of the 25 items selected for the draft joabpeq ; for this , test - retest reliability was ascertained . altogether , 460 of the 829 japanese board - certified spine surgeons were randomly selected , and each was asked to recruit two patients to evaluate the joabpeq between january and june 2004 . the recruited patients were scheduled to reply to the questionnaire twice at a 2-week interval . patient criteria were as follows : ( 1 ) patients could be any age of either sex ; ( 2 ) patients had any lumbar spine disorder and were currently visiting an outpatient clinic ; ( 3 ) the severity of the symptoms was expected to be at the same level between the two interviews . exclusion criteria were the presence of : ( 1 ) other musculoskeletal diseases requiring medical treatment ; ( 2 ) psychiatric disease ( e.g. , dementia ) , potentially leading to inappropriate answers ; ( 3 ) a postoperative condition ; ( 4 ) having participated in previous surveys of the related study . each patient was asked to complete the same questionnaire twice at an interval of 2 weeks ( 3 days ) . the attending surgeon filled out the patient information on the diagnosis and the presence or absence of concomitant diseases , followed by judging the severity of symptoms using a three - step rating scale ( mild , moderate , severe ) . symptom severity was determined subjectively by the attending surgeon , who was asked not to select a similar patient solely on the basis of severity . patients who had the same level of severity as judged by all surgeons were then selected and analyzed to verify the reliability of the questionnaire . this study was approved by the ethics committee of the japanese society for spine surgery and related research . the reliability of the questionnaire was evaluated by determining the extension of the kappa coefficients . the kappa and weighted kappa coefficients were calculated based on a formula using microsoft office excel 2003 . kappa and weighted kappa coefficients of 0.4 or above were judged to be reliable.9 the 95% confidence intervals ( 95% ci ) were calculated for all reliability coefficients using the bootstrap method . altogether , 460 of the 829 japanese board - certified spine surgeons were randomly selected , and each was asked to recruit two patients to evaluate the joabpeq between january and june 2004 . the recruited patients were scheduled to reply to the questionnaire twice at a 2-week interval . patient criteria were as follows : ( 1 ) patients could be any age of either sex ; ( 2 ) patients had any lumbar spine disorder and were currently visiting an outpatient clinic ; ( 3 ) the severity of the symptoms was expected to be at the same level between the two interviews . exclusion criteria were the presence of : ( 1 ) other musculoskeletal diseases requiring medical treatment ; ( 2 ) psychiatric disease ( e.g. , dementia ) , potentially leading to inappropriate answers ; ( 3 ) a postoperative condition ; ( 4 ) having participated in previous surveys of the related study . each patient was asked to complete the same questionnaire twice at an interval of 2 weeks ( 3 days ) . the attending surgeon filled out the patient information on the diagnosis and the presence or absence of concomitant diseases , followed by judging the severity of symptoms using a three - step rating scale ( mild , moderate , severe ) . symptom severity was determined subjectively by the attending surgeon , who was asked not to select a similar patient solely on the basis of severity . patients who had the same level of severity as judged by all surgeons were then selected and analyzed to verify the reliability of the questionnaire . this study was approved by the ethics committee of the japanese society for spine surgery and related research . the reliability of the questionnaire was evaluated by determining the extension of the kappa coefficients . the kappa and weighted kappa coefficients were calculated based on a formula using microsoft office excel 2003 . kappa and weighted kappa coefficients of 0.4 or above were judged to be reliable.9 the 95% confidence intervals ( 95% ci ) were calculated for all reliability coefficients using the bootstrap method . a total of 350 patients participated in this study and completed the questionnaire twice following the project s plan . however , 135 patients were excluded because the severity of their symptoms had changed between the two interviews or they violated the interval period . of the remaining 215 patients , 54 were ineligible because of other musculoskeletal diseases , such as knee and hip osteoarthrosis . as a result , a total of 161 patients were available for the analysis in this study : 86 men and 75 women with a mean age of 57.7 years ( sd 16.3 years ) . the clinical diagnosis included degenerative lumbar canal stenosis in 49 patients , lumbar disc herniation in 44 , spondylolisthesis in 20 , spondylosis in 16 , degenerative disc disease in 13 , mechanical low back pain in 11 , and miscellaneous in 8 . the patients age varied from their twenties to their eighties , and symptom severity varied from mild to severe ( table 2 ) . neurological and physical status was evaluated for each patient using the current joa score rating system and finger - floor distance ( table 3 ) . neurological deficits varied from mild to severe , and trunk flexibility varied among the subjects as well . table 2distribution of age and severity of symptoms in the patient analyzed ( n = 161)no . of patients , by severity of symptomsage ( years)mildmoderateseveretotalmen20223730441940220450791176013832470618024800011total3443986women2023053045094061185094114607111197089118801102total3734475total no.717713161table 3current japanese orthopaedic association score rating system and fi nger to fl oor distance for the patients analyzed ( n = 161)parameterno.slr testnormal124307035<302motor functionnormal113slight weakness ( mmt : good)38severe weakness ( mmt : less than good)10sensory functionnormal80slight disturbance59severe disturbance22bladder functionnormal147mild dysuria12severe dysuria2finger to floor distance ( cm)-151 - 14-517 - 4-441514401524322534935447455475564465741immeasurable2total no.161slr , straight leg raising ; mmt , manual muscle testing distribution of age and severity of symptoms in the patient analyzed ( n = 161 ) current japanese orthopaedic association score rating system and fi nger to fl oor distance for the patients analyzed ( n = 161 ) slr , straight leg raising ; mmt , manual muscle testing face validity was checked in terms of the completion rate for filling out the questionnaire . the distribution of the answers for all question items was then checked to ensure that there were no biased answers . items remaining unanswered accounted for less than 5% in the first test , and there was no skewed distribution , such as floor and ceiling effects ( table 4 ) . table 4reproducibility of each item ( n = 161)choices for answeritem12345no answerq1 - 111743172.7%26.7%0.6%q1 - 232127219.9%78.9%1.2%q1 - 37683247.2%51.6%1.2%q1 - 44211926.1%73.9%q1 - 5778447.8%52.2%q1 - 63113019.3%80.7%q1 - 7689342.2%57.8%q1 - 86595140.4%59.0%0.6%q1 - 9728944.7%55.3%q1 - 10877454.0%46.0%q1 - 1135122421.7%75.8%2.5%q1 - 1241119125.5%73.9%0.6%q1 - 133612522.4%77.6%q1 - 1411545171.4%28.0%0.6%q2 - 118596616211.2%36.6%41.0%9.9%1.2%q2 - 215925229.3%57.1%32.3%1.2%q2 - 3259338515.5%57.8%23.6%3.1%q2 - 4357055121.7%43.5%34.2%0.6%q2 - 5151285351319.3%7.5%52.8%21.7%8.1%0.6%q2 - 6133666321138.1%22.4%41.0%19.9%6.8%1.9%q2 - 712879392217.5%5.0%49.1%24.2%13.7%0.6%q2 - 882788241225.0%16.8%54.7%14.9%7.5%1.2%q2 - 98427431515.0%26.1%46.0%19.3%3.1%0.6%q2 - 10135942341218.1%36.6%26.1%21.1%7.5%0.6%q2 - 111748562811110.6%29.8%34.8%17.4%6.8%0.6% reproducibility of each item ( n = 161 ) the test - retest reliability was confirmed by calculating the kappa and weighted kappa coefficients for each item ( tables 5a , 5b ) . both kappa and weighted kappa were more than 0.50 in all items , except in one item with 0.48 . the lower 95% ci exceeded 0.4 in all items , except in two items with 0.39 . this implied that the test - retest reliability of joabpeq was acceptable as a measurement of outcome . table 5akappa coefficient with 95% ci for items q1 - 1 to q1 - 14item95% ciq1 - 10.690.600.77q1 - 20.620.510.73q1 - 30.670.600.75q1 - 40.650.560.75q1 - 50.480.390.57q1 - 60.550.430.66q1 - 70.650.570.73q1 - 80.550.470.64q1 - 90.710.640.78q1 - 100.630.550.72q1 - 110.500.390.61q1 - 120.560.460.65q1 - 130.650.550.74q1 - 140.720.640.80ci , confidence intervaltable 5bweighted kappa coefficient with 95% ci for items q2 - 1 to q2 - 11itemweighted 95% ciq2 - 10.510.430.57q2 - 20.610.520.68q2 - 30.570.490.64q2 - 40.730.680.78q2 - 50.540.470.60q2 - 60.610.550.67q2 - 70.530.460.59q2 - 80.550.480.61q2 - 90.540.460.60q2 - 100.540.470.61q2 - 110.530.460.60 kappa coefficient with 95% ci for items q1 - 1 to q1 - 14 ci , confidence interval weighted kappa coefficient with 95% ci for items q2 - 1 to q2 - 11 a total of 350 patients participated in this study and completed the questionnaire twice following the project s plan . however , 135 patients were excluded because the severity of their symptoms had changed between the two interviews or they violated the interval period . of the remaining 215 patients , 54 were ineligible because of other musculoskeletal diseases , such as knee and hip osteoarthrosis . as a result , a total of 161 patients were available for the analysis in this study : 86 men and 75 women with a mean age of 57.7 years ( sd 16.3 years ) . the clinical diagnosis included degenerative lumbar canal stenosis in 49 patients , lumbar disc herniation in 44 , spondylolisthesis in 20 , spondylosis in 16 , degenerative disc disease in 13 , mechanical low back pain in 11 , and miscellaneous in 8 . the patients age varied from their twenties to their eighties , and symptom severity varied from mild to severe ( table 2 ) . neurological and physical status was evaluated for each patient using the current joa score rating system and finger - floor distance ( table 3 ) . neurological deficits varied from mild to severe , and trunk flexibility varied among the subjects as well . table 2distribution of age and severity of symptoms in the patient analyzed ( n = 161)no . of patients , by severity of symptomsage ( years)mildmoderateseveretotalmen20223730441940220450791176013832470618024800011total3443986women2023053045094061185094114607111197089118801102total3734475total no.717713161table 3current japanese orthopaedic association score rating system and fi nger to fl oor distance for the patients analyzed ( n = 161)parameterno.slr testnormal124307035<302motor functionnormal113slight weakness ( mmt : good)38severe weakness ( mmt : less than good)10sensory functionnormal80slight disturbance59severe disturbance22bladder functionnormal147mild dysuria12severe dysuria2finger to floor distance ( cm)-151 - 14-517 - 4-441514401524322534935447455475564465741immeasurable2total no.161slr , straight leg raising ; mmt , manual muscle testing distribution of age and severity of symptoms in the patient analyzed ( n = 161 ) current japanese orthopaedic association score rating system and fi nger to fl oor distance for the patients analyzed ( n = 161 ) slr , straight leg raising ; mmt , manual muscle testing face validity was checked in terms of the completion rate for filling out the questionnaire . the distribution of the answers for all question items was then checked to ensure that there were no biased answers . items remaining unanswered accounted for less than 5% in the first test , and there was no skewed distribution , such as floor and ceiling effects ( table 4 ) . table 4reproducibility of each item ( n = 161)choices for answeritem12345no answerq1 - 111743172.7%26.7%0.6%q1 - 232127219.9%78.9%1.2%q1 - 37683247.2%51.6%1.2%q1 - 44211926.1%73.9%q1 - 5778447.8%52.2%q1 - 63113019.3%80.7%q1 - 7689342.2%57.8%q1 - 86595140.4%59.0%0.6%q1 - 9728944.7%55.3%q1 - 10877454.0%46.0%q1 - 1135122421.7%75.8%2.5%q1 - 1241119125.5%73.9%0.6%q1 - 133612522.4%77.6%q1 - 1411545171.4%28.0%0.6%q2 - 118596616211.2%36.6%41.0%9.9%1.2%q2 - 215925229.3%57.1%32.3%1.2%q2 - 3259338515.5%57.8%23.6%3.1%q2 - 4357055121.7%43.5%34.2%0.6%q2 - 5151285351319.3%7.5%52.8%21.7%8.1%0.6%q2 - 6133666321138.1%22.4%41.0%19.9%6.8%1.9%q2 - 712879392217.5%5.0%49.1%24.2%13.7%0.6%q2 - 882788241225.0%16.8%54.7%14.9%7.5%1.2%q2 - 98427431515.0%26.1%46.0%19.3%3.1%0.6%q2 - 10135942341218.1%36.6%26.1%21.1%7.5%0.6%q2 - 111748562811110.6%29.8%34.8%17.4%6.8%0.6% reproducibility of each item ( n = 161 ) the test - retest reliability was confirmed by calculating the kappa and weighted kappa coefficients for each item ( tables 5a , 5b ) . both kappa and weighted kappa were more than 0.50 in all items , except in one item with 0.48 . the lower 95% ci exceeded 0.4 in all items , except in two items with 0.39 . this implied that the test - retest reliability of joabpeq was acceptable as a measurement of outcome . table 5akappa coefficient with 95% ci for items q1 - 1 to q1 - 14item95% ciq1 - 10.690.600.77q1 - 20.620.510.73q1 - 30.670.600.75q1 - 40.650.560.75q1 - 50.480.390.57q1 - 60.550.430.66q1 - 70.650.570.73q1 - 80.550.470.64q1 - 90.710.640.78q1 - 100.630.550.72q1 - 110.500.390.61q1 - 120.560.460.65q1 - 130.650.550.74q1 - 140.720.640.80ci , confidence intervaltable 5bweighted kappa coefficient with 95% ci for items q2 - 1 to q2 - 11itemweighted 95% ciq2 - 10.510.430.57q2 - 20.610.520.68q2 - 30.570.490.64q2 - 40.730.680.78q2 - 50.540.470.60q2 - 60.610.550.67q2 - 70.530.460.59q2 - 80.550.480.61q2 - 90.540.460.60q2 - 100.540.470.61q2 - 110.530.460.60 kappa coefficient with 95% ci for items q1 - 1 to q1 - 14 ci , confidence interval weighted kappa coefficient with 95% ci for items q2 - 1 to q2 - 11 measurement of the outcome is generally divided into two categories : generic and disease - specific measures.5,10 sf-36 has been commonly used as representative of a measurement of generic health status.5,7,10 the rdq and the oswestry disability index are widely used as disease - specific measurements for back pain.8,11 the joa score rating system for low back pain , developed in 1986 , was also a disease - specific measuring instrument for back disorders and injuries and has been widely utilized in clinical research and the decision - making process in japan . however , this is not a patient - based outcome measure reliable enough to describe the objective status of the function and quality of life ( qol ) of patients with low - back disorders . there has , to date , been insufficient psychometric analysis to confirm the validity and reliability of this joa score rating system . the project for developing the new questionnaire , joabpeq , was initiated to create a self - administered , disease - specific method for measuring low back pain . this instrument should include functions of the lumbar spine as well as health - related qol . the reliability of the questionnaire that includes the 25 suggested items was evaluated using psychometric analysis as part 2 of this project . kappa and weighted kappa coefficient were utilized to verify the test - retest reliability.12,13 in terms of external validity , biased data were inevitable because one criterion that was included was that the severity of the symptoms was expected to be at the same level between the two interviews . however , there was no bias on the choices of answer to each question . this implies that test - retest reliability was acceptable even if the subjects had symptoms of different severity . the older the patients were , the worse was the interpretation of each question . there were small numbers of patients of younger generations , such as those in their thirties and forties , in this study . thus , the reliability would not deteriorate even if the number of young people were to increase . in terms of english expression , there is a possibility of ambiguity in questions 12 and 111 , where double negatives ( two no s in the answer ) may be confusing . it is necessary to reconsider and revise the english expression so it is more easily understood by native english - language users . the number of choices for the answer in all questions varied from two to five , which is also a point to be reconsidered in the future . the current study demonstrated that the 25 items had enough reliability to describe the qol in patients suffering low back disorders . however , further studies are needed to complete the project , including a factor analysis to determine the underlying cluster of the questionnaire items , a formula for calculating the severity score , and confirmation of the responsiveness to the questionnaire . the tentative joabpeq with 25 items was confirmed to be reliable enough to describe the qol of patients suffering low back disorders .	backgroundthe project to develop a new japanese orthopaedic association ( joa ) score rating system for low back disorders , the joa back pain evaluation questionnaire ( joabpeq ) , is currently in progress . part 1 of the study selected 25 candidate items for use on the joabpeq . the purpose of this current part 2 of the study was to verify the reliability of the questionnaire.methodsa total of 161 patients with low - back disorders of any type participated in the study . each patient was interviewed twice at an interval of 2 weeks using the same questionnaire . the reliability of the questionnaire was evaluated by determining the extension of the kappa and weighted kappa coefficients.resultsboth kappa and weighted kappa were more than 0.50 for all but one item , which was 0.48 . the lower 95% confidence interval exceeded 0.4 in all but two items , which was 0.39 . this implied that the test retest reliability of joabpeq was acceptable as a measure of outcome.conclusionsthe tentative questionnaire of the joabpeq with 25 items was confirmed to be reliable enough to describe the quality of life of patients who suffer low back disorders .
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Proceed to summarize the following text: breast cancer , first described as early as 3000 b.c . by edwin smith papyrus of egypt , comprises 23% of all female malignancies ( excluding non melanomatous skin cancer ) [ 1 , 2 ] . in 2008 , the number of deaths from breast cancer totaled 460,000 patients ; it is a global concern accounting for 14% of all cancer deaths in females . it is the most common invasive cancer in women with an incidence that ranges between 19.3 per 100,000 in eastern africa to 89.7 per 100,000 in western europe [ 4 , 5 ] . the mortality in breast cancer patients is attributed to metastatic disease [ 3 , 6 ] . it is known to metastasize to numerous organs including lymph nodes , lung , bone , liver , skin , kidneys , brain , adrenal , thyroid , and heart [ 3 , 6 , 7 , 8 ] . breast cancer metastasizing to the urinary bladder has only been reported sporadically totaling 41 cases in the english medical literature [ 3 , 913 ] . bladder metastasis from breast cancer as the only organ involved is very rare , with only eight cases reported worldwide [ 3 , 11 , 12 , 14 ] . we herein present a patient who presented with bladder metastasis from breast cancer with the bladder being the only organ involved . a 64year old female patient , a non smoker known to have hypertension , diabetes mellitus , dyslipidemia , supraventricular tachycardia , and osteoporosis was diagnosed in 2005 with left breast intraductal carcinoma . ten out of 23 nodes were positive ; she was staged as t2 ( 4 cm ) n3 m0 disease . the patient was treated with eight cycles of chemotherapy , adriamycin , cytoxan , and taxol and 25 sessions of radiation therapy ( total dose of 50 grays ) , and completed a 5year treatment with an aromatase inhibitor . she had no evidence of disease until march 2010 , when she presented to our clinic with recurrent urinary tract infections and urinary incontinence that failed to resolve with antibiotics alone . workup included a computed tomography ( ct ) scan of her abdomen and pelvis , which showed diffuse thickening of the urinary bladder wall with surrounding fat streaking of the pelvis and retroperitoneum ( figure 1 ) . there was evidence of mild right hydroureteronephrosis with no evidence of obstructing urinary tract calculi ( figure 1 ) . a , b. diffuse thickening of the urinary bladder wall ( arrows ) with surrounding fat streaking of the pelvis . a pocket of gas is noted c , d. mild right hydroureteronephrosis ( arrowheads ) . pathology showed an unremarkable bladder mucosa , but a submucosal nest of carcinoma cells was found . the cells were plasmacytoid with marked nuclear pleomorphism , frequent mitotic figures and multiple foci suggestive of lymphovascular invasion were present . the tumor cells were positive for estrogen receptor and e cadherin ; they were negative for progesterone receptors . a. h&e sections of the urinary bladder biopsy revealing a dense submucosal infiltrate ( mag . b. the cells are cohesive , plasmacytoid with an abundant eosinophilic cytoplasm and eccentric nucleus ( mag . the cells demonstrate positive immunostaining with anti estrogen receptor ( er ) antibody ( c , mag . chest ct scan and bone scan were performed as part of the full work up and failed to show any evidence of other distant metastasis . one week after receiving the first cycle of chemotherapy , the patient developed hematuria and clot retention . several attempts of irrigation failed so the decision was made to perform cystoscopy in order to fulgurate all bleeders . the cystoscopy identified a large clot in the bladder , which was removed and all bleeding areas were fulgurated . . however , a few days after the third chemotherapy cycle she developed severe dyspnea and was found to have pneumonia that progressed to septic shock . the patient passed away from cardiorespiratory arrest one year after the diagnosis of the bladder metastasis . until 2012 , 41 cases of bladder metastasis from breast cancer have been reported and they were mostly associated with systemic dissemination and multiple organ involvement . only eight cases of solitary bladder metastasis from primary breast cancer have been documented and our case represents the ninth in the english medical literature [ 3 , 11 , 12 , 14 ] ( table i , in bold ) . macroscopically , bladder metastasis may appear as a mass , irregular lesion , mucosal nodularity , abnormally thickened bladder wall , or plaque with telangiectasias . cystoscopy was performed based on the suspicious ct scan findings , and it revealed an abnormal bladder wall thickening and inflammation , which were both subsequently biopsied . abbreviations : gu genitourinary , nr not reported , mets metastasis , ln lymph node , sb small bowels , lb large bowels , ilc invasive lobular carcinoma , idc invasive ductal carcinoma , er estrogene receptors , pr progesterone receptors the breast primary tumor subtype was invasive intraductal carcinoma while in the previously published reports , the most common histology of the breast primary was invasive lobular ( 10 out of the 15 cases where the breast cancer subtype was determined ) . bates and baithun reported 4.5% incidence of secondary bladder metastasis among all bladder cancer [ 9 , 15 ] , with secondary metastasis to the bladder from breast cancer being approximately 3% [ 16 , 17 ] . when autopsy and pathology are used as mainstay for diagnosis , the incidence ranges from 0% to 7% . bladder metastasis from previously diagnosed breast cancer is reported in the literature to vary from 2% to 14% . the most common primary tumors metastasizing to the bladder are : stomach , lung , skin / melanoma , and breast . it is only when the mucosa is involved by the disease will symptoms become clinically detectable . since mucosal involvement is the last stage of metastatic invasiveness into the bladder , the prognosis is very poor with a mean survival of two to three years , although a 5year survival of two patients out of the 41 was reported in the literature [ 14 , 20 ] . as a consequence , early stages of breast cancer metastasizing to the bladder the most common findings in patients with secondary bladder metastasis are lower urinary tract symptoms ( luts ) , flank or abdominal pain , hydronephrosis , and the painless hematuria that is in many cases the most common initial symptom ( microscopic being more frequent than gross ) . hematuria as a sign of bladder involvement following primary breast cancer is considered sensitive , but not specific for tumor metastasis . gross hematuria with a history of breast cancer needs to be thoroughly investigated , keeping in mind the side effects of cyclophosphamide as treatment of the primary breast cancer , regardless of time or duration of treatment . suprapubic and bilateral flank pain was later the major disabling symptom that warranted the investigation through ct scan imaging . ca 153 is one method to follow up breast cancer recurrence or metastasis , but strong evidence are lacking on its clinical usefulness . the positron emission tomography ( pet ) scan has been showed to have increasing usage after suspecting bladder involvement in a breast cancer patient ; however , its cost effectiveness is yet to be determined . breast metastasis to the bladder has been shown to have a worse prognosis than metastasis to bone . the time interval between primary tumor diagnosis and detection of metastasis is highly variable between 0 month and 30 years with an average of 6.2 years ( table 1 ) . bladder metastases in our patient were identified five years after the initial diagnosis of primary breast carcinoma . conduction of the proper investigations prevented the delay in the diagnosis of the metastatic disease . the patient survived one year from the time she first presented with urinary symptoms , and there was no evidence that her death was related to the bladder metastasis . only 8% of all breast cancer is lobular carcinoma , however , it is the most common type of breast cancer type involving the bladder ( 33% of secondary bladder metastasis ) followed by ductal carcinoma , which accounts for the majority of primary breast cancer ( 66% ) and metastasizes mostly to the lung parenchyma . one hypothesis is that lobular carcinoma is of the serosal type , which gives it a predilection to spread to the gastrointestinal and gynecological systems . it is part of the seeding soil hypothesis : the interaction of tumor with specific host factors in the metastasized organ . another culprit is concomitant steroid therapy , which is thought to be due to exacerbation of the immunosuppressive effect . estrogen , progesterone , and her2 receptors are the three main receptors studied in breast cancer . discrepancy between receptors is not uncommon between the primary and the secondary tumor ( reported between 30 and 39% ) . bladder metastases from our case were positive for estrogen receptors , which was also true for the patient 's known primary cancer of the breast . however , progesterone receptors were only present in the malignant breast cells and not the secondary bladder metastasis . one hypothesis is that the polyclonicity of breast tumor cells is affected by treatment modalities ( hormonal therapy may select some and suppress others ) , which manifests itself later in case of bladder metastasis . it has been shown that if receptors convert from positive in the breast to negative in the bladder , it is associated with decrease survival [ 17 , 24 ] . even with receptor negative secondary bladder metastasis , a trial of anti receptor therapy has been used with promising results in controlling disease . reported cases of bladder metastasis were managed through surgery , chemotherapy , radiotherapy , hormonal therapy , or a combination of those . in our patient , we report a rare case of breast cancer with solitary urinary bladder metastasis that was diagnosed several years after the initial presentation . secondary malignancies of the bladder are difficult to distinguish from non transitional cell primary bladder cancer . a high level of suspicion and extensive investigation are warranted if a known primary cancer already exists . we emphasize the need to be more aware of the possible metastatic nature of every urinary symptom that shows in a breast cancer patient .	breast cancer is the most common malignancy in woman . the urinary bladder is an unusual site for metastasis from primary tumors of the breast , especially when it is the only organ involved . we present the case of a female patient with known breast cancer stage t2n3m0 who developed isolated bladder metastasis five years after the primary diagnosis . we reviewed the literature for similar cases and discussed the clinical presentation , pathophysiology , and prognosis of this entity .
You are an expert at summarizing long articles. Proceed to summarize the following text: eukaryotic genomic dna is assembled into a highly compacted state with an equal mass of protein to form chromatin . the repeating unit of chromatin is the nucleosome , which is formed by wrapping ~150 base pairs of dna around a core histone octamer ( two molecules each of histone h2a , h2b , h3 , and h4 ) . nucleosomes are connected by short linker dna segments to form long arrays , which undergo short - range intra and long - range inter - nucleosomal interactions to contribute to the high degree of compaction observed in interphase chromosomes . this condensation of genomic dna into chromatin has profound implications for the regulation of key dna processes such as transcription . for example , this compaction and occupancy of canonical nucleosomes at the tss of genes transcribed by rna polymerase ii prevents access to the transcription machinery . it therefore has been assumed that for transcription to proceed , a nucleosome free ( nfr ) or depleted region ( ndr ) must be created at the tss , and the experimentally observed increase in accessibility to micrococcal nuclease digestion has been used as evidence to support this notion . however , such sensitivity to micrococcal nuclease digestion at the tss does not necessarily imply a loss of nucleosomes but could also be the consequence of the formation of a highly unstable nucleosome . indeed , our work and the studies of others indicate that this is the case and that histone variants play an important role in assembling unstable nucleosomes at the tss . among the core histones , the h2a family shows the greatest divergence in primary sequence leading to the greatest number of variants known . the key amino acid differences between histone h2a variants and histone h2a are strategically placed within the nucleosome to affect nucleosome stability and also on the surface of the nucleosome ( the acidic patch ) to regulate the interactions between neighboring nucleosomes thereby determining the extent chromatin compaction . h2a.z has a range of functions including being required for early metazoan development , chromosome organization and inheritance , as well as regulating promoter chromatin architecture . notably in mammalian cells , the apparent ndr of active genes at the tss is flanked on both sides by h2a.z containing - nucleosomes ( the 2 and + 1 nucleosome , respectively ) . a long - held view has been that chromatin - based information at the promoter , including h2a.z , are stable marks that provide transcriptional memory so that following cell division , a previously active pol ii promoter can be readily re - activated . we tested this hypothesis by examining how the two h2a.z - containing nucleosomes that flank the tss ( in particular the h2a.z nucleosome located just downstream of the tss , which is referred to as the + 1 nucleosome ) are restored following the highly disruptive process of dna replication ( s phase ) and whether they remain with chromosomes as they condense during metaphase ( m phase ) . we employed mouse trophoblast stem ( ts ) cells as a model system , given that h2a.z is required for their viability . experimentally , we synchronized cells at the g1 , s and m phases of the cell cycle and then performed h2a.z and histone h3 chip - seq experiments as well as examining global gene expression at these three stages of the cell cycle using whole mouse genome expression microarrays . unexpectedly , compared with histone h3 , there was a loss of h2a.z at active promoters in s and m vs. g1 for all groups of genes irrespective of whether they were more highly expressed at g1 , s or m phase . the next question therefore became why was h2a.z not fully restored at promoters following s phase to g1 levels ? to address this problem we then investigated how h2a.z - containing nucleosomes were inherited following s phase by determining whether the number of copies of h2a.z per nucleosome changed ( a h2a.z nucleosome could contain either one or two copies of h2a.z ) . specifically , we investigated whether at the promoter there was an increase in heterotypic h2a.z - h2a nucleosomes or would the ratio of heterotypic h2a.z - h2a to homotypic h2a.z - h2a.z remain unchanged following s phase . to investigate this issue we performed h2a.z chip followed by h2a rechip assays . basically , chip assays were first performed using affinity purified h2a.z antibodies to immunoprecipitate all h2a.z - containing nucleosomes followed by a second chip employing h2a antibodies to pull down heterotypic h2a.z - h2a nucleosomes . significantly , we observed that only 5% of h2a.z nucleosomes at g1 were heterotypic but by m phase , this dramatically increased to 35% . at active promoters , there was a clear increase in heterotypic h2a.z - h2a nucleosomes at s and m compared with g1 phase ( compare fig . this allowed us to conclude that at g1 , homotypic h2a.z - h2a.z nucleosomes flanked both sides of the tss but following the disruptive dna replication process , these nucleosomes became heterotypic , which can explain , at least in part , why h2a.z was not fully restored at promoters to g1 levels following s phase . ( a ) four h2a.z chip - h2a rechip profiles were generated for the g1 phase . each individual line represents a group of 4200 genes and represents the normalized tag counts at each base pair , aligned between 1 and + 1 kb from the tss . the line color reflects the average gene expression rank of the contained genes as shown . ( b ) h2a.z chip - h2a rechip normalized tag counts at s phase for 4 groups of 4200 genes aligned with the tss . ( c ) h2a.z normalized tag counts at m phase for 4 groups of 4200 genes aligned with the tss . ( d ) we chose the top 100 differentially expressed genes for genes more highly expressed in g1 phase vs. m phase and represented them as a single line of normalized h2a.z chip - h2a rechip counts at each base pair , aligned with the tss . how this profile changes as the ts cells progresses from g1 to m is shown . what is clearly observed is that h2a.z - h2a heterotypic nucleosomes form at the promoter at s phase and that this profile remains similar at m phase . surprisingly , we found that heterotypic h2a.z - h2a nucleosomes do also exist at g1 and most notably these nucleosomes were located on the tss of active promoters ( fig . this demonstration that the tss is not nucleosome free in ts cells is supported by a previous finding showing that a double histone variant nucleosome comprised of h2a.z and h3.3 was located at the tss of active genes in hela cells . moreover , while it was revealed that this double variant nucleosome was unstable , the structural basis for its instability remained unknown . our demonstration that this unstable nucleosome located at the tss is heterotypic in respect with h2a.z may offer a molecular explanation for its instability . based on the crystal structure of a homotypic h2a.zh2a.z nucleosome , it was predicted that the replacement of h2a with only one molecule of h2a.z will cause a major structural clash between their l1 loop regions , which is sufficient to destabilize it . consistent with this heterotypic nucleosome being labile , we also found it at other dynamic genomic loci including ctcf binding sites and dnase i hypersensitive sites . in addition , we observed displacement of this heterotypic h2a.z / h2a nucleosome following the passage through s phase at all of these sites . on the other hand , / h2a.z nucleosome is more stable than a canonical nucleosome , which can be attributed to an enhanced interaction between the l1 loop regions of each h2a.z molecule compared with h2a . why a heterotypic h2a.z / h2a nucleosome only exists at the tss in g1 is unclear but probably reflects a different chromatin configuration at the tss for genes expressed in g1 vs. genes expressed in m and s. finally , we addressed the question of why heterotypic h2a.z - h2a nucleosomes formed during s phase was not fully restored to the homotypic state by m phase ( fig . . another important function of h2a.z is to maintain the structural integrity of the centromere and surrounding constitutive heterochromatin . we therefore wondered whether the loss of h2a.z at promoters was coincident with a gain of h2a.z at centromeres and indeed this was found to be the case . taken together , this dynamic net transfer of h2a.z from promoters to the centromere at m phase brings into question whether h2a.z has a role in providing transcriptional memory during the cell cycle . interestingly , it has also been shown that histone post - translational modifications and other histone variants are likewise restored slowly after dna replication , and in some cases requiring more than one round of cell division to be fully re - established . while h2a.z is ubiquitously expressed , human h2a.bbd ( h2a.b ) is expressed primarily in the adult testis and brain ( ncbi geo data sets ) . it is also aberrantly expressed in a number of tumors and cancer cell lines ( ncbi geo data sets ) . in contrast to h2a.z which promotes nucleosome stability and chromatin compaction , h2a.bbd-containing histone octamers and mononucleosomes are unstable and the latter only protects 120 base pairs of dna from micrococcal digestion compared with 145 base pairs for canonical nucleosome core particles . this is consistent with fret , afm , and cryo - em studies showing the unwrapping of nucleosomal dna from the nucleosome surface at the dna entry and exit points . assembly of nucleosomal arrays with h2a.bbd completely inhibits array folding and compaction leading to a de - repression of chromatin mediated transcription . this ability of h2a.bbd to prevent chromatin compaction is due to the loss of three acidic amino acid residues , which partially neutralizes the acidic patch located on the surface of the nucleosome . to gain new insights into the possible role(s ) of h2a.bbd and its orthologs , we turned to the mouse where we identified four major we designated these variants as h2a.lap 14 ( lack of an acidic patch ) ( h2a.b.1 , h2a.l.1 , h2a.l.2 , and h2a.m , respectively ) to more accurately reflect their partially neutralized acidic patch , with h2a.lap1 being most similar to h2a.bbd . intriguingly though , while h2a.lap1 also prevented chromatin compaction and also only protected 120 base pairs of dna from micrococcal digestion in vitro , it was less efficient in decompacting chromatin compared with h2a.bbd indicating that these histone variants are not completely functionally equivalent ( which may explain why the mouse has four major h2a.bbd-like proteins whereas human has only one protein ) . a rapidly dividing pool of stem cell spermatogonia is located at the outer edge of these tubules . some spermatogonia stop dividing and differentiate into primary spermatocytes , which enter the first meiotic prophase . the first meiotic prophase consists of five sequential stages : leptotene ( condensation of interphase chromosomes ) , zygotene ( synapsis begins ) , pachytene ( recombination and crossing over occurs ) , diplotene ( desynapsis initiates ) , and diakinesis ( transition to metaphase i ) . cells then complete division i of meiosis to produce secondary spermatocytes , which then undergo meiotic division ii to produce haploid round spermatids . differentiation ( spermiogenesis ) continues toward the center of the tubule where mature spermatozoa ultimately escape into the lumen . to determine precisely which mouse testis cell types express h2a.lap1 , we performed indirect immunofluorescence analyses using affinity purified h2a.lap1 antibodies on testis sections , displaying all 12 stages of the cell cycle of the seminiferous tubule , as well as on surface spreads of individual spermatocytes and spermatids . we found that h2a.lap1 is not ubiquitously expressed throughout spermatogenesis but is expressed in a temporally specific manner , between pachytene and the round spermatid stage of spermatogenesis . to investigate the genomic location of h2a.lap1 and its link with transcription , we performed h2a.lap1 chip - seq experiments and examined global gene expression using whole mouse expression microarrays in the 30 d testis ( where the vast majority of tubules contained round spermatids ) . the striking observation was that h2a.lap1 was located at the tss of active genes analogous to the position of heterotypic h2a.z / h2a nucleosomes in ts cells ( fig . this allowed us to conclude that h2a.lap1 is a new component of genes expressed during spermatogenesis and moreover , confirmed the view that the tss of an active gene is not nucleosome free . taken together , our results show that h2a.z or a h2a.lap1-containing nucleosome can be precisely positioned at the tss dependent upon their physiological context . each individual line represents a group of 4,700 genes representing the normalized tag counts at each base pair , aligned between -1 and + 1 kb from the tss . ( b ) using published round spermatid x chromosome expression data , four individual h2a.lap1 chip profiles were generated . ( c ) a model depicting the specific targeting of h2a.lap1 to the tss of the small number of genes on the x chromosome that become specifically activated during the latter stages of round spermatid differentiation . we propose that the replacement of h2a with h2a.lap1 destabilizes chromatin at the tss to facilitate the recruitment of the pol ii machinery . our immunofluorescence analysis of round spermatids revealed an unexpected observation ; while h2a.lap1 was largely excluded from heterochromatin regions , as we expected , it was targeted to the inactive x or y chromosome specifically during the latter stages of round spermatid differentiation . in mammals , the x or y chromosome are subjected to meiotic sex chromosome inactivation during pachytene and significantly , this silencing of the sex chromosomes persists postmeiotically throughout spermatogenesis . however , namekawa and colleagues showed that a small number of genes on the x chromosome , required for spermatogenesis , are reactivated in round spermatids ( ~13% ) . based on this knowledge , our hypothesis therefore became that h2a.lap1 was specifically targeted to these x - linked and round spermatid specific genes to enable their activation . to test this hypothesis , we used our h2a.lap1 chip - seq data and combined it with published x chromosome gene expression data in round spermatids . confirming our hypothesis , we found that h2a.lap1 was not present on inactive genes but selectively enriched at the tss of x chromosome genes that were only active in round spermatids ( fig . moreover , maximal transcription was coincident with the highest h2a.lap1 signal at their transcription start site at the latter stages of round spermatid differentiation suggesting that h2a.lap1 is directly involved in the transcriptional activation process . combined with our structural data , this allowed us to propose a model whereby h2a.lap1 coordinately activates transcription of genes on the x chromosome by creating an open chromatin configuration at the tss ( fig . 2c ) . a major problem for the future is to uncover the specific h2a.lap1 chaperone and/or atp - dependent remodelling complex that targets h2a.lap1 to the tss ( see below ) . given our observation that heterotypic h2a.z - h2a nucleosomes exist at an active tss in ts cells ( fig . 1a ) , we wondered whether such heterotypic nucleosomes might also be present on promoters activated during spermatogenesis and if so , whether they are on active promoters during the early stages of spermatogenesis before being replaced by h2a.lap1 at pachytene . alternatively , perhaps both types of histone variant - containing nucleosomes can be present at same time during and following the pachytene stage but on different genes involved in distinct biological processes . both possibilities are not mutually exclusive , however . to answer the first question we performed h2a.z chip h2a rechip assays using 30 d testes and combined this data with our gene expression analysis . all mouse genes were separated into quartiles according to their expression level . a clear positive correlation between the abundance of a heterotypic h2a.z - h2a nucleosome at the tss and the level of expression is clearly observed analogous to active promoters in ts cells ( fig . interestingly , the heterotypic h2a.z - h2a nucleosome is not uniquely positioned at the tss but is present at several locations around the tss including at nucleosome position 2 and + 1 indicating a different chromatin structure for genes active in the mouse testis compared with ts cells . indeed , we previously performed h2a.z chip experiments in the 30 d testis and reported the unexpected observation that while h2a.z was broadly enriched at the 2 nucleosome position , it was depleted at the + 1 position . this suggests that in addition to the tss , the + 1 nucleosome is also unstable being predominantly in the heterotypic form . we conclude that a heterotypic h2a.z - h2a nucleosome is present at the tss in the testis arguing that this special heterotypic nucleosome may be a universal feature of an active mammalian gene . ( a ) four h2a.z chip - h2a rechip profiles were generated for the 30 d testis . each individual line represents a group of 3200 genes and represents the normalized tag counts at each base pair , aligned between 1 and + 1 kb from the tss . the line color reflects the average gene expression rank of the contained genes as shown . ( b ) log2 change in gene expression during selected stages of spermatogenesis ( sta , spermatogonium type a ; stb , spermatogonium type b ; pyt , pachytene ; rs , round spermatid ) for the top 250 genes containing the strongest enrichment signals for either h2a.lap1 or h2a.z - h2a at their tss . blue lines and boxes are enriched for h2a.lap1 ; black lines and boxes are enriched for heterotypic h2a.z - h2a nucleosomes . box plots show the upper and lower quartiles , and the median expression at each stage . we used published gene expression data from namekawa and colleagues as described previously . to address the second possibility that an active promoter can selectively choose between h2a.lap1 or h2a.z , we took the top 250 genes in the 30 d testis enriched with either a h2a.lap1-containing nucleosome or a heterotypic h2a.z - h2a - containing nucleosome at their tss , and examined the relative level of expression of these 250 genes at four stages of spermatogenesis ( spermatogonia stage a , spermatogonia stage b , pachytene , and the round spermatid stage ) normalized to the level of expression at spermatogonia stage a using published gene expression data ( fig . significantly , these top 250 heterotypic h2a.z - h2a or h2a.lap1-nucleosome containing genes were different ( table 1 ) . items marked with ( f ) were major components of functional clusters observed by david . we find that heterotypic nucleosomes are present on genes active at different stages of spermatogenesis with the normalized mean expression for the top 250 heterotypic h2a.z - h2a - containing genes peaking at the pachytene stage ( fig . the normalized mean expression of h2a.lap1-containing genes also increases at pachytene but this increase is more significant compared with heterotypic containing genes . in addition , this level of expression is maintained in the round spermatid stage , which is expected given that h2a.lap1 is expressed between pachytene and the round spermatid stage ( fig . it is worth highlighting that the presence of h2a.lap1 at the tss of genes active in the round spermatid stage is not due to a simple mass action effect because the expression of h2a.z protein actually peaks in round spermatids . given that the genes that contained either h2a.lap1 or h2a.z - h2a were different , we wondered whether their respective genes were involved in different biological processes . to investigate this possibility , we performed gene ontology analysis on these two groups of genes . while h2a.z - h2a heterotypic nucleosomes were associated with genes that display a range of different functions , such as metabolic processes , no single process displayed a significant enrichment of h2a.z - h2a indicating that this histone variant nucleosome has a more ubiquitous distribution not involved in any specific biological process ( table 1 ) . on the other hand , processes involving transcription , rna metabolism , and spermatogenesis were significantly overrepresented among h2a.lap1 enriched genes ( table 1 ) . taken together , these new observations show that during the same stages of spermatogenesis the tss of active genes can selectively contain either a h2a.z - h2a heterotypic or a h2a.lap1-containing nucleosome . this argues that specific mechanisms exist that can determine whether h2a.z or h2a.lap1 is targeted to the tss . these mechanisms most likely involve histone variant specific chaperones and/or atp - dependent remodelling complexes and promoter specific transcription factors . experiments are in progress to identify the nature of such putative complexes specific for h2a.lap1 . concerning h2a.z , our hypothesis is that atp - dependent complexes that load h2a.z into chromatin ( e.g. , p400 or srcap ) will exchange only a single copy of h2a with h2a.z when a nucleosome is positioned at the tss ( perhaps regulated by h3.3 incorporation and/or histone acetylation ) . alternatively , or in addition , atp - dependent complexes that are capably of removing h2a.z ( e.g. , ino80 ) may function to exchange h2a.z with h2a at the tss to create an unstable heterotypic nucleosome . the reason why the nucleosome composition at the tss is regulated in this manner is unclear but the specific targeting of h2a.lap1 may ensure genes are activated in the correct temporal and spatial manner and/or might facilitate higher levels of transcription compared with a h2a.z - h2a heterotypic nucleosome . indeed , the highest level of overall transcription does occur between pachytene and the round spermatid stage , which is when h2a.lap1 is expressed . moreover , it is particularly intriguing that h2a.lap1 is associated with genes that are , themselves , required for gene expression . in conclusion , we believe that this selectivity of histone variants at the tss adds another layer of complexity to the process of transcriptional regulation .	considerable attention has been given to the understanding of how nucleosomes are altered or removed from the transcription start site of rna polymerase ii genes to enable transcription to proceed . this has led to the view that for transcriptional activation to occur , the transcription start site ( tss ) must become depleted of nucleosomes . however , we have shown that this is not the case with different unstable histone h2a variant - containing nucleosomes occupying the tss under different physiological settings . for example , during mouse spermatogenesis we found that the mouse homolog of human h2a.bbd , h2a.lap1 , is targeted to the tss of active genes expressed during specific stages of spermatogenesis . on the other hand , we observed in trophoblast stem cells , a h2a.z - containing nucleosome occupying the tss of genes active in the g1 phase of the cell cycle . notably , this h2a.z - containing nucleosome was different compared with other promoter specific h2a.z nucleosomes by being heterotypic rather than being homotypic . in other words , it did not contain the expected two copies of h2a.z per nucleosome but only one ( i.e. , h2a.z / h2a rather than h2a.z / h2a.z ) . given these observations , we wondered whether the histone variant composition of a nucleosome at an active tss could in fact vary in the same cell type . to investigate this possibility , we performed h2a.z chip - h2a rechip assays in the mouse testis and compared this data with our testis h2a.lap1 chip - seq data . indeed , we find that different promoters involved in the expression of genes involved in distinct biological processes can contain either h2a.z / h2a or h2a.lap1 . this argues that specific mechanisms exist , which can determine whether h2a.z or h2a.lap1 is targeted to the tss of an active gene .
You are an expert at summarizing long articles. Proceed to summarize the following text: in forensic practice , it is difficult to distinguish between accidental and intentionally inflicted head trauma in an injured child . the key question is often whether the head injury was the consequence of a fall or a blow from caretakers suspected of child abuse . computer - based finite element modeling of the head can be used to simulate the events associated with traumatic head injury and quantify biomechanical responses within the brain , but the utility of these models depends upon the accuracy of cranial material property inputs . determining the material properties of pediatric bones and sutures , such as elastic modulus , ultimate stress , and ultimate strain , and elucidating the factors that affect them will not only provide insight into the reaction of the pediatric cranial bones and sutures to loading , but will improve the accuracy of computational and experimental models simulating falls or impacts to a pediatric head [ 47 ] . the dynamic material properties of human adult or animal skull tissues are well documented [ 816 ] , but little information is available about the dynamic properties of human infant cranial bones and sutures . testing speed , strain rate , and cranial sampling position reportedly have significant effects on some or all of the computed mechanical parameters of adult cranial bones . a modest correlation was also found between percent bone volume and the elastic modulus and the maximum bending stress . furthermore , the frontal bone ( fb ) was thicker and less porous and had a higher percent bone volume than the parietal bone ( pb ) ; thus , it fractured under higher forces and absorbed more energy prior to fracturing . coats and margulies were the first to investigate pediatric bone and suture material properties at high rates ( 1.22.8 m / sec ) related to fetal head molding in three - point bending and tension . in addition , pediatric cranial suture deforms 30 times more before failure than pediatric cranial bone . the pb was found to be significantly stiffer and have a higher ultimate stress than occipital bone . mclaughlin et al . reported no significant difference in ultimate stress at failure between the coronal and sagittal sutures of 1-week - old wistar rats . the elastic modulus of coronal and sagittal sutures was 13 mpa and 14 mpa , respectively , while the estimated ultimate strain values were 160% and 120% for the sagittal and coronal sutures , respectively . however , no data currently exist in the literature regarding whether there is a difference in the material properties between coronal and sagittal sutures of human infants . few studies have reported differences in bending properties the purpose of this study was to examine the differences in the material properties of human infants ( 12 years old ) in different bone and suture sampling positions . these results could be used in future computational models simulating the infant head during accidental and intentionally inflicted impact . the cadaver specimens were obtained from deceased infants donated by their families to the southern medical university in china . human infant cranial bones and sutures were obtained from seven fresh - frozen human infant cadavers ( females , three ; males , four ; 1.50.5 years old ) . all materials were thoroughly examined to ensure a lack of skull fractures and malformations and refrigerated at 20c until the preparation and testing , thus minimizing the number of freeze - thaw cycles to prevent bone damage . we then thawed out the subjects in saline no more than 6 hours before the experiment . eight specimens ( 6 cm long , 1 cm wide ) were obtained from each subject : two fb , two pb , two parietal parietal suture ( sagittal sutures ) samples , and two parietal frontal suture ( coronal sutures ) samples . the latter two contained suture and bone . we used a vertical band saw and gently filed the cut faces to ensure accurate dimensions . the positions and directions of the samples from the eight skulls were kept as uniform as possible to allow for realistic future comparisons ( figure 1 ) . the width and thickness of each test specimen were measured adjacent to the cross - section where the fracture occurred . the samples ( n=56 ; eight each from seven infant cadavers ) were subjected to a three - point bending test using a bose material testing machine ( elf-3510at ; bose , inc . , chicago , usa ) at 1.5 mm / s after micro - computed tomography measurements . each specimen was thawed at room temperature in saline no more than 4 h prior to testing . prior to testing , each specimen was instrumented with support structures ( figure 2 ) made from a rigid two - part epoxy resin to prevent erroneous slippage during testing and provide a flat surface on which to rest the specimens . this reduced the effective span length for the tests to 4 cm ( the distance between the two epoxy supports ) but still allowed specimen rotation and bending . all data were collected using the computer data acquisition system consisting of the bose material testing machine and a computer . during the mechanical testing , the euler - bernoulli beam theory is applicable only in cases in which the ratio of span length to thickness is at least 8 . in our study , the average span length to thickness ratios of the tested specimens was > 8 . therefore , the euler - bernoulli equation could be used to calculate the elastic modulus ( e ) : where f/ is the force - displacement ratio in the linear elastic region of a three - point bending trace ( figure 3 ) , l is the span of the beam , and i is the moment of inertia of the rectangular cross - section of the beam . the flexural strain ( f ) from three - point bending was calculated from the relationship : where is the maximum deflection in the center of the beam , t is the thickness of the sample , and l is the span . ultimate strain ( ult ) was selected as the flexural strain corresponding to the ultimate stress . in - plane stress ( xx ) was calculated using timoshenko s corrected version of the beam theory equation , which accounts for radial tensile forces within the beam as the result of an applied concentrated load to the center of the beam : ( 3)xx=3fl8wc3y-0.133fwc where f is the measured force , w is the width of the specimen , l is the span , c is half of the thickness , and y is the location of interest along the y - axis ( outer surfaces at y=c ) in the center of the beam . the ultimate stress ( ult ) the statistical significance of the differences between groups was determined by one - way analysis of variance followed by student - newman - keuls post hoc multiple comparison tests ( two - means comparison ) . correlations were measured using the pearson coefficient and two - tailed p values . the significance level for all analyses was set as p<0.05 and all statistical analyses were performed using spss 21.0 ( spss , inc . , human infant cranial bones and sutures were obtained from seven fresh - frozen human infant cadavers ( females , three ; males , four ; 1.50.5 years old ) . all materials were thoroughly examined to ensure a lack of skull fractures and malformations and refrigerated at 20c until the preparation and testing , thus minimizing the number of freeze - thaw cycles to prevent bone damage . we then thawed out the subjects in saline no more than 6 hours before the experiment . eight specimens ( 6 cm long , 1 cm wide ) were obtained from each subject : two fb , two pb , two parietal parietal suture ( sagittal sutures ) samples , and two parietal frontal suture ( coronal sutures ) samples . the latter two contained suture and bone . we used a vertical band saw and gently filed the cut faces to ensure accurate dimensions . the positions and directions of the samples from the eight skulls were kept as uniform as possible to allow for realistic future comparisons ( figure 1 ) . the width and thickness of each test specimen were measured adjacent to the cross - section where the fracture occurred . the samples ( n=56 ; eight each from seven infant cadavers ) were subjected to a three - point bending test using a bose material testing machine ( elf-3510at ; bose , inc . , chicago , usa ) at 1.5 mm / s after micro - computed tomography measurements . each specimen was thawed at room temperature in saline no more than 4 h prior to testing . prior to testing , each specimen was instrumented with support structures ( figure 2 ) made from a rigid two - part epoxy resin to prevent erroneous slippage during testing and provide a flat surface on which to rest the specimens . this reduced the effective span length for the tests to 4 cm ( the distance between the two epoxy supports ) but still allowed specimen rotation and bending . all data were collected using the computer data acquisition system consisting of the bose material testing machine and a computer . during the mechanical testing , the force - displacement curves of the 56 tested specimens were recorded . the euler - bernoulli beam theory is applicable only in cases in which the ratio of span length to thickness is at least 8 . in our study , the average span length to thickness ratios of the tested specimens was > 8 . therefore , the euler - bernoulli equation could be used to calculate the elastic modulus ( e ) : where f/ is the force - displacement ratio in the linear elastic region of a three - point bending trace ( figure 3 ) , l is the span of the beam , and i is the moment of inertia of the rectangular cross - section of the beam . the flexural strain ( f ) from three - point bending was calculated from the relationship : where is the maximum deflection in the center of the beam , t is the thickness of the sample , and l is the span . ultimate strain ( ult ) was selected as the flexural strain corresponding to the ultimate stress . in - plane stress ( xx ) was calculated using timoshenko s corrected version of the beam theory equation , which accounts for radial tensile forces within the beam as the result of an applied concentrated load to the center of the beam : ( 3)xx=3fl8wc3y-0.133fwc where f is the measured force , w is the width of the specimen , l is the span , c is half of the thickness , and y is the location of interest along the y - axis ( outer surfaces at y=c ) in the center of the beam . the ultimate stress ( ult ) the statistical significance of the differences between groups was determined by one - way analysis of variance followed by student - newman - keuls post hoc multiple comparison tests ( two - means comparison ) . correlations were measured using the pearson coefficient and two - tailed p values . the significance level for all analyses was set as p<0.05 and all statistical analyses were performed using spss 21.0 ( spss , inc . , according to the calculated mechanical properties of pb and fb ( figure 3 ) , both ultimate stress and elastic modulus in fb ( 99.7511.08 mpa and 1265.65120.90 mpa , respectively ) were higher than those in pb ( 87.12 10.58 mpa , p<0.05 and 1103.01112.77 mpa , p<0.01 , respectively ) . no significant difference was found in ultimate strain between pb and fb ( p>0.05 for both ) . the order of ultimate stress and elastic modulus from high to low in different positions of cranial bone was : fb ( 99.7511.08 mpa and 1265.65120.90 mpa , respectively ) > pb ( 87.1210.58 mpa and 1103.01112.77 mpa , respectively ) > coronal suture ( 52.448.71 mpa and 354.8344.86 mpa , respectively ) > sagittal suture ( 44.7510.13 mpa and 408.1259.08 mpa , respectively ) . the order of ultimate strain from high to low was : coronal suture ( 0.20850.0529 mm / mm ) > sagittal suture ( 0.18760.0348 mm / mm ) > fb ( 0.09720.0424 mm / mm ) > pb ( 0.08660.0270 mm / mm ) . significant differences were found in ultimate stress , elastic modulus , and ultimate strain between cranial bones ( pb , fb ) and cranial sutures ( sagittal suture , coronal suture , p<0.01 for elastic modulus between both fb , pb and sagittal suture , coronal suture ; p<0.05 for all others ) . no significant difference was found in ultimate stress , elastic modulus , or ultimate strain between sagittal and coronal sutures ( p>0.05 for all ) . a few human infant finite element models have been constructed to perform a series of virtual experiments [ 6,2429 ] . however , there has been no fe model for infants aged 12 years until now . an important reason for this is that no mechanical properties are documented for 12-year - old infants . the aim of this paper is to study the mechanical properties of the cranial bones and sutures of 12-year - old infants to better understand how the pediatric calvarium differs from that of adults under loading stress . although the mechanical properties of adult cranial bone including ultimate stress and elastic modulus did not differ , it was reported that pediatric cranial bones < 13 months old were significantly stiffer and have a higher ultimate stress than occipital bone . however , no studies have reported the differences in material properties between human infant pb and fb , while few studies have reported the material properties of human pediatric cranial sutures , especially in 12-year - old infants . in our study , the ultimate stress and elastic modulus values of the fb were higher than those of the pb , which was consistent with the findings of coats and margulies in which both ultimate stress and elastic modulus differed between the pb and occipital bone . this result might be related to the fact that the average fb thickness was greater than that of the pb . in addition , the ultimate stress of the pb was higher than those of the sagittal and coronal sutures . the elastic modulus of the pb was also higher than those of the sagittal and coronal sutures , while the ultimate strain of the pb was lower than those of the sagittal and coronal sutures . sutures are more energy absorbent than cranial bone ; thus , they are able to reduce the amount of stress experienced by the surrounding bones . this effect is partially due to the much lower young s modulus of the suture than of the bone , which allows the suture to act as a cushion within the skull . whether there are differences in the material properties of the human pediatric cranial suture remains unknown . these data are also important in the development of an accurate pediatric head injury computational model . as was reported that pediatric pb and occipital bones had different material properties such as ultimate stress and elastic modulus , we wondered whether the material properties would differ from coronal and sagittal sutures . to our surprise , we found no significant difference between coronal and sagittal sutures in ultimate stress , elastic modulus , or ultimate strain . mclaughlin et al . demonstrated that sagittal and coronal sutures in 7-day - old rats had elastic moduli of 13 and 14 mpa , respectively . the difference was not significant , however , which was in very good agreement with the results of the present study . besides , their result was obtained in rats , which differ from humans . since location plays an important role in ultimate stress and elastic modulus , skulls should not be considered homogeneous in the creation of computational models to investigate impact events in pediatric head trauma . however , our study has some limitations . first , the data were obtained from only seven subjects , which is a small sample . second , the assumptions that were made about the geometry of each bending specimen were not large enough to be significant . the analysis method used in this paper assumed that each specimen behaved like a linear beam of a uniform cross - section . although it was observed that the bone samples from different individuals were curved in the same direction , the initial radius of curvature was eight times greater than the thickness of each specimen . despite these limitations , our results provide a valuable reference for relevant research . to the best of our knowledge , the current mechanical data in the literature do not include studies on the differentiating mechanical response of 12-year - old pediatric cranial sagittal and coronal sutures on the mechanical properties of pediatric cranial bones and sutures . the results of our study might extend the scope of our knowledge and aid further studies of the mechanical properties of 12-year - old infant cranial bones and sutures . this study examined the effects of cranial sampling position and bone microstructural parameters on the calculated mechanical properties of pediatric cranial bones and sutures . first , ultimate stress and elastic modulus values of the fb were higher than those of the pb in 12-year - old infants . second , no differences were found between coronal and sagittal sutures in terms of ultimate stress , elastic modulus , or ultimate strain . third , the ultimate stress and elastic modulus values of the fb and pb were higher than those of the sagittal and coronal sutures , while the ultimate strain findings were opposite .	backgroundthe mechanical properties of 12-year - old pediatric cranial bones and sutures and their influential factors were studied to better understand how the pediatric calvarium reacts to loading.material/methodscranial bone and suture specimens were extracted from seven fresh - frozen human infant cadavers ( 1.50.5 years old ) . eight specimens were obtained from each subject : two frontal bones , two parietal bones , two sagittal suture samples , and two coronal suture samples . the specimens were tested in a three - point bend setup at 1.5 mm / s . the mechanical properties , such as ultimate stress , elastic modulus , and ultimate strain , were calculated for each specimen.resultsthe ultimate stress and elastic modulus of the frontal bone were higher than those of the parietal bone ( p<0.05 ) . no differences were found between the coronal and sagittal sutures in ultimate stress , elastic modulus , or ultimate strain ( p>0.05 ) . the ultimate stress and elastic modulus of the frontal and parietal bones were higher than those of the sagittal and coronal sutures ( p<0.05 ) , whereas the opposite ultimate strain findings were revealed ( p<0.05).conclusionsthere was no significant difference in ultimate stress , elastic modulus , or ultimate strain between the sagittal and coronal sutures . however , there were significant differences in ultimate stress , elastic modulus , and ultimate strain between the frontal and parietal bones as well as between the cranial bones and sutures .
You are an expert at summarizing long articles. Proceed to summarize the following text: early postoperative gastrointestinal motility dysfunction is a frequent complication of laparotomy , especially after gastrointestinal operation . it may cause small bowel obstruction , serious infection , hepatic dysfunction , or reoperation with prolonged hospitalization and increased perioperative expenses . in patients with treatment , continuous parenteral nutrition or enteral feeding is necessary , but the outcomes of such approaches are variable . during parenteral nutrition or enteral nutrition , numerous hormones secreted by the gut influence gastrointestinal motor , and appear to contribute to the pathogenesis of gastrointestinal motility dysfunction . it is well established that manipulation of the bowel induces the secretion of proinflammatory cytokines , including interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , and tissue necrosis factor- ( tnf- ) , and then to impaired muscle function . and cyclooxygenase-2 ( cox-2 ) is the key enzyme of prostaglandin synthesis in inflamed tissues . it can affect the motor activity of gastrointestinal tract in postoperative ileus , and cox-2 inhibitors could help to improve the outcome of this gut disorder [ 3 , 4 ] . supplementation of enteral nutritional formulas and parenteral nutrition lipid emulsions with -3 fatty acid could help reduce inflammation [ 5 , 6 ] , but the relationship of -3 fatty acid and gastrointestinal motility still remains unknown . our research was designed to study whether -3 fatty acid could stimulate gastrointestinal motility after abdominal operation and its mechanisms of action . 54 wistar rats with the average body weight 250 20 g were randomly divided into normal saline group , intralipid group , and -3 fatty acid group ( n = 18/group ) . each group was divided into pod 1 group , pod 3 group , and pod 6 group ( n = 6/group ) according to the time of sacrifice . abdominal operation . wistar rats were anaesthetized with 1% pentobarbital ( 40 mg / k ) . then , the rats were perfused normal saline ( 12.5 ml / d ) , intralipid ( 12.5 ml / d , 5 g / kgd ) , or -3 fatty acid ( 12.5 ml / d , 5 g / kgd ) through gastric feeding tube after operation . on pod 1 , pod 3 , and pod 6 , the rat gastric emptying rate , small bowel propulsion rate , serum gastrin ( gas ) , il-1 , il-6 , tnf- , cox-2 were measured with elisa methods ( uscnlife science & technoilgy company , usa . ) . mtl ria kit ( china institute of atomic energy , china ) and multigamma 1261 instrument ( finland ) were used . phenolsulfonphthalein paste ( 0.1 mg / ml phenolsulfonphthalein , 10% gelatin ) was injected into stomach through stomach - tube 25 min before rats were sacrificed . gastric emptying rate , small bowel propulsion rate ( see tables 1 and 2 ) . serum gas and mtl levels ( see tables 3 and 4 ) . serum il-1 , il-6 , tnf- levels ( see tables 5 , 6 , 7 and 8) . on pod 3 , gastric emptying rate , small bowel propulsion rate in -3 fatty acid group were higher than those in normal saline group and intralipid group . serum il-1 , il-6 , tnf- , cox-2 levels in -3 fatty acid group were lower than those in normal saline group and intralipid group . serum gas and mtl levels in -3 fatty acid group were higher than those in normal saline group . -3 fatty acid include eicosapentaenoic acid ( epa ) , ducosahexaenoic acid ( dha ) , and alpha - linolenic acid ( ala ) , which play an important role in the regulation of inflammation . gas is secreted from neuroendocrine g cells which are principally located in the antrum of the stomach . our research showed that on pod 3 , the serum gas and mtl levels in -3 fatty acid group were higher than those in normal saline group , but there was no difference between -3 fatty acid group and intralipid group . this indicates the mechanism that -3 fatty acid promotes the recovery of gastrointestinal motility does not lie in the increase of serum gas and mtl levels . both -3 fatty acid and intralipid belong to fatty acids ; their effects to stimulating gastrointestinal hormones ' secretion are similar , but more powerful than normal saline . ( 2 ) -3 fatty acid and intralipid can inhibit the secretion of gastric acid , reduce ph value in stomach , then stimulate the secretion of gas . ( 3 ) -3 fatty acid and intralipid can also stimulate the secretion of mtl . as we know , intestinal handling induces the secretion of il-1 , il-6 and tnf- , triggers mast cell activation and inflammation associated with prolonged postoperative ileus . cox-2 catalyzes the first step of conversion of its common substrate , arachidonic acid ( aa ) , into a number of biologically active derivatives , designated as prostaglandins ( pgs , including pg - e2 , pg - d2 , pgf-2 , and pg - i2 ) and thromboxane ( tx ) a2 , which are major participants in rodent postoperative ileus . in our research , on pod 3 , the serum il-1 , il-6 , tnf- , cox-2 levels in -3 fatty acid group were lower than those in normal saline group and intralipid group , p < ( 1 ) after consumption , -3 fatty acid can be incorporated into cell membranes and can reduce the amount of aa available for the synthesis of proinflammatory eicosanoids ( e.g. , pgs , leukotrienes ( lts ) ) . likewise , -3 fatty acid can also reduce the production of inflammatory cytokines , such as il-1 , il-6 , and tnf- . ( 2 ) with regard to inflammatory processes , the main fatty acids of interest are the aa , which is the precursor of inflammatory eicosanoids like pg - e2 and lt - b4 , but -3 fatty acid gives rise to mediators ( pg - e3 , lt - b5 ) that are less inflammatory than those produced from aa . ( 3 ) in addition to modifying the lipid mediator profile , -3 fatty acid exerts effects on other aspects of inflammation like leukocyte chemotaxis and inflammatory cytokine production . some of these effects are likely due to changes in gene expression , as a result of altered transcription factor activity . ( 4 ) cox-2 is the key enzyme of prostaglandin synthesis and -3 fatty acid can reduce the expression of cox-2 mrna , in this respect , -3 fatty acid could inhibit inflammation [ 11 , 12 ] . ( 5 ) recently , resolvins and protectins , novel -3 fatty - acids - derived mediators were identified . these mediators can signal for potent counter - regulatory effects on leukocyte functions , including preventing uncontrolled neutrophil swarming , decreasing the generation of cytokines , chemokines , and reactive oxygen species and promoting clearance of apoptotic neutrophils from inflamed tissues . in this way , -3 fatty acids could reduce gastrointestinal inflammation . because postoperative inflammation inhibits gastrointestinal mobility significantly , our study showed that enteral nutrition with -3 fatty acid compared to intralipid could accelerate the recovery of gastrointestinal mobility after caecectomy in rats . the mechanism of action is that -3 fatty acid could decrease serum inflammatory facts levels and relieve postoperative inflammation . -3 fatty acid could accelerate the recovery of gastrointestinal mobility after abdominal operation in rats , mainly by decreasing serum levels of il-1 , il-6 , tnf- , and cox-2 and by relieving postoperative inflammation .	objective . to investigate whether -3 fatty acid could stimulate gastrointestinal motility after abdominal operation . method . wistar rats were randomly divided into 3 group ( normal saline group , intralipid group , and -3 fatty acid group , n = 18/group ) after partial caecectomy and gastrostomosis , each group was divided into 3 groups ( pod1 , pod3 , and pod6 , n = 6/group ) . serum gastrin ( gas ) , motilin ( mtl ) , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , tissue necrosis factor- ( tnf- ) , cyclooxygenase-2 ( cox-2 ) , gastric emptying rate , and small bowel propulsion rate were measured . results . on pod 3 , gastric emptying rate and small bowel propulsion rate in -3 fatty acid group were higher than those in normal saline group and intralipid group . serum gas and mtl levels in -3 fatty acid group were higher than those in normal saline group , but serum il-1 , il-6 , tnf- , and cox-2 levels were lower than those in normal saline group and intralipid group . conclusion . -3 fatty acid could accelerate the recovery of gastrointestinal mobility after abdominal operation in rats , mainly by relieving postoperative inflammation .
You are an expert at summarizing long articles. Proceed to summarize the following text: the implantablecardioverter defibrillator ( icd ) is an established therapy for primary and secondary prevention of sudden cardiac death.1 , 2 the totally subcutaneous implantable defibrillator ( sicd ; boston scientific ) has been introduced as a new alternative to the conventional transvenous defibrillator system . the obvious advantages of this system are marketed as a minimization of lead complications and systemic infections . this innovative system can particularly be considered in patients with congenital heart disease,3 other rare entities impeding transvenous lead implantation,4 or electrical heart disease.5 , 6 the early results of the worldwide evaluation of factors impacting clinical outcome and cost effectiveness ( effortless ) registry suggested an appropriate system performance . occurrence of arrhythmic events and inappropriate shocks resembled those reported for conventional transvenous icd systems.7 these results were confirmed in the 2year followup of the same cohort.8 common problems in the sicd patient population requiring surgical revision or changes in device settings are lead migration , inappropriate sensing due to muscular noise , and twave oversensing.9 , 10 , 11 , 12 , 13 ineffective shocks have also been described previously.14 the results of the recently published shockless implant evaluation ( simple ) trial question the value of perioperative defibrillation testing in individuals undergoing icd implantation.15 in this singleblinded , randomized , multicenter noninferiority trial , routine defibrillation testing at the time of implantation did not improve shock efficacy or reduce arrhythmic death.15 the lack of longterm experience with the sicd system and the described cases of ineffective shock deliveries underline the necessity of intraoperative defibrillation testing in sicd recipients . thus , in the present study , the singlecenter experience of 102 consecutive patients undergoing intraoperative defibrillation tests was systematically evaluated . the study conforms to the declaration of helsinki and later amendments and was approved by an institutional review board . patients gave informed consent for sicd implantation and testing . between july 2010 and june 2015 , the left parasternal position of the shock coil with the pulse generator positioned over the sixth rib in the left midaxillary line was recognized as the optimal configuration.16 all patients underwent a standardized intraoperative defibrillation test . before induction of ventricular fibrillation by using a dc burst for 4 seconds , the detection rate was lowered to a minimal value of 170 bpm . the first shock energy was programmed to 65 j , resulting in a safety margin of at least 15 j. in case of an ineffective first shock delivery , the second shock energy was programmed to 80 j in reversed polarity . an ineffective first shock required further tests in either reverse polarity or after repositioning of the subcutaneous lead and/or the pulse generator . data are presented as percentages including 95% ci . left ventricular ejection fraction ( lvef ) and body mass index ( bmi ) are presented as meansd . one hundred of 102 consecutive sicd recipients underwent intraoperative defibrillation testing . in 1 patient , defibrillation test not done because of fibrotic adhesions on a dialysis catheter , while another patient was not tested because of severe comorbidities . two of these 100 patients were not inducible despite numerous attempts with dc bursts of different lengths . in 74 ( 75% ; 95% ci 6683% ) of 98 patients , ventricular fibrillation was effectively terminated by the first programmed internal shock . in 24 ( 25% ; 95% ci 2244% ) of 98 patients , the first internal shock was ineffective and further internal or external shock deliveries were required ( figures 1 and 2a ) . representative example of an ineffective shock delivery during intraoperative defibrillation test : 2 ineffective internal shocks . a , another ineffective internal shock in the same patient after programming of reversed polarity without altering the position of the pulse generator and subsequent external defibrillation . b , example of an effective internal shock delivery during defibrillation testing in the same patient after repositioning of the pulse generator in a more cranial position . in 14 of 24 patients with ineffective first shock of 65 j , further defibrillation with reversed polarity and the same energy was effective ( table 2 ) . therefore , the first shock was programmed with reversed polarity in these patients . in 6 patients with ineffective first shock of 65 j , further defibrillation attempts with reversed polarity and maximum energy were not effective . these patients underwent a fluoroscopic examination in which a potential for improvement of the shock vector was diagnosed . therefore , repositioning of the sensing lead and/or the pulse generator under fluoroscopic control was performed . in 4 of these patients , the can was moved to a more cranial position ( figure 2b ) , while in 1 patient , the can was positioned more caudal . in 1 additional patient , the sensing lead was moved to a right parasternal position . in 4 additional patients with ineffective first shock , further attempts with reversed polarity and increased shock energy ( either 70 or 80 j ) effectively terminated ventricular fibrillation . in 2 of these patients , ventricular fibrillation could not be further induced for a second test . therefore , a decreased safety margin of < 10 was accepted in these 4 patients . one hundred of 102 consecutive sicd recipients underwent intraoperative defibrillation testing . in 1 patient , defibrillation test not done because of fibrotic adhesions on a dialysis catheter , while another patient was not tested because of severe comorbidities . two of these 100 patients were not inducible despite numerous attempts with dc bursts of different lengths . in 74 ( 75% ; 95% ci 6683% ) of 98 patients , ventricular fibrillation was effectively terminated by the first programmed internal shock . in 24 ( 25% ; 95% ci 2244% ) of 98 patients , the first internal shock was ineffective and further internal or external shock deliveries were required ( figures 1 and 2a ) . representative example of an ineffective shock delivery during intraoperative defibrillation test : 2 ineffective internal shocks . a , another ineffective internal shock in the same patient after programming of reversed polarity without altering the position of the pulse generator and subsequent external defibrillation . b , example of an effective internal shock delivery during defibrillation testing in the same patient after repositioning of the pulse generator in a more cranial position . in 14 of 24 patients with ineffective first shock of 65 j , further defibrillation with reversed polarity and the same energy was effective ( table 2 ) . therefore , the first shock was programmed with reversed polarity in these patients . in 6 patients with ineffective first shock of 65 j , further defibrillation attempts with reversed polarity and maximum energy were not effective . these patients underwent a fluoroscopic examination in which a potential for improvement of the shock vector was diagnosed . therefore , repositioning of the sensing lead and/or the pulse generator under fluoroscopic control was performed . in 4 of these patients , the can was moved to a more cranial position ( figure 2b ) , while in 1 patient , the can was positioned more caudal . in 1 additional patient , the sensing lead was moved to a right parasternal position . in 4 additional patients with ineffective first shock , further attempts with reversed polarity and increased shock energy ( either 70 or 80 j ) effectively terminated ventricular fibrillation . in 2 of these patients therefore , a decreased safety margin of < 10 was accepted in these 4 patients . in the present study , in a significant proportion of sicd recipients who underwent routine intraoperative defibrillation testing , the first shock of 65 j did not effectively terminate ventricular fibrillation . in most cases , either programming of a reversed shock polarity or repositioning of the sensing lead solved this problem and resulted in successful defibrillation . however , in a small percentage of these patients , a reduced safety margin of < 10 j had to be accepted . with regard to the recently published results of the simple trial that questions the clinical value of routine defibrillation testing in patients receiving conventional transvenous icd,15 the results of the present study underline that defibrillation tests are currently necessary to evaluate system functionality . routine defibrillation tests in patients with the use of modern conventional icd systems are almost always effective in the first programmed shock configuration.17 in a study that included 1530 icd recipients , only 3.9% did not meet the 10j safety margin criterion . after programming of reversed shocking polarity , lead repositioning , or addition of a subcutaneous array , a 10j safety margin could be achieved in all patients.17 in the simple study , icd implantation without defibrillation testing was not inferior to intraoperative defibrillation testing regarding longterm efficacy of the icd or total mortality . these results were judged as rather robust by the authors and could simplify the implantation procedure of transvenous icd systems in the future . of note , an increased mortality rate compared with other icd studies was described in the simple population.18 , 19 this aspect was attributed to the fact that the patient population in the simple study presented many comorbidities . however , small patient groups that might have a higher risk for ineffective shock deliveries such as patients with hypertrophic cardiomyopathy might not be sufficiently encompassed in this study . the results of the no regular defibrillation testing in cardioverter defibrillator implantation ( nordicicd ) trial , also randomized , further underlined that intraoperative defibrillation testing during routine leftsided icd implantation does not improve defibrillation efficacy.20 , 21 ineffective shock deliveries have been described in the literature . these episodes can also occur in patients who undergo successful intraoperative defibrillation testing.14 the cited cases mostly resulted in subsequent system explantation and implantation of a transvenous icd . in case of defibrillation threshold problems , comparable positions of the subcutaneous lead as described in the present study have been suggested previouslye.22 these attempts to improve the shock vector can only be minimal because complex repositioning manoeuvers are impeded by the rather strict localization of the sicd system . further , other possible interventions to improve shock efficacy that are available in transvenous icd systems , such as implantation of a subcutaneous array , can not be used . the present study is a singlecenter experience of intraoperative defibrillation testing of sicd systems with a small sample size compared with former multicenter trials with transvenous icd systems . of note , the patient population fundamentally differs from patients in former trials because in the present study mostly young patients with channelopathies and patients who underwent extraction of transvenous icd systems as a results of infections received sicd systems , while the majority of icd recipients in previous studies consists of older patients with ischemic or nonischemic heart disease . further , predictors of successful termination of ventricular fibrillation could not be identified because of the limited sample size . routine defibrillation tests in patients with the use of modern conventional icd systems are almost always effective in the first programmed shock configuration.17 in a study that included 1530 icd recipients , only 3.9% did not meet the 10j safety margin criterion . after programming of reversed shocking polarity , lead repositioning , or addition of a subcutaneous array , a 10j safety margin could be achieved in all patients.17 in the simple study , icd implantation without defibrillation testing was not inferior to intraoperative defibrillation testing regarding longterm efficacy of the icd or total mortality . these results were judged as rather robust by the authors and could simplify the implantation procedure of transvenous icd systems in the future . of note , an increased mortality rate compared with other icd studies was described in the simple population.18 , 19 this aspect was attributed to the fact that the patient population in the simple study presented many comorbidities . however , small patient groups that might have a higher risk for ineffective shock deliveries such as patients with hypertrophic cardiomyopathy might not be sufficiently encompassed in this study . the results of the no regular defibrillation testing in cardioverter defibrillator implantation ( nordicicd ) trial , also randomized , further underlined that intraoperative defibrillation testing during routine leftsided icd implantation does not improve defibrillation efficacy.20 , 21 these episodes can also occur in patients who undergo successful intraoperative defibrillation testing.14 the cited cases mostly resulted in subsequent system explantation and implantation of a transvenous icd . in case of defibrillation threshold problems , comparable positions of the subcutaneous lead as described in the present study have been suggested previouslye.22 these attempts to improve the shock vector can only be minimal because complex repositioning manoeuvers are impeded by the rather strict localization of the sicd system . further , other possible interventions to improve shock efficacy that are available in transvenous icd systems , such as implantation of a subcutaneous array , can not be used . the present study is a singlecenter experience of intraoperative defibrillation testing of sicd systems with a small sample size compared with former multicenter trials with transvenous icd systems . of note , the patient population fundamentally differs from patients in former trials because in the present study mostly young patients with channelopathies and patients who underwent extraction of transvenous icd systems as a results of infections received sicd systems , while the majority of icd recipients in previous studies consists of older patients with ischemic or nonischemic heart disease . further , predictors of successful termination of ventricular fibrillation could not be identified because of the limited sample size . the results of the present study underline that in a significant proportion of sicd recipients ineffective shock deliveries may occur during routine intraoperative defibrillation testing . in the majority of these cases , the shock vector could be improved by programming a reversed shock polarity or repositioning of the sensing lead or the pulse generator . these results imply that ineffective shock deliveries are more common in sicd patients than in patients with conventional transvenous icds . in addition , revision of programmed shock polarity of repositioning of the lead or pulse generator is often necessary to ensure adequate system functionality . therefore , the recently published study data most likely can not be directly transferred to sicd recipients and patients who receive these innovative devices should still be tested during the implantation procedure . drs frommeyer , dechering , zumhagen , kbe , eckardt , and reinke received travel grants and lecture honoraria from biotronik , boston scientific , medtronic , sorin group , and st . drs larbig , bettin , and lher received travels grants from biotronik , boston scientific , medtronic , sorin group , and st .	backgroundthe results of the recently published randomized simple trial question the role of routine intraoperative defibrillation testing . however , testing is still recommended during implantation of the entirely subcutaneous implantable cardioverterdefibrillator ( sicd ) system . to address the question of whether defibrillation testing in sicd systems is still necessary , we analyzed the data of a large , standardofcare prospective singlecenter sicd registry.methods and resultsin the present study , 102 consecutive patients received an sicd for primary ( n=50 ) or secondary prevention ( n=52 ) . defibrillation testing was performed in all except 4 patients . in 74 ( 75% ; 95% ci 0.660.83 ) of 98 patients , ventricular fibrillation was effectively terminated by the first programmed internal shock . in 24 ( 25% ; 95% ci 0.220.44 ) of 98 patients , the first internal shock was ineffective and further internal or external shock deliveries were required . in these patients , programming to reversed shock polarity ( n=14 ) or repositioning of the sensing lead ( n=1 ) or the pulse generator ( n=5 ) led to successful defibrillation . in 4 patients , a safety margin of < 10 j was not attained . nevertheless , in these 4 patients , ventricular arrhythmias were effectively terminated with an internal 80j shock.conclusionsalthough it has been shown that defibrillation testing is not necessary in transvenous icd systems , it seems particular important for sicd systems , because in nearly 25% of the cases the primary intraoperative test was not successful . in most cases , a successful defibrillation could be achieved by changing shock polarity or by optimizing the shock vector caused by the pulse generator or lead repositioning .
You are an expert at summarizing long articles. Proceed to summarize the following text: a number of factors - including preparation design , occlusal forces , oral hygiene , and the restoration materials used - influence the success of fixed dental prostheses . nevertheless , the key factor for success is the choice of suitable luting cement and cementing procedure.1 cementing is the final stage for indirect restoration and the most critical step for ensuring the long - term success of the dental restoration.1 dental luting cement is a substance that bonds juxtaposed surfaces together and provides thermal , electrical , and chemical insulation . the main purpose of such bond is to achieve marginal sealing , adaptation , and stable adhesion between the different types of substrate , as well as adequate retention and resistance.1234 radiopacity is one of the main requirements for luting cements , particularly when used for cementing indirect restorations , as radiographs enable dentists to complete their diagnoses.5 in a radiograph , a sufficiently radiopaque material allows proper assessment of its bond with the dental tissues , particularly in the areas of difficult access.2 the radiopacity also assists the detection of secondary caries under the restoration6 and marginal defects and enables the observation of periodontal effects of the cement overhangs . therefore , when the luting agent is not radiopaque enough , it is impossible to detect voids,6 inadequate marginal adaptation , and interfacial gaps.6789101112 for many years in the past , zinc phosphate cement had been the most commonly used luting material.131415 however , despite its good radiopacity - attar et al.16 showed it to be the most radiopaque cement - it has certain undesirable properties , such as solubility in the oral environment , which have prompted the search for an alternative with ideal characteristics . in recent years , the use of resin cements and resin - modified glass ionomer cements has increased significantly . these materials have the great advantage of adhering not only to the tooth structure but also to ceramics , compound resins and metal alloys . they widely used in restorations with little frictional retention , owing to their high bonding strength.21318 however , despite the scientific advances they bring to dentistry , few studies have addressed their radiopacity . the purposes of the present study were to compare the radiopacity of six resin cements used in dentistry with that of human enamel and dentin , verify whether they meet the requirements of the iso 4049/2000 standard and ansi / ada specification no . 27/1993 , and assess whether their radiopacity is influenced by the thickness of the cement employed . their chemical composition and characteristics are shown in table 1 . in order to standardize the specimens , a stainless steel wedge was made to have 3 steps of thicknesses ( 0.5 , 1 and 1.5 mm thick ) , and a light silicon mould was cast from this wedge . the moulds were filled with the cements , covered with microscopy slides to remove excess material and allow the passage of light , and cured with the led curing lamp ( bluephase c8 , ivoclarvivadent ) with a power of 800 mw / cm and wavelength of 380 - 515 nm , as verified by a radiometer . specimens of more than the desired thickness ( 0.01 mm ) were polished to get the correct size and were then cleaned with 70% ethyl alcohol . for each cement , 3 specimens in the form of wedges of the previously mentioned thicknesses were prepared . to prepare of tooth specimens , sections of a third molar were cut with a low - speed diamond disc ( bredent , senden , germany ) to obtain 0.5 , 1 and 1.5 mm ( 0.01 mm ) mesiodistal specimens of enamel and dentin . the final thickness of each tooth specimen was adjusted by sandpapering and checked with a digital calibrator , allowing a critical tolerance level of 0.01 mm . the aluminum step wedge employed was machined from a single block of aluminum ( alcoa inespal , avils , asturias , spain ) with a purity of 99.889% al , 0.0202% si , 0.0687% fe and 0.0001% cu . it had 10 steps or thickness levels , rising in 0.5 mm increments , and its overall dimensions were 10 mm wide by 20 mm long . afterwards , each specimen was x - rayed 5 times at 65 kv and 10 ma for 0.30 seconds at a focal object distance of 30 cm , with the cement specimen , the aluminum step wedge and the three enamel - dentin specimens placed on the sopix 2 digital sensor ( satelec , la ciotat , france ) ( fig . the images were then imported into digora for windows 2.5 software ( soredex , tuusula , finland ) in order to measure the specimens ' radiopacity in pixels , using the density measurement tool . only the regions that were free of air bubbles or other defects were analyzed ( fig . the radiopacity of the enamel and dentin was also measured in 5 different regions of each thickness specimen , with 15 exposures in each region . the density of each of the 10 thicknesses of the aluminum step wedge was measured by 15 random radiographs , each yielding 5 radiopacity values . a density calibration curve was constructed from the mean values for each step of the aluminum wedge , making it possible to convert the mean radiopacity value of each luting cement into millimeters of aluminum . statistical analysis of the data collected in this study was performed with spss software at a 95% confidence level . a t - test for equality of means was used to examine the differences in radiopacity between the different materials , and an anova , with post - hoc tests , was carried out to study the influence of the thickness of the materials on the radiopacities measured in this study . firstly , the independent specimens t - test for equality of means was used to discover any statistically significant differences between the mean radiopacity of each cement and that of the enamel and dentin at the three thicknesses studied . at every thickness level , statistically significant differences ( p < .05 ) between the radiopacities of the enamel and dentin and those of all the cements were encountered . the results also demonstrate that the calibra , block out , ultrabond plus , insure and rely x ultimate cements were significantly more radiopaque than the enamel and dentin at each of the study thicknesses , while the radiopacity of the variolink veneer cement with medium value 0 shade was lower than that of the tooth specimen at all the thicknesses studied . to verify whether the materials comply with current standards , the independent specimens t - test for equality of means was used . the mean radiopacity of all the materials differed significantly from that of aluminum ( p < .001 ) . observing the mean differences column values , it may also be stated that calibra , block out , ultrabond plus , insure and rely x ultimate show a positive difference compared to the value for aluminum - in other words , their mean radiopacity is higher than that of aluminum - while the mean radiopacity of the variolink veneer cement with medium value 0 shade is significantly lower than that of aluminum at all the thicknesses tested . this demonstrates that all the cements except variolink veneer cement with medium value 0 shade comply with the current standards . the present study also examined whether the radiopacity values presented statistically significant differences depending on the thickness of the cement ( table 2 ) . for all the materials studied , the anova and welch statistic findings provide statistical evidence that the radiopacity of the cements depends to a significant degree on its thickness , as the radiopacity results fall into three groups , significantly differentiated by thickness , and the thicker the material , the greater its radiopacity ( fig . in the present study , the radiopacity of six resin luting cements was compared with human enamel and dentin and with the mean values of the aluminum step wedge employed as a standard control material . the radiopacity of dentin and enamel specimens can vary , but pure aluminum provides a constant reference value.69 it should be pointed out that it is difficult to compare the findings of the present study with those of previous researchers who examined older luting cements . advances in materials are constantly being made and the present study examined cements that have only recently come on the market . enamel is a radiopaque structure , as it has a 90% mineral content , whereas dentin is less mineralized ( 75% ) and therefore less radiopaque.19 van dijken19 showed that the radiopacity of dentin is approximately equivalent to that of aluminum of the same thickness , while enamel has approximately twice the radiopacity of aluminium . these findings agree with the values reported by salzedas et al.11 the present study yielded similar results for the three thicknesses examined , as the enamel values were approximately double those of dentin . some authors consider that to allow correct diagnosis , these restoration materials should be at least as radiopaque as the same thickness of dentin , and have highlighted the importance of using sections of dental tissues as a secondary standard.6712 papers by altintas et al . 20 present similarities with the present study as regards encountering similar or higher radiopacity to that of dentin in most of the cements they studied . however , other researchers consider that restoration materials need to be slightly more radiopaque than enamel.912 pedrosa et al.10 state that materials with higher levels of radiopacity than enamel facilitate diagnosis as the contrast differentiates the dental structures , whereas radiopacity values between those of enamel and dentin or lower than in dentin tend to confuse the examiner , so there is a greater possibility of false positive diagnoses of secondary caries . along the same lines , takuma tsuge4 studied 8 resin cements and reported that 5 were more radiopaque than enamel and 3 less radiopaque , as was found with the variolink veneer cement medium value 0 shade in the present study . according to the iso and ansi / ada standards,2122 the minimum acceptable radiopacity for a restoration material is equivalent to that of the same thickness of aluminium . the results of the present study fell within the radiopacity requirements required for cementing agents included in the standards issued by the iso 4049 and ansi / ada,2122 that it to say , the radiopacity of any thickness of the resin cement materials studied is significantly higher than that of aluminum , with the exception of variolink veneer medium value 0 , which is less radiopaque than the standard . according to the bibliography , the radiopacity of resin cement is typically attributed to its inorganic load , with little contribution from the matrix.23720 the physical properties of dental cements can vary considerably as a result of differences in the quantity and quality of their chemical components . including elements with a high atomic weight among the filler particles of a resin - based cement helps to increase the radiopacity of these materials.313 furtos et al.20 verified that the cements with the highest filler content were the most radiopaque ( a direct correlation between filler content and radiopacity ) , and that level of radiopacity was significantly higher than that this of enamel . however , the cement with the lowest percentage load was still significantly more radiopaque than dentin . information on the correct , detailed chemical composition of resin cements is scarce and most manufacturers consider these data confidential . owing to the limited data available on this aspect of the materials studied , it has not proved possible to construct a correlation between filler content , organic matrix and type of radiopacity . at each of the study thicknesses , the calibra , block out , ultrabond plus , insure and rely x ultimate cements are significantly more radiopaque than enamel and dentin , while variolink veneer medium value 0 is significantly less radiopaque than either at all these thicknesses . with the exception of variolink veneer medium value 0 , all the cements studied comply with the iso and ansi / ada standards at all three thicknesses . furthemore , the radiopacity of all these luting cements depends to a significant degree on their thickness : the thicker the material , the greater its radiopacity .	purposethe aim of this study was to compare the radiopacity of 6 modern resin cements with that of human enamel and dentine using the digora digital radiography system , to verify whether they meet the requirements of ansi / ada specification no . 27/1993 and the iso 4049/2000 standard and assess whether their radiopacity is influenced by the thickness of the cement employed.materials and methodsthree 3-thickness samples ( 0.5 , 1 and 1.5 mm ) were fabricated for each material . the individual cement samples were radiographed on the ccd sensor next to the aluminium wedge and the tooth samples . five radiographs were made of each sample and therefore five readings of radiographic density were taken for each thickness of the materials . the radiopacity was measured in pixels using digora 2.6 software . the calibration curve obtained from the mean values of each step of the wedge made it possible to obtain the equivalent in mm of aluminium for each mm of the luting material.resultswith the exception of variolink veneer medium value 0 , all the cements studied were more radiopaque than enamel and dentin ( p<.05 ) and complied with the iso and ansi / ada requirements ( p<.001 ) . the radiopacity of all the cements examined depended on their thickness : the thicker the material , the greater its radiopacity.conclusionall materials except variolink veneer medium value 0 yielded radiopacity values that complied with the recommendations of the iso and ansi / ada . variolink veneer medium value 0 showed less radiopacity than enamel and dentin .
You are an expert at summarizing long articles. Proceed to summarize the following text: in korea , dioscoreae rhizoma(dr ) is widely used as a herbal medicine or food product with potential health benefits [ 1 , 2 ] . the major components of dr are known to be saponins and sapogenins , starch , purine derivatives , and mucilage . dr extract was approved by the korean food and drug administration ( kfda ) in 2011 . the kfda claimed that the extract at a dose of 900 mg / day effectively regulated the blood sugar level . in addition , the protective effect of dr against diabetic neuropathy has been demonstrated by induction of nerve growth factor ( ngf ) . diabetic neuropathy is the most common condition associated with peripheral neuropathy , which is the most common form of nerve damage and may be caused by diseases or trauma to the nerves or by the side effects of systemic illness . frequently , the causes and treatments of neuropathy can not be identified , and it is designated as being idiopathic . in this report , a literature search was performed in medline and three korean medical databases ( korean studies information , dbpia - database periodical information academic , and korean pharmacopuncture institute information ) up to april 2013 without language restriction . all studies evaluating either the effects of dr monopreparations ( dioscoreae rhizoma or sanyak ) on peripheral neuropathy or the safety of those dr monopreparations were considered . articles that appeared to be appropriate were then investigated in full by two authors , who then discussed the articles and made the decision to include or exclude them . randomized controlled clinical trials ( rct ) with dr pharmacopuncture was published to evaluate its safety in 2013 . a total of 25 participants were divided into the following three groups by a medical statistics expert according to block randomization : 10 participants for the distilled saline dr group ( ddg ) , 10 participants for the alcohol extracted dr group ( adg ) , and five participants for the control ( normal saline ) group ( nsg ) . each treatment was performed three times a week , for a total of 10 times . the participants fasted for at least 14 hr and underwent a liver function test , a complete blood cell count , and urinalysis before the injection and after completing 10 rounds of pharmacopuncture . after injections , the total protein level in the liver function tests and the hematocrits changed within normal limits . patient 1 complained of severe urticaria and angioedema following indigestion of dr with water as a health supplement . she had been suffering from multiple food allergies , including shellfish and peaches , and from allergic rhinitis . patient 2 had been working as a merchant dealing in several herbal materials including dr . she was allergic to food - stuffs , including chestnuts and potatoes , and was suffering from allergic rhinitis . therefore , long - term use of dr or its use by an allergy - sensitive person should be treated with extra caution . the protective effects of dr extract against diabetic neuropathy neuropathy were shown by induction of nerve growth factor ( ngf ) . ngf is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system . mice ( icr mice , 7-week - old ) were administered samples by po . control group ( n = 3 ) : 0.1 ml vehicle po , dr extract 1 group ( n = 3 ) : 1 mg / kg dr extract po , dr extract 3 group ( n = 3 ) : 3 mg / kg dr extract po , dr extract 10 group ( n = 3 ) : 10 mg / kg dr extract po , dr extract 30 group ( n = 3 ) : 30 mg/ kg dr extract po , dr extract 100 group ( n = 3 ) : 100 mg / kg dr extract po , and dr extract 300 group ( n = 3 ) : 300 mg/ kg dr extract po . sciatic nerves and salivary glands were collected after 16-h administrations of the samples by po . the ngf levels in the mice were investigated by using the enzyme - linked immunosorbent assay ( elisa ) method in each section . dr extract increased the endogenous ngf levels in the salivary gland and the sciatic nerve of a mouse , although not in proportion . type-2 diabetes mice ( db / db mice , 7-week - old ) were administered samples by po . control group ( n = 5 ) : 0.1 ml vehicle po , dr extract 10 group ( n = 5 ) : 10 mg / kg dr extract po , and dr extract 100 group ( n = 5 ) : 100 mg / kg dr extract po . . the sensory nerve conductivity velocity ( sncv ) , the motor nerve conductivity velocity ( mncv ) and thermal hyperalgesia increased in the dr - extract - treated mice compared with the control group . dr extract ( 5 l of 10 mg / ml ) or an equivalent volume of saline ( 0.9% nacl ) was directly applied into the crush sites of the sciatic nerve for comparison . focal application of dr extract at the injury site increased gap-43 and cdc2 protein levels in the distal portion of the injured nerve . dr was demonstrated to be effective for the regeneration of the peripheral sciatic nerve after crush injury in rats by using biochemical and histological analyses . gap-43 is strongly induced at the gene expression level in neurons after axonal injury and is transported into the growth cone involved in the process of regrowth [ 14 , 15 ] . cdc2 is the prototypical cell cycle protein and plays a critical role in the transition from growth phase 2 to the mitotic phase . furthermore , a quantitative analysis of axonal regeneration by counting dii - labeled axons of nerurons undergoing axonal regeneration revealed that dr treatment increased axonal regeneration . the authors of this study conducted a retrospective investigation for a total 70 cases of patients , who were diagnosed as having peripheral facial paralysis by physical examination . for comparative analysis , the patients were divided into 11 patients treated with acupuncture and alcohol extracted dr pharmacopuncture ( adg ) , 25 patients treated with acupuncture and distillation dr pharmacopuncture ( ddg ) and 34 patients treated with acupuncture and non - dr pharmacopuncture ( ndg ) . the changed house - brackmann grades indicated significant improvements in all three groups , and the adg and the ddg showed significant results after two weeks of treatment compared to the ndg . the change in yanagihara s score showed significant improvements in all three groups , and the adg group showed significant results after 10 and 15 days of treatment when compared to the ndg . dr pharmacopuncture did not cause any severe physical responses or subjective symptoms except pain after application of pharmacopuncture . randomized controlled clinical trials ( rct ) with dr pharmacopuncture was published to evaluate its safety in 2013 . a total of 25 participants were divided into the following three groups by a medical statistics expert according to block randomization : 10 participants for the distilled saline dr group ( ddg ) , 10 participants for the alcohol extracted dr group ( adg ) , and five participants for the control ( normal saline ) group ( nsg ) . each treatment was performed three times a week , for a total of 10 times . the participants fasted for at least 14 hr and underwent a liver function test , a complete blood cell count , and urinalysis before the injection and after completing 10 rounds of pharmacopuncture . after injections , the total protein level in the liver function tests and the hematocrits changed within normal limits . patient 1 complained of severe urticaria and angioedema following indigestion of dr with water as a health supplement . she had been suffering from multiple food allergies , including shellfish and peaches , and from allergic rhinitis . patient 2 had been working as a merchant dealing in several herbal materials including dr . she was allergic to food - stuffs , including chestnuts and potatoes , and was suffering from allergic rhinitis . therefore , long - term use of dr or its use by an allergy - sensitive person should be treated with extra caution . randomized controlled clinical trials ( rct ) with dr pharmacopuncture was published to evaluate its safety in 2013 . a total of 25 participants were divided into the following three groups by a medical statistics expert according to block randomization : 10 participants for the distilled saline dr group ( ddg ) , 10 participants for the alcohol extracted dr group ( adg ) , and five participants for the control ( normal saline ) group ( nsg ) . each treatment was performed three times a week , for a total of 10 times . the participants fasted for at least 14 hr and underwent a liver function test , a complete blood cell count , and urinalysis before the injection and after completing 10 rounds of pharmacopuncture . after injections , the total protein level in the liver function tests and the hematocrits changed within normal limits . patient 1 complained of severe urticaria and angioedema following indigestion of dr with water as a health supplement . she had been suffering from multiple food allergies , including shellfish and peaches , and from allergic rhinitis . patient 2 had been working as a merchant dealing in several herbal materials including dr . she was allergic to food - stuffs , including chestnuts and potatoes , and was suffering from allergic rhinitis . therefore , long - term use of dr or its use by an allergy - sensitive person should be treated with extra caution . the protective effects of dr extract against diabetic neuropathy neuropathy were shown by induction of nerve growth factor ( ngf ) . ngf is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system . control group ( n = 3 ) : 0.1 ml vehicle po , dr extract 1 group ( n = 3 ) : 1 mg / kg dr extract po , dr extract 3 group ( n = 3 ) : 3 mg / kg dr extract po , dr extract 10 group ( n = 3 ) : 10 mg / kg dr extract po , dr extract 30 group ( n = 3 ) : 30 mg/ kg dr extract po , dr extract 100 group ( n = 3 ) : 100 mg / kg dr extract po , and dr extract 300 group ( n = 3 ) : 300 mg/ kg dr extract po . sciatic nerves and salivary glands were collected after 16-h administrations of the samples by po . the ngf levels in the mice were investigated by using the enzyme - linked immunosorbent assay ( elisa ) method in each section . dr extract increased the endogenous ngf levels in the salivary gland and the sciatic nerve of a mouse , although not in proportion . type-2 diabetes mice ( db / db mice , 7-week - old ) were administered samples by po . control group ( n = 5 ) : 0.1 ml vehicle po , dr extract 10 group ( n = 5 ) : 10 mg / kg dr extract po , and dr extract 100 group ( n = 5 ) : 100 mg / kg dr extract po . vehicle and samples were administered once a day for 30 days . the sensory nerve conductivity velocity ( sncv ) , the motor nerve conductivity velocity ( mncv ) and thermal hyperalgesia increased in the dr - extract - treated mice compared with the control group . dr extract ( 5 l of 10 mg / ml ) or an equivalent volume of saline ( 0.9% nacl ) was directly applied into the crush sites of the sciatic nerve for comparison . focal application of dr extract at the injury site increased gap-43 and cdc2 protein levels in the distal portion of the injured nerve . dr was demonstrated to be effective for the regeneration of the peripheral sciatic nerve after crush injury in rats by using biochemical and histological analyses . gap-43 is strongly induced at the gene expression level in neurons after axonal injury and is transported into the growth cone involved in the process of regrowth [ 14 , 15 ] . cdc2 is the prototypical cell cycle protein and plays a critical role in the transition from growth phase 2 to the mitotic phase . furthermore , a quantitative analysis of axonal regeneration by counting dii - labeled axons of nerurons undergoing axonal regeneration revealed that dr treatment increased axonal regeneration . the authors of this study conducted a retrospective investigation for a total 70 cases of patients , who were diagnosed as having peripheral facial paralysis by physical examination . for comparative analysis , the patients were divided into 11 patients treated with acupuncture and alcohol extracted dr pharmacopuncture ( adg ) , 25 patients treated with acupuncture and distillation dr pharmacopuncture ( ddg ) and 34 patients treated with acupuncture and non - dr pharmacopuncture ( ndg ) . the changed house - brackmann grades indicated significant improvements in all three groups , and the adg and the ddg showed significant results after two weeks of treatment compared to the ndg . the change in yanagihara s score showed significant improvements in all three groups , and the adg group showed significant results after 10 and 15 days of treatment when compared to the ndg . dr pharmacopuncture did not cause any severe physical responses or subjective symptoms except pain after application of pharmacopuncture . the protective effects of dr extract against diabetic neuropathy neuropathy were shown by induction of nerve growth factor ( ngf ) . ngf is essential for the development and phenotypic maintenance of neurons in the peripheral nervous system . control group ( n = 3 ) : 0.1 ml vehicle po , dr extract 1 group ( n = 3 ) : 1 mg / kg dr extract po , dr extract 3 group ( n = 3 ) : 3 mg / kg dr extract po , dr extract 10 group ( n = 3 ) : 10 mg / kg dr extract po , dr extract 30 group ( n = 3 ) : 30 mg/ kg dr extract po , dr extract 100 group ( n = 3 ) : 100 mg / kg dr extract po , and dr extract 300 group ( n = 3 ) : 300 mg/ kg dr extract po . sciatic nerves and salivary glands were collected after 16-h administrations of the samples by po . the ngf levels in the mice were investigated by using the enzyme - linked immunosorbent assay ( elisa ) method in each section . dr extract increased the endogenous ngf levels in the salivary gland and the sciatic nerve of a mouse , although not in proportion . type-2 diabetes mice ( db / db mice , 7-week - old ) were administered samples by po . control group ( n = 5 ) : 0.1 ml vehicle po , dr extract 10 group ( n = 5 ) : 10 mg / kg dr extract po , and dr extract 100 group ( n = 5 ) : 100 mg / kg dr extract po . vehicle and samples were administered once a day for 30 days . the sensory nerve conductivity velocity ( sncv ) , the motor nerve conductivity velocity ( mncv ) and thermal hyperalgesia increased in the dr - extract - treated mice compared with the control group . dr extract ( 5 l of 10 mg / ml ) or an equivalent volume of saline ( 0.9% nacl ) was directly applied into the crush sites of the sciatic nerve for comparison . focal application of dr extract at the injury site increased gap-43 and cdc2 protein levels in the distal portion of the injured nerve . dr was demonstrated to be effective for the regeneration of the peripheral sciatic nerve after crush injury in rats by using biochemical and histological analyses . gap-43 is strongly induced at the gene expression level in neurons after axonal injury and is transported into the growth cone involved in the process of regrowth [ 14 , 15 ] . cdc2 is the prototypical cell cycle protein and plays a critical role in the transition from growth phase 2 to the mitotic phase . furthermore , a quantitative analysis of axonal regeneration by counting dii - labeled axons of nerurons undergoing axonal regeneration revealed that dr treatment increased axonal regeneration . the authors of this study conducted a retrospective investigation for a total 70 cases of patients , who were diagnosed as having peripheral facial paralysis by physical examination . for comparative analysis , the patients were divided into 11 patients treated with acupuncture and alcohol extracted dr pharmacopuncture ( adg ) , 25 patients treated with acupuncture and distillation dr pharmacopuncture ( ddg ) and 34 patients treated with acupuncture and non - dr pharmacopuncture ( ndg ) . the changed house - brackmann grades indicated significant improvements in all three groups , and the adg and the ddg showed significant results after two weeks of treatment compared to the ndg . the change in yanagihara s score showed significant improvements in all three groups , and the adg group showed significant results after 10 and 15 days of treatment when compared to the ndg . dr pharmacopuncture did not cause any severe physical responses or subjective symptoms except pain after application of pharmacopuncture . dr is a member of the dioscoreaceae or yam family , frequently used to treat diarrhea , cough , spermatorrhea , leucorrhea , urinary frequency , and arthritis . in the theories of traditional korean medicine , the function of dr is delineated as helping to remove dampness and to promote healthy body condition . several studies have shown that dr decreases damage in renal tubules , inflammation in the central vein , and necrosis in the liver through its anti - inflammatory action . the inhibitory effevts of dioscorealide b , a naphthofuranoxepin isolated from dr , on no and tnf - production were also reported . recently in korea , the hypoglycemic effect and the preventive effect of dr extract were examined in a rodent model [ 1 , 2 ] . the protective effect of dr against diabetic neuropathy has been demonstrated . in this study , we demonstrated the effects of dr on the nerve , as well as its safety , by reviewing previous studies . one randomized controlled trial was performed to evaluate the safety of using dr pharmacopuncture on healthy persons . after injections , the total protein level in liver function tests and hematocrits changed within normal limits , and dr pharmacopuncture did not cause any severe physical responses or subjective symptoms in this study . another clinical study with 70 cases of peripheral facial paralysis reported that dr pharmacopuncture did not cause any severe physical responses or subjective symptoms . though dr allergy cases are very rare , two cases with dr allergy to multiple food allergies , as well as allergic rhinitis , were evaluated . therefore long - term use of dr or use by allergy - sensitive persons should be treated with extra caution . protective effects of dr extract against diabetic neuropathy were shown by induction of ngf . in this study , dr extract po increased the endogenous ngf level in the salivary gland and the sciatic nerve of a normal mouse . the sensory nerve conductivity velocity , motor nerve conductivity velocity and thermal hyperalgesia were increased in the dr - extract - treated type-2 db / db mice compared with the control group . another study with focal application of dr extract ( 5 l of 10 mg / ml ) at the injury site of the sciatic nerve showed that dr was effective for the regeneration of the peripheral nerve by using biochemical and histological analyses of gap-43 , cdc2 , dii - labeled neurons and nf-200 . clinical research with 70 cases the changes in the house - brackmann grades and the yanagihara scores in dr - pharmacopuncture - treated group indicated significant improvements compared with normal saline group . there were three studies - dr extract per os ( po ) on diabetic neuropathy in mice , dr extract injection on the peripheral sciatic nerve after crush injury in rats and dr extract injection to patients with peripheral facial paralysis . in these studies , dr treatments were identified to be effective for the treatment of nerve injuries , but studies addressing direct factors related to the nerve still remain insufficient .	objectives : this study aimed to evaluate the evidence available in the literature for the safety and efficacy of dioscoreae rhizoma(dr ) for the treatment of peripheral neuropathy.methods:literature searches were performed in medline and three korean medical databases up to april 2013 . all studies evaluating the effects on peripheral neuropathy or the safety of dr monopreparations were considered.results:three studies - dr extract per os ( po ) on diabetic neuropathy in mice , dr extract injection on the peripheral sciatic nerve after crush injury in rats and dr extract injection to patients with peripheral facial paralysis proved that dr treatments were effective for the treatment of nerve injuries.conclusions:in conclusion , we found the dr has a strong positive potential for the treatment of peripheral neuropathy , but studies addressing direct factors related to the nerve still remain insufficient .
You are an expert at summarizing long articles. Proceed to summarize the following text: adult ( 12 months ) and aged ( 25 months ) c57bl/6 male mice were purchased from charles river and housed in the specific pathogen - free ( spf ) facility at the university of liverpool for at least 2 weeks before use . all experiments were performed in accordance with united kingdom home office guidelines under the united kingdom animals ( scientific procedures ) act 1986 . a gastrocnemius muscle from each mouse was placed immediately in a thiol blocking buffer containing ( 25 mm d(0 ) nem , 50 mm ammonium bicarbonate , ph 8) for redox proteomic analysis . briefly protein extracts for redox analysis were prepared in the presence of thiol blocking buffer containing d(0 ) nem under anaerobic conditions . homogenized protein lysates were cleared by centrifugation at 15,000 g for 10 min at 4 c and protein concentrations were calculated by bradford assay ( biorad ) using bsa as a standard . protein extracts for redox analysis were desalted using zeba spin desalting columns ( thermo ) and protein concentrations were re - calculated as before . 200 g in 160 l of 25 mm ammonium bicarbonate of the desalted protein extract was denatured by addition of 10 l of 1% w / v rapigest ( waters , manchester , uk ) in 25 mm ammonium bicarbonate followed by incubation at 80 c for 10 min . reversibly oxidized cys residues were reduced by the addition of 10 l of 100 mm tcep and incubated at 60 c for 10 min . newly reduced cys residues were subsequently alkylated with 10 l of 200 mm d(5 ) nem and incubated at room temperature for 30 min . trypsin ( sigma , poole , uk ) was reconstituted in 50 mm acetic acid and 2 g added to the samples followed by incubation overnight at 37 c . the digestion was terminated and rapigest removed by acidification ( 3 l of tfa and incubation at 37 c for 45 min ) and centrifugation ( 15,000 g for 15 min ) . the data - dependent label - free analysis was performed using an ultimate 3000 rslc nano system coupled to a qexactive mass spectrometer ( thermo scientific ) . the sample ( 5 l corresponding to 200 ng of protein ) was loaded onto the trapping column ( thermo scientific , pepmap100 , c18 , 75 m20 mm ) , using partial loop injection , for 7 min at a flow rate of 4 l / min with 0.1% ( v / v ) tfa . the sample was resolved on the analytical column ( easy - spray c18 75 m500 mm2 m column ) using a gradient of 97% a ( 0.1% formic acid ) 3% b ( 99.9% acn 0.1% formic acid ) to 60% a 40% b over 120 min at a flow rate of 300 nl / min . data dependent acquisition consisted of a 70,000 resolution full - scan ms scan ( agc set to 106 ions with a maximum fill time of 250 ms ) . the 10 most abundant peaks were selected for ms / ms using a 17,000 resolution scan ( agc set to 5104 ions with a maximum fill time of 250 ms ) with an ion selection window of 3 m / z and a normalized collision energy of 30 . to avoid repeated selection of peptides for ms / ms the program used a 30 s dynamic exclusion window . raw spectra were converted to mascot generated files ( mgf ) using proteome discoverer software ( thermo scientific ) . the resulting mgf files were searched against uniprot mouse database sequence database ( 12/05/2012 , 16376 sequences ) using an in - house mascot server ( matrix science , london , uk ) . search parameters used were : peptide mass tolerances , 10 ppm ; fragment mass tolerance , 0.01 da , 1 + , 2 + and 3 + ions ; missed cleavages , 1 ; instrument type , esi - trap . variable modifications included were : d(0 ) nem , d(5 ) nem , mono- , di- and tri - oxidation of cys residues and oxidation of methionine . label - free relative quantification software peaks7 was used to analyze raw data files against the same mouse protein database used for identifications with mascot . proteins were considered significantly changed between adult and aged samples using a -10logp score of 20 ( equivalent to a p value of 0.01 ) , a fold change 1.5 , using a quality value of 0.8 and fdr set to 1% . supplementary file 1 contains additional information on the search and result parameters used including a volcano plot for peptides , the distribution of feature vector ratio by quality , distribution of feature vector ratio by intensity , retention time shift distribution and m / z shift distribution . a volcano plot of the expression of proteomic data is provided in fig . 1 and a list of proteins identified and quantified is included in supplementary table 1 . an example of a peptide containing a redox sensitive cys residue ( cys385 from aconitase ) is presented in fig . analysis of redox peptides identified with both d(0 ) nem and d(5 ) nem was performed using skyline and the relative quantification of the reversible oxidation state of cys residues ( reduced : reversibly oxidized ) was calculated from the intensity of precursor ions and is included in supplementary table 2 . adult ( 12 months ) and aged ( 25 months ) c57bl/6 male mice were purchased from charles river and housed in the specific pathogen - free ( spf ) facility at the university of liverpool for at least 2 weeks before use . all experiments were performed in accordance with united kingdom home office guidelines under the united kingdom animals ( scientific procedures ) act 1986 . a gastrocnemius muscle from each mouse was placed immediately in a thiol blocking buffer containing ( 25 mm d(0 ) nem , 50 mm ammonium bicarbonate , ph 8) for redox proteomic analysis . briefly protein extracts for redox analysis were prepared in the presence of thiol blocking buffer containing d(0 ) nem under anaerobic conditions . homogenized protein lysates were cleared by centrifugation at 15,000 g for 10 min at 4 c and protein concentrations were calculated by bradford assay ( biorad ) using bsa as a standard . protein extracts for redox analysis were desalted using zeba spin desalting columns ( thermo ) and protein concentrations were re - calculated as before . 200 g in 160 l of 25 mm ammonium bicarbonate of the desalted protein extract was denatured by addition of 10 l of 1% w / v rapigest ( waters , manchester , uk ) in 25 mm ammonium bicarbonate followed by incubation at 80 c for 10 min . reversibly oxidized cys residues were reduced by the addition of 10 l of 100 mm tcep and incubated at 60 c for 10 min . newly reduced cys residues were subsequently alkylated with 10 l of 200 mm d(5 ) nem and incubated at room temperature for 30 min . trypsin ( sigma , poole , uk ) was reconstituted in 50 mm acetic acid and 2 g added to the samples followed by incubation overnight at 37 c . the digestion was terminated and rapigest removed by acidification ( 3 l of tfa and incubation at 37 c for 45 min ) and centrifugation ( 15,000 g for 15 min ) . the data - dependent label - free analysis was performed using an ultimate 3000 rslc nano system coupled to a qexactive mass spectrometer ( thermo scientific ) . the sample ( 5 l corresponding to 200 ng of protein ) was loaded onto the trapping column ( thermo scientific , pepmap100 , c18 , 75 m20 mm ) , using partial loop injection , for 7 min at a flow rate of 4 l / min with 0.1% ( v / v ) tfa . the sample was resolved on the analytical column ( easy - spray c18 75 m500 mm2 m column ) using a gradient of 97% a ( 0.1% formic acid ) 3% b ( 99.9% acn 0.1% formic acid ) to 60% a 40% b over 120 min at a flow rate of 300 nl / min . data dependent acquisition consisted of a 70,000 resolution full - scan ms scan ( agc set to 106 ions with a maximum fill time of 250 ms ) . the 10 most abundant peaks were selected for ms / ms using a 17,000 resolution scan ( agc set to 5104 ions with a maximum fill time of 250 ms ) with an ion selection window of 3 m / z and a normalized collision energy of 30 . to avoid repeated selection of peptides for ms / ms the program used a 30 s dynamic exclusion window . raw spectra were converted to mascot generated files ( mgf ) using proteome discoverer software ( thermo scientific ) . the resulting mgf files were searched against uniprot mouse database sequence database ( 12/05/2012 , 16376 sequences ) using an in - house mascot server ( matrix science , london , uk ) . search parameters used were : peptide mass tolerances , 10 ppm ; fragment mass tolerance , 0.01 da , 1 + , 2 + and 3 + ions ; missed cleavages , 1 ; instrument type , esi - trap . variable modifications included were : d(0 ) nem , d(5 ) nem , mono- , di- and tri - oxidation of cys residues and oxidation of methionine . label - free relative quantification software peaks7 was used to analyze raw data files against the same mouse protein database used for identifications with mascot . proteins were considered significantly changed between adult and aged samples using a -10logp score of 20 ( equivalent to a p value of 0.01 ) , a fold change 1.5 , using a quality value of 0.8 and fdr set to 1% . supplementary file 1 contains additional information on the search and result parameters used including a volcano plot for peptides , the distribution of feature vector ratio by quality , distribution of feature vector ratio by intensity , retention time shift distribution and m / z shift distribution . a volcano plot of the expression of proteomic data is provided in fig . 1 and a list of proteins identified and quantified is included in supplementary table 1 . an example of a peptide containing a redox sensitive cys residue ( cys385 from aconitase ) is presented in fig . 2 . analysis of redox peptides identified with both d(0 ) nem and d(5 ) nem was performed using skyline and the relative quantification of the reversible oxidation state of cys residues ( reduced : reversibly oxidized ) was calculated from the intensity of precursor ions and is included in supplementary table 2 .	the data provides information in support of the research article , differential cysteine labeling and global label - free proteomics reveals an altered metabolic state in skeletal muscle aging , journal of proteome research , 2014 , 13 ( 11 ) , 200821 [ 1 ] . raw data is available from proteomexchange [ 2 ] with identifier pdx001054 . the proteome of gastrocnemius muscle from adult and old mice was analyzed by global label - free proteomics and the relative quantification of specific reduced and reversibly oxidized cysteine ( cys ) residues was performed using skyline [ 3 ] . briefly , reduced cysteine ( cys ) containing peptides was alkylated using n - ethylmalemide ( d0-nem ) . samples were desalted and reversibly oxidized cys residues were reduced using tris(2-carboxyethyl)phosphine ( tcep ) and the newly formed reduced cys residues were labeled with heavy nem ( d5-nem ) . label - free analysis of the global proteome of adult ( n=5 ) and old ( n=4 ) gastrocnemius muscles was performed using peaks7 mass spectrometry data analysis software [ 4 ] . relative quantification of cys containing peptides that were identified as reduced ( d(0 ) nem labeled ) and reversibly oxidized d(5)nem labeled was performed using the intensity of their precursor ions in skyline . results indicate that muscles from old mice show reduced redox flexibility particularly in proteins involved in the generation of precursor metabolites and energy metabolism , indicating a loss in the flexibility of the redox energy response .
You are an expert at summarizing long articles. Proceed to summarize the following text: twelve patients with type 2 diabetes ( 9 males and 3 females ) , managed by diet alone , were studied after they had provided written informed consent . the mean se age was 66.2 1.4 years , bmi 28.9 1.0 kg / m , hba1c 6.6 0.2% ( 48.9 2.5 mmol / mol ) , and duration of known diabetes 4.9 1.1 years . none had significant comorbidities , were smokers , or were taking any medication known to affect gastrointestinal function . the protocol was approved by the human research ethics committee of the royal adelaide hospital and was conducted in accordance with the principles of the declaration of helsinki as revised in 2000 . each subject was studied on four occasions , separated by at least 3 days , in randomized double - blind fashion . on the evening before each study day ( 1900 h ) , each subject consumed a standardized evening meal ( mccain s frozen beef lasagna , 2,170 kj ; mccain foods proprietary , victoria , australia ) . at 2200 h , subjects took a tablet of either 100 mg sitagliptin ( januvia ; merck sharp & dohme ) or matching placebo , and compliance was reinforced with a reminder phone call that evening . subjects then fasted until the following morning , when they attended the laboratory at 0800 h and were seated comfortably for the duration of the study . an intravenous cannula was inserted into an antecubital vein for repeated blood sampling . on each study day , between t = 40 and 38 min , they consumed a 200-ml preload drink containing either 50 g d - xylose or 80 mg sucralose ( a control of equivalent sweetness , which we have shown not to stimulate glp-1 secretion or slow gastric emptying in healthy humans ) , so that the four treatments were sitagliptin plus d - xylose ( sx ) , sitagliptin plus control ( sc ) , placebo plus d - xylose ( px ) , and placebo plus control ( pc ) . forty minutes later ( between t = 0 and 5 min ) , they ate a solid meal consisting of 65 g powdered potato ( deb instant mashed potato ; continental , epping , australia ) and 20 g glucose , reconstituted with 200 ml water and 1 egg yolk containing 100 l c - octanoic acid . breath samples were collected immediately before and every 5 min after meal ingestion in the first hour and every 15 min for a further 3 h for the measurement of gastric emptying . venous blood samples and additional breath samples before the preload drink ( at t = 40 min ) and at t = 20 , 0 , 15 , 30 , 60 , 90 , 120 , and 240 min were taken for the measurements of blood glucose , insulin , intact glp-1 , and breath hydrogen . blood samples for insulin were collected in serum tubes . for the measurement of intact glp-1 , venous blood was collected into ice - chilled edta tubes containing dpp-4 inhibitor ( dpp4 - 010 ; linco research , st . samples were mixed six times by gentle inversion and stored on ice before centrifugation at 3,200 rpm for 15 min at 4c within 15 min of collection . blood glucose concentrations were measured immediately using a glucometer ( medisense precision qid ; abbott laboratories , bedford , ma ) . the accuracy of the method has been validated against the hexokinase technique ( 4 ) . mu / l and the coefficient of variation was 2.1% within assays and 6.6% between assays . plasma intact glp-1 was measured by radioimmunoassay using a commercially available kit ( glp1a-35hk ; millipore , billerica , ma ) , which allows quantification of biologically active forms of glp-1 ( i.e. , 7 - 36 amide and 7 - 37 ) in plasma and other biological media . pmol / l , and intra- and interassay coefficients of variation were 3.4 and 9.1% , respectively . co2 concentrations in breath samples were measured by an isotope ratio mass spectrometer ( abca 2020 ; europa scientific , crewe , u.k . ) with an online gas chromatographic purification system . the half - emptying time ( t50 ) this method has been validated against scintigraphy for the measurement of gastric emptying ( 24 ) . hydrogen concentrations in breath samples were measured using quintron microlyzer sc ( quintron instrument , milwaukee , wi ) and were corrected for co2 levels ( 25 ) . the incremental areas under the curve ( iaucs ) were calculated using the trapezoidal rule ( 26 ) for blood glucose , serum insulin , plasma intact glp-1 , and breath hydrogen and analyzed using one - factor repeated - measures anova . these variables were also assessed by repeated - measures anova , with treatment and time as factors . the amplitude of glycemic excursion ( age ) ( postprandial glycemic peak minus the nadir ) and j index [ j = 0.324 ( mean blood glucose + sd ) ] were calculated as measures of glycemic variability , as previously described ( 27 ) , and these together with gastric emptying ( t50 ) were compared using one - factor anova . post hoc comparisons , adjusted for multiple comparisons by bonferroni - holm correction , were performed if anovas showed significant effects . all analyses were performed with spss statistics ( version 19.0 ; ibm , armonk , ny ) . data are presented as means se ; p < 0.05 was considered statistically significant . blood glucose concentrations were measured immediately using a glucometer ( medisense precision qid ; abbott laboratories , bedford , ma ) . the accuracy of the method has been validated against the hexokinase technique ( 4 ) . mu / l and the coefficient of variation was 2.1% within assays and 6.6% between assays . plasma intact glp-1 was measured by radioimmunoassay using a commercially available kit ( glp1a-35hk ; millipore , billerica , ma ) , which allows quantification of biologically active forms of glp-1 ( i.e. , 7 - 36 amide and 7 - 37 ) in plasma and other biological media . pmol / l , and intra- and interassay coefficients of variation were 3.4 and 9.1% , respectively . co2 concentrations in breath samples were measured by an isotope ratio mass spectrometer ( abca 2020 ; europa scientific , crewe , u.k . ) with an online gas chromatographic purification system . the half - emptying time ( t50 ) this method has been validated against scintigraphy for the measurement of gastric emptying ( 24 ) . hydrogen concentrations in breath samples were measured using quintron microlyzer sc ( quintron instrument , milwaukee , wi ) and were corrected for co2 levels ( 25 ) . the incremental areas under the curve ( iaucs ) were calculated using the trapezoidal rule ( 26 ) for blood glucose , serum insulin , plasma intact glp-1 , and breath hydrogen and analyzed using one - factor repeated - measures anova . these variables were also assessed by repeated - measures anova , with treatment and time as factors . the amplitude of glycemic excursion ( age ) ( postprandial glycemic peak minus the nadir ) and j index [ j = 0.324 ( mean blood glucose + sd ) ] were calculated as measures of glycemic variability , as previously described ( 27 ) , and these together with gastric emptying ( t50 ) were compared using one - factor anova . post hoc comparisons , adjusted for multiple comparisons by bonferroni - holm correction , were performed if anovas showed significant effects . all analyses were performed with spss statistics ( version 19.0 ; ibm , armonk , ny ) . data are presented as means se ; p < 0.05 was considered statistically significant . three subjects reported mild transient loose stools after completion of the study on the d - xylose days . fasting blood glucose concentrations did not differ between the four study days ( pc 7.4 0.3 mmol / l , px 7.5 0.3 mmol / l , sc 7.1 0.3 mmol / l , and sx 7.5 0.4 , blood glucose concentrations increased slightly when the d - xylose preload was given ( i.e. , px and sx ) in contrast to the control days , so that the iauc ( 40 to 0 min ) was greater for px and sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) . after the meal , blood glucose concentrations increased on each day before returning to baseline . the postprandial glycemic peak , age , and j index were all lower after px and sc than pc ( p < 0.01 for each ) and were lowest after sx ( p < 0.05 for each ) ( table 1 ) . there was a significant treatment effect on the overall iauc for blood glucose ( p = 0.008 ) ( table 1 ) , such that blood glucose was lower after sx compared with pc ( p < 0.05 ) ( fig . postprandial glycemic peak , j index , age , iauc for blood glucose , serum insulin , and glp-1 in response to a carbohydrate meal after a preload of either d - xylose or sucralose ( control ) with or without 100 mg sitagliptin ( n = 12 ) effects of d - xylose or sucralose ( control ) with or without sitagliptin on blood glucose ( a ) , serum insulin ( b ) , insulin - to - glucose ratio ( c ) , and plasma intact glp-1 ( d ) in response to a carbohydrate meal ( n = 12 ) . the four treatments were sx , sc , px , and pc . repeated - measures anova was used to determine statistical difference . p = 0.000 for each treatment time interaction ; * p < 0.05 , px vs. pc ; # p < 0.05 , sc vs. pc ; p < 0.05 , sx vs. pc ; p < 0.05 , sx vs. px ; p < 0.05 , sx vs. sc . before the meal , insulin concentrations increased slightly when d - xylose was given ( i.e. , px and sx ) in contrast to the control days , so that the iauc ( 40 to 0 min ) was greater for px and sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) . after the meal , serum insulin concentrations increased on each day , but d - xylose and sitagliptin alone and in combination resulted in a lower postprandial serum insulin than pc ( p = 0.000 for treatment time interaction , with significant differences at t = 30 , 60 , and 90 min for px vs. pc and sx vs. pc ; t = 90 min for sc vs. pc ; and t = 30 min for sx vs. sc [ p < 0.05 for each ] ) . there was also a significant treatment effect on the overall iauc for serum insulin ( p = 0.009 ) ( table 1 ) , such that insulin concentrations were lower after sx than pc ( p < 0.05 ) ( fig . 1b ) . before the meal , the insulin - to - glucose ratio ( fig . 1c ) remained unchanged after both d - xylose and control preloads , but the ratio increased after the meal and was lower from t = 30 to 90 min after the d - xylose preload compared with control ( p = 0.000 for a treatment time interaction : px vs. pc and sx vs. sc [ p < 0.05 for each ] ) without any effect of sitagliptin . fasting plasma intact glp-1 concentrations did not differ between the four study days . before the meal , glp-1 concentrations increased when the d - xylose preload was given , so that the iauc ( 40 to 0 ) was greater for sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) , although the difference between px and pc was not significant . after the meal , intact glp-1 increased on the control days , but the combination of the d - xylose preload with sitagliptin resulted in more sustained elevation of plasma intact glp-1 than on the other days ( p = 0.000 for a treatment time interaction , with significant differences at t = 15 , 60 , 90 , and 120 min for sx vs. pc and during t = 30120 for sx vs. px [ p < 0.05 for each ] ) . there was also a treatment effect on the overall iauc for plasma intact glp-1 ( p = 0.003 ) ( table 1 ) , such that glp-1 was greatest after sx ( sx vs. pc , px , and sc , p < 0.05 for each ) ( fig . fasting breath hydrogen approximated 0 ppm and did not differ between the four study days . after the d - xylose drink , breath hydrogen increased slightly before the meal ( t = 40 to 0 min ) and continued to rise to a plateau afterward , while it remained unchanged after the control preload and was unaffected by sitagliptin ( p = 0.000 for a treatment time interaction ) ( fig . effects of d - xylose or sucralose ( control ) with or without sitagliptin on breath hydrogen production in response to a carbohydrate meal ( n = 12 ) . the four treatments were sx ( ) , sc ( ) , px ( ) , and pc ( ) . repeated - measures anova was used to determine statistical difference . * p = 0.000 , px vs. pc and sc ; # p = 0.000 , sx vs. pc and sc . data are means sem . there was a treatment effect for gastric emptying ( p = 0.000 ) , such that the half - emptying time was greater after d - xylose than control ( t50 for px 238.2 26.4 min and sx 256.9 23.1 min vs. pc 152.3 6.0 min and sc166.3 11.0 min [ p < 0.01 for each ] ) without any effect of sitagliptin ( fig . effects of d - xylose or sucralose ( control ) with or without sitagliptin on gastric emptying ( half - emptying time [ t50 ] ) ( n = 12 ) . p = 0.000 for a treatment effect ; * p < 0.01 , px vs. pc and sc ; # p < 0.001 , sx vs. pc and sc . when data from the four study visits were pooled , the magnitude of the postprandial rise in blood glucose from baseline at t = 30 , 60 , and 90 min was inversely related to the t50 ( p < 0.01 for each ) and at t = 240 min directly related to the t50 ( p = 0.001 ) . given that plasma intact glp-1 would be more sensitive as a measure of glp-1 secretion when dpp-4 was inhibited , data from the sx day alone were examined for a relationship between breath hydrogen production and glp-1 secretion . intact glp-1 concentrations were found to be related directly to breath hydrogen production ( for iauc40 to 0 , r = 0.72 , p = 0.009 ; for iauc40 to 240 , r = 0.74 , p = 0.006 ) . fasting blood glucose concentrations did not differ between the four study days ( pc 7.4 0.3 mmol / l , px 7.5 0.3 mmol / l , sc 7.1 0.3 mmol / l , and sx 7.5 0.4 , blood glucose concentrations increased slightly when the d - xylose preload was given ( i.e. , px and sx ) in contrast to the control days , so that the iauc ( 40 to 0 min ) was greater for px and sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) . after the meal , blood glucose concentrations increased on each day before returning to baseline . the postprandial glycemic peak , age , and j index were all lower after px and sc than pc ( p < 0.01 for each ) and were lowest after sx ( p < 0.05 for each ) ( table 1 ) . there was a significant treatment effect on the overall iauc for blood glucose ( p = 0.008 ) ( table 1 ) , such that blood glucose was lower after sx compared with pc ( p < 0.05 ) ( fig . postprandial glycemic peak , j index , age , iauc for blood glucose , serum insulin , and glp-1 in response to a carbohydrate meal after a preload of either d - xylose or sucralose ( control ) with or without 100 mg sitagliptin ( n = 12 ) effects of d - xylose or sucralose ( control ) with or without sitagliptin on blood glucose ( a ) , serum insulin ( b ) , insulin - to - glucose ratio ( c ) , and plasma intact glp-1 ( d ) in response to a carbohydrate meal ( n = 12 ) . p = 0.000 for each treatment time interaction ; * p < 0.05 , px vs. pc ; # p < 0.05 , sc vs. pc ; p < 0.05 , sx vs. pc ; p < 0.05 , sx vs. px ; p < 0.05 , sx vs. sc . before the meal , insulin concentrations increased slightly when d - xylose was given ( i.e. , px and sx ) in contrast to the control days , so that the iauc ( 40 to 0 min ) was greater for px and sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) . after the meal , serum insulin concentrations increased on each day , but d - xylose and sitagliptin alone and in combination resulted in a lower postprandial serum insulin than pc ( p = 0.000 for treatment time interaction , with significant differences at t = 30 , 60 , and 90 min for px vs. pc and sx vs. pc ; t = 90 min for sc vs. pc ; and t = 30 min for sx vs. sc [ p < 0.05 for each ] ) . there was also a significant treatment effect on the overall iauc for serum insulin ( p = 0.009 ) ( table 1 ) , such that insulin concentrations were lower after sx than pc ( p < 0.05 ) ( fig . 1c ) remained unchanged after both d - xylose and control preloads , but the ratio increased after the meal and was lower from t = 30 to 90 min after the d - xylose preload compared with control ( p = 0.000 for a treatment time interaction : px vs. pc and sx vs. sc [ p < 0.05 for each ] ) without any effect of sitagliptin . before the meal , glp-1 concentrations increased when the d - xylose preload was given , so that the iauc ( 40 to 0 ) was greater for sx than for pc and sc ( p < 0.05 for each ) ( table 1 ) , although the difference between px and pc was not significant . after the meal , intact glp-1 increased on the control days , but the combination of the d - xylose preload with sitagliptin resulted in more sustained elevation of plasma intact glp-1 than on the other days ( p = 0.000 for a treatment time interaction , with significant differences at t = 15 , 60 , 90 , and 120 min for sx vs. pc and during t = 30120 for sx vs. px [ p < 0.05 for each ] ) . there was also a treatment effect on the overall iauc for plasma intact glp-1 ( p = 0.003 ) ( table 1 ) , such that glp-1 was greatest after sx ( sx vs. pc , px , and sc , p < 0.05 for each ) ( fig . fasting breath hydrogen approximated 0 ppm and did not differ between the four study days . after the d - xylose drink , breath hydrogen increased slightly before the meal ( t = 40 to 0 min ) and continued to rise to a plateau afterward , while it remained unchanged after the control preload and was unaffected by sitagliptin ( p = 0.000 for a treatment time interaction ) ( fig . effects of d - xylose or sucralose ( control ) with or without sitagliptin on breath hydrogen production in response to a carbohydrate meal ( n = 12 ) . the four treatments were sx ( ) , sc ( ) , px ( ) , and pc ( ) . repeated - measures anova was used to determine statistical difference . * p = 0.000 , px vs. pc and sc ; # p = 0.000 , sx vs. pc and sc . there was a treatment effect for gastric emptying ( p = 0.000 ) , such that the half - emptying time was greater after d - xylose than control ( t50 for px 238.2 26.4 min and sx 256.9 23.1 min vs. pc 152.3 6.0 min and sc166.3 11.0 min [ p < 0.01 for each ] ) without any effect of sitagliptin ( fig . effects of d - xylose or sucralose ( control ) with or without sitagliptin on gastric emptying ( half - emptying time [ t50 ] ) ( n = 12 ) . p = 0.000 for a treatment effect ; * p < 0.01 , px vs. pc and sc ; # p < 0.001 , sx vs. pc and sc . when data from the four study visits were pooled , the magnitude of the postprandial rise in blood glucose from baseline at t = 30 , 60 , and 90 min was inversely related to the t50 ( p < 0.01 for each ) and at t = 240 min directly related to the t50 ( p = 0.001 ) . given that plasma intact glp-1 would be more sensitive as a measure of glp-1 secretion when dpp-4 was inhibited , data from the sx day alone were examined for a relationship between breath hydrogen production and glp-1 secretion . intact glp-1 concentrations were found to be related directly to breath hydrogen production ( for iauc40 to 0 , r = 0.72 , p = 0.009 ; for iauc40 to 240 , r = 0.74 , p = 0.006 ) . the main observations made in this study of patients with type 2 diabetes managed by diet were that 1 ) consumption of the low - energy pentose d - xylose in advance of a high - carbohydrate meal attenuates the postprandial glycemic excursion in association with stimulation of glp-1 secretion before the meal and slowing of gastric emptying , 2 ) a single dose of the dpp-4 inhibitor sitagliptin increases postprandial intact glp-1 concentrations and reduces postprandial glycemia without slowing gastric emptying or stimulating postprandial insulin secretion , and 3 ) the combination of a d - xylose preload with sitagliptin reduces the postprandial glycemic excursion more than either treatment alone . the magnitude of the reduction in postprandial blood glucose achieved by the combination of d - xylose preload and sitagliptin in our group of patients with well - controlled type 2 diabetes was substantial ( i.e. , reduction in peak blood glucose of 2.5 mmol / l ) , and moreover , there was a marked reduction in indices of glycemic variability ; the latter is associated with oxidative stress and may independently increase cardiovascular risk ( 27 ) . we chose d - xylose as the preload , since it is incompletely absorbed and poorly metabolized ( 16 ) and , accordingly , additional energy intake is minimized in contrast to preloads such as fat ( 12 ) and protein ( 13 ) . consistent with our previous report in healthy older subjects ( 17 ) , ingestion of d - xylose resulted in a modest increase in blood glucose and serum insulin , possibly as a result of enhanced gluconeogenesis . sucralose was selected as a sweet - tasting negative control , since we have shown that this artificial sweetener when given acutely has no effect on either the secretion of glp-1 or gastric emptying ( 21,22 ) , and indeed in the current study , there was no increase in intact glp-1 before the meal on the days when sucralose was given . the osmolarity of the preloads could not readily be matched without changing the taste and this may have contributed to the effect of the d - xylose preload on gastric emptying ( 28 ) but is unlikely to account for differences in glp-1 secretion ( 22 ) . as expected , d - xylose was associated with stimulation of glp-1 in advance of the meal . it has been postulated that intestinal fermentation to form free fatty acids represents an important mechanism for the stimulation of glp-1 release ( 29 ) ; the latter could be indirectly quantified by resultant hydrogen production in breath samples ( 16 ) . our demonstration of a relationship between glp-1 concentrations and hydrogen production is consistent with this concept . however , the fact that glp-1 concentrations began to increase within 20 min of d - xylose ingestion suggests that other mechanisms , involving , for example , the passage of d - xylose through the monosaccharide transporters glut2 and glut3 ( 30 ) , are likely involved . it may appear surprising that plasma intact glp-1 levels after the potato meal did not differ between the d - xylose and control preload study days , when sitagliptin was not given . however , the magnitude of glp-1 secretion is dependent on the rate ( 31 ) and load ( 32 ) of nutrient entry to the small intestine , so that the slowing of gastric emptying induced by the d - xylose preload may well have attenuated the component of the postprandial glp-1 response attributable to the meal . we observed that the magnitude of the initial postprandial glycemic excursion was inversely related to the gastric half - emptying time , consistent with evidence that the rate of gastric emptying is a major determinant of postprandial glycemia ( 4,5 ) . in the absence of sitagliptin , the reduction in postprandial glycemia by d - xylose is probably attributable mainly to the slowing of gastric emptying , since postprandial intact glp-1 concentrations were no greater than on the control day . the observed decrease in postprandial insulin concentrations after d - xylose in contrast to control , particularly when corrected for differences in blood glucose ( i.e. , the insulin - to - glucose ratio ) , is consistent with what would be expected when gastric emptying of the potato meal is slower ( 12 ) . the slowing of gastric emptying after the d - xylose preload was associated with stimulation of glp-1 in advance of the meal , although postprandial intact glp-1 concentrations were not increased in the absence of sitagliptin . since endogenous glp-1 is known to slow gastric emptying ( 9 ) , the elevated glp-1 may have contributed to the slower gastric emptying after d - xylose , at least during the initial phase . nevertheless , the combination with sitagliptin , which increased plasma concentrations of intact glp-1 , had no additional effect on the rate of gastric emptying a finding that is consistent with our recent report of the lack of effect of 2-day dosing with sitagliptin on gastric emptying ( 33 ) . for example , acute hyperglycemia , even within the physiological range , is known to slow gastric emptying ( 34 ) , and sitagliptin , particularly when combined with d - xylose , potently decreased postprandial glycemia . moreover , peptide yy ( pyy ) , which is cosecreted with glp-1 from enteroendocrone l cells , has the capacity to slow gastric emptying . pyy 1 - 36 and 3 - 36 are the predominant biologically active forms in the circulation ; the latter is formed from degradation of pyy 1 - 36 by dpp-4 and is reportedly more potent at retarding emptying ( 35 ) . therefore , dpp-4 inhibition might , to some extent , blunt pyy- mediated slowing of gastric emptying . in contrast , addition of sitagliptin did not affect gastric emptying but was associated with lowering of postprandial glycemia . despite the fact that plasma intact glp-1 concentrations were increased after sitagliptin , particularly when given in combination with d - xylose , the insulin response to the meal did not increase in parallel . this is partly accounted for by the fact that the insulinotropic effect of glp-1 is glucose dependent ( 36 ) , but there is also evidence that mechanisms other than insulin secretion are likely to be important in mediating the glucose - lowering effect of glp-1 ; the latter include suppression of both glucagon secretion and endogenous glucose production ( 37,38 ) and enhancement of peripheral glucose uptake ( 39 ) . it is noteworthy that there has been little consideration of how dietary intake interacts with the actions of dpp-4 inhibitors . the current study is therefore novel in demonstrating how consumption of a specific nutrient can improve the efficacy of a dpp-4 inhibitor for reducing postprandial glycemia . although the number of subjects studied was relatively small , the observed effects were consistent between subjects and were in keeping with previous observations relating to the slowing of gastric emptying by d - xylose ( 17 ) and glucose lowering by sitagliptin ( 20 ) . our study represents an acute intervention in a relatively well - controlled group of patients with type 2 diabetes managed by diet alone . in view of the positive outcomes , it would be of particular interest to investigate this approach in type 2 diabetic patients taking metformin , given the synergistic effect of dpp-4 inhibitors with metformin for increasing intact glp-1 and improving glycemia ( 40 ) , and to extend our observations to larger and more diverse groups of type 2 diabetic patients over a longer duration .	objectivemacronutrient preloads can reduce postprandial glycemia by slowing gastric emptying and stimulating glucagon - like peptide-1 ( glp-1 ) secretion . an ideal preload would entail minimal additional energy intake and might be optimized by concurrent inhibition of dipeptidyl peptidase-4 ( dpp-4 ) . we evaluated the effects of a low - energy d - xylose preload , with or without sitagliptin , on gastric emptying , plasma intact glp-1 concentrations , and postprandial glycemia in type 2 diabetes.research design and methodstwelve type 2 diabetic patients were studied on four occasions each . after 100 mg sitagliptin ( s ) or placebo ( p ) and an overnight fast , patients consumed a preload drink containing either 50 g d - xylose ( x ) or 80 mg sucralose ( control [ c ] ) , followed after 40 min by a mashed potato meal labeled with 13c - octanoate . blood was sampled at intervals . gastric emptying was determined.resultsboth peak blood glucose and the amplitude of glycemic excursion were lower after px and sc than pc ( p < 0.01 for each ) and were lowest after sx ( p < 0.05 for each ) , while overall blood glucose was lower after sx than pc ( p < 0.05 ) . the postprandial insulin - to - glucose ratio was attenuated ( p < 0.05 ) and gastric emptying was slower ( p < 0.01 ) after d - xylose , without any effect of sitagliptin . plasma glp-1 concentrations were higher after d - xylose than control only before the meal ( p < 0.05 ) but were sustained postprandially when combined with sitagliptin ( p < 0.05).conclusionsin type 2 diabetes , acute administration of a d - xylose preload reduces postprandial glycemia and enhances the effect of a dpp-4 inhibitor .
You are an expert at summarizing long articles. Proceed to summarize the following text: fibromyalgia syndrome ( fms ) is a disorder of chronic widespread pain ( cwp ) associated with fatigue , sleep disturbance , depression , anxiety , cognitive dysfunction and muscle and joint stiffness.1 fms affects patients worldwide , with an estimated prevalence in developed countries that ranges from 0.5% to 5.8%.2 fms can exist as a primary disorder or may occur secondarily in association with a variety of chronic medical conditions . while fms is not an inflammatory disorder , it is commonly seen in association with inflammatory autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.3,4 the most widely used criteria for identifying fms patients is the american college of rheumatology ( acr ) 1990 classification criteria.5 however use of the acr criteria for fms diagnosis is discouraged,6 since the acr criteria lack sensitivity and fail to recognize associated fms symptoms that must be addressed to optimally manage the disorder.7 recently proposed fms clinical diagnostic criteria that identify patients based on the number of somatic symptoms and painful body areas along with severity of symptoms of fatigue , sleep disturbance and cognitive dysfunction may eventually improve fms diagnosis.8 pending finalization of diagnostic criteria , we recommend assessment and treatment of all patients with cwp for associated fms symptoms . these symptoms can be recalled using the fibro mnemonic , where f = fatigue and/or fibrofog ( cognitive dysfunction ) , i = insomnia ( nonrestorative sleep ) , b = blues ( depression and/or anxiety ) , r = rigidity ( stiffness in muscles and/or joints ) and o = ow ! ( pain and work difficulty).9 questionnaires such as the mvasfiq9 or the siqr10 can be used to assess fibro symptoms and provide the basis for an individualized , symptom - based treatment approach to fms management consistent with evidence - based guidelines.7 three general points should be kept in mind when treating fms patients . first , primary disorders that can mimic fms must be ruled out before symptomatic therapies are used . these include vitamin deficiencies , anemia , and metabolic , oncologic , inflammatory or sleep disorders . second , since the majority of fms patients suffer from multiple medication intolerances , medications should be started individually at low dose and slowly uptitrated and/or combined . finally , while this review focuses on pharmacologic treatment of fms with milnacipran , medications have a limited role in fms management . medications work best when they are used to provide symptomatic relief so that patients can participate in nonpharmacologic modalities that provide long - term disease coping strategies including aerobic and resistance exercise , education , and cognitive behavioral therapies . while fms was initially considered a disorder of peripheral myofascial tissue , this has been proven incorrect . fms is a neurologic disorder caused by aberrant processing of pain and other sensory information within the central nervous system ( cns).11 the concept of fms as a centrally - mediated disorder has arisen from numerous scientific studies including functional neuroimaging of fms patients showing that brain regions involved in pain processing are hyperactive compared to controls.1214 serotonin and norepinephrine are known to provide important inhibitory signals in regulating pain processing pathways,15 and fms patients have reduced levels of norepinephrine and serotonin in their cerebrospinal fluid.16 augmentation of serotonin and norepinephrine cns activity is thought to be the mechanisms by which norepinephrine serotonin reuptake inhibitor ( nsri ) medications like milnacipran and serotonin norepinephrine reuptake inhibitor ( snri ) medications like duloxetine decrease fms pain . however , fms patients are very heterogeneous and it is unlikely that a single pathogenic mechanism is responsible for causing fms in all patients . no single treatment has been found that is effective for all fms symptoms,17 and individual fms patients often respond best to different therapies . also , the serotonin selective reuptake inhibitors ( ssris ) have shown mixed results in managing fms.18 it is generally accepted that these mixed results are due to the fact that different ssris have widely varying concomitant norepinephrine reuptake inhibition ( nri ) activity , and it is thought to be the nri activity that is responsible for analgesia . norepinephrine has an important role in pain modulation that is supported by research showing that snris have analgesic effects in animal models of central pain that highly selective ssris lack.19 the balanced ratio of serotonin : norepinephrine reuptake inhibition provided by milnacipran is the likely reason for its analgesic properties ( table 1).20,21 milnacipran is one of three food and drug administration ( fda)-approved medications for the management of fms . milnacipran is characterized as an nsri because in vitro studies have shown milnacipran has a three - fold greater efficacy for inhibiting norepinephrine reuptake compared to serotonin , which differentiates it from the snris which are more serotonin active ( table 1).23 however , measurement of relative reuptake inhibition is difficult and variability exists for reported values in the literature . also , the relative reuptake inhibition observed in vitro may not have physiologic relevance in vivo . for instance , while duloxetine is considered a dual reuptake inhibitor based on its in vitro activity , one study showed no effect on norepinephrine reuptake in vivo as determined by response to tyramine infusion in normal subjects.24 our clinical experience with dual reuptake inhibitors has been that individual fms patients tend to respond best to a specific snri and the relative reuptake inhibition ratio does not appear to predict therapeutic response ( ie , some patients who have no response to duloxetine do well on milnacipran and vice versa ) . unfortunately , the inability to predict treatment response usually necessitates multiple therapeutic trials before an efficacious drug is found . a rational method for drug selection will likely require improved understanding of the mechanism(s ) of action of dual reuptake inhibitors together with patient pharmacogenomic data . milnacipran has limited hepatic metabolism.25 approximately 50% of administered drug is excreted unchanged , 30% undergoes glucuronidation and 20% is oxidatively metabolized . the interaction between milnacipran and the cytochrome p450 ( cyp ) isoenzymes is limited , with no interaction with cyp2d6 or cyp2c19 pathways and minimal interaction with cyp1a2 , cyp2c19 , cyp2d6 and cyp3a4 . this is in contrast to duloxetine , which is extensively metabolized by the liver and has interactions with numerous cyp isoenzymes.26 the differences in cyp interactions are the reason that no dose adjustment is necessary for milnacipran use in patients with hepatic insufficiency,27 whereas duloxetine use in patients with hepatic insufficiency is not recommended.28 however , there have been cases of increased liver enzymes and reports of severe liver injury with milnacipran use in foreign post - marketing experience . because of this , we recommend routine lab monitoring and discontinuation of milnacipran in patients who demonstrate evidence of liver dysfunction . concomitant use of hepatotoxic substances with milnacipran should be avoided , and milnacipran should not be prescribed to patients with substantial alcohol use or chronic liver disease . milnacipran can be used without dosage adjustment in patients with mild to moderate renal insufficiency ( ie , a creatinine clearance ( crcl ) 30 ml / min).29 milnacipran can also be used in patients with severe renal impairment ( ie , a crcl between 5 and 29 ml / min ) if the dose is reduced by 50% . this is an important difference between milnacipran and duloxetine , as duloxetine use is not recommended in patients with severe renal impairment.28 also , due to its limited hepatic metabolism and lower protein binding in plasma ( 13% versus > 90% , respectively ) , milnacipran has fewer drug - drug interactions compared with duloxetine.28,30 the treatment - emergent adverse events ( teaes ) seen in the milnacipran fms trials are related to nsri activity and are similar to those seen with the snris.30 gastrointestinal disorders are the most frequent teaes , with nausea occurring in 35% and 39% of patients treated with 100 and 200 mg / day of milnacipran , respectively , compared to 20% of placebo - treated patients . nervous system disorders were also commonly seen teaes , with headache reported by 18% of milnacipran - treated patients versus 14% of placebo controls . the noradrenergic activity of milnacipran is apparent in the increased rates of hot flushes , hyperhidrosis and palpitations reported by milnacipran treated patients versus that seen in controls ( 12% , 9% and 7% for milnacipran treated patients , respectively , versus a 2% rate for all these teaes in controls ) . while heart rate and blood pressure increases were seen at an increased rate in milnacipran - treated patients ( 6 and 3% for milnacipran treated patients versus 1% in controls for both teaes ) , these changes were mild and not deemed clinically significant on average . however , since the effect of milnacipran on blood pressure and heart rate in individual patients can not be predicted , it is recommended that blood pressure and heart rate be regularly monitored in patients treated with milnacipran . other common teaes ( incidence 5% and twice the placebo rate ) include constipation , vomiting and dry mouth . in placebo - controlled fms trials , 23% of patients treated with milnacipran 100 mg / day and 26% of patients treated with milnacipran 200 mg / day discontinued prematurely due to teaes , compared to only 12% of patients treated with placebo.30 nausea was the most frequently cited reason for discontinuation in milnacipran treatment groups ( 6% ) , followed by palpitations ( 3% ) , and headache ( 2% ) . milnacipran treated patients tended to have weight loss during clinical trials that was higher than that seen in placebo - treated patients , however the difference was small ( 0.8 versus 0.2 kg , respectively ) . it is likely the weight loss seen was due to the high prevalence of nausea seen in milnacipran treated patients . nausea is a common teae seen with snri use in fms patients , and its prevalence and severity can be lessened by having patients take milnacipran with food and through gradual dose escalation.31,32 one of the milnacipran fms trials specifically mentioned that teaes were dose related and typically resolved within 1 to 2 weeks of continued therapy,33 an observation consistent with our clinical experience . the indicated dose of milnacipran for management of fms is 100 or 200 mg divided twice daily . however , we have found that many patients have good efficacy with better tolerability when milnacipran is slowly uptitrated . we start with 12.5 mg once daily in the morning for one week , increase to 25 mg once daily in the morning for 2 weeks , and then 50 mg once daily in the morning . some patients have good efficacy with less than the indicated dose and we do not up - titrate unless necessary . if insufficient clinical efficacy is seen but the drug remains well tolerated , we will add an evening dose with slow uptitration as above . we will discuss other tolerability observations along with a review of milnacipran efficacy in the next section . to determine what evidence exists for the efficacy of milnacipran in treating fms , we performed a search of medline and cochrane library databases through september 2009 for randomized - control trials using the key words milnacipran and fibromyalgia limiting results to those in the english language . there were four published studies identified by the search,3336 the results of which are summarized below and in table 2 . it should be noted that none of the milnacipran trials utilized a placebo run - in phase to exclude placebo responders , as was done in trials of pregabalin , since these run - in phases can have important implications for external validity . studies 1 and 2 : the first two studies will be discussed together since they are separate reports of the same study.34,35 we will limit our discussion to results from the later report by gendreau et al since a more conservative analysis of the data was used than that in the earlier report.35 the study was a 12 week double - blind , randomized , placebo - controlled , dose - ranging , multi - center ( 14 sites experienced in treating fms ) trial performed in the us comparing milnacipran to placebo in patients with fms . inclusion criteria required adult patients to meet the 1990 acr classification criteria for fms,5 have a pain score of 10 on the 20-point gracely logarithmic pain scale , and be willing to discontinue all cns - active therapies ( although stable doses of aspirin , acetaminophen and nsaids were allowed during the study ) . exclusion criteria included psychosis ; active suicidality ; alcohol or substance abuse ; concurrent auto - immune , inflammatory , infectious , or malignant disease ; known sleep apnea ; or abnormal baseline liver or kidney function tests . the subjects recorded pain during a 2-week baseline phase and were then randomized in a 3:3:2 ratio to receive milnacipran 25 mg once daily , milnacipran 12.5 mg twice daily , or placebo with all patients receiving capsules twice daily . milnacipran dose was uptitrated as tolerated in weekly intervals up to a maximum of 200 mg / day over 4 weeks with the ability to decrease the dose if dose - limiting toxicities ( dlt ) occurred . the primary endpoint was change in average daily patient self - reported pain scores over the final 2 weeks of the trial compared to the 2-week baseline period . to eliminate bias involved in asking individuals to recall symptoms and to improve compliance,37 an innovative system using palm - based electronic diaries ( e - diaries ) was employed to measured pain level through gracely s anchored logarithmic scale ( 0 to 20).38 patients were asked to rate pain level in the e - diaries each morning , retrospectively once weekly , and in response to 4 to 6 random prompts . patients also completed standard paper questionnaires to assess pain in monthly intervals using the mcgill pain questionnaire and gracely pain intensity scale . at the end of the study , patients completed the patient global impression of change ( pgic ) scale and numerous secondary endpoints were assessed to determine the impact of once - daily and twice - daily milnacipran on fatigue , mood , physical function , sleep quality and global function as determined by the fibromyalgia impact questionnaire,39 the medical outcomes study short form 36 ( sf-36),40 the beck depression inventory,41 the jenkins sleep scale,42 and the arizona sexual experience scale.43 an intention to treat analysis with last observation carried forward was used for data analysis . subjects receiving milnacipran dosed twice daily reported significantly better pain relief than those receiving milnacipran once daily , but in other parameters the once - daily and twice - daily dosing regimens were similar . thirty - seven percent of milnacipran subjects dosed twice daily reported at least a 50% reduction in pain intensity compared with only 22% of subjects receiving milnacipran once daily and 14% of those receiving placebo . there was a statistically significant difference seen when comparing the percent of subjects who rated some degree of improvement in endpoint pgic ratings between milnacipran and placebo treatment ( 73% for twice daily and 77% for once - daily milnacipran treatment versus 38% for placebo ) . while no differences were seen for changes in fiq global scores , milnacipran - treated subjects reported significant increases in the number of days they felt good and the fiq vass for pain , fatigue and stiffness were significantly improved by milnacipran treatment along with improvements in physical function scores that neared statistical significance ( p = 0.074 ) . this is in contrast to results from the sf-36 , jenkins and asex scores which showed no significant improvements in self - reported physical function , sleep quality , or sexual function , respectively . however , it must be noted that randomization of patients with baseline comorbid depression as determined by the mini international neuropsychiatric interview ( mini ) was not consistent across treatment groups ( placebo 32% , milnacipran twice daily 16% and once daily 7% ) . this may have significantly affected the results as , similar to numerous other studies,44 the authors found depressed subjects had significantly higher placebo response rates and it is likely the differences would have been larger if depressed patients had been equally distributed . the main importance of this study was that it proved the efficacy of milnacipran for managing fms and that twice - daily dosing was more efficacious than once - daily dosing . it also showed that milnacipran was well tolerated , with no significant difference in dropout rates between active drug and placebo . nearly all patients completing the trial ( 81% of once daily and 92% of twice - daily participants ) achieved dose escalation to the maximum of 200 mg daily . these results set the stage for future phase 3 trials that ultimately led to the fda approval of milnacipran for fms management . study 3 : this 15-week , multicenter ( 86 centers in us ) , double - blind , placebo - controlled , fixed - dose trial compared milnacipran 100 mg and 200 mg divided twice daily.36 as in the gendreau et al study,35 subjects were required to meet the acr 1990 criteria for fms;5 in contrast , a linear rather than logarithmic vas pain scale was used to ensure included subjects had significant pain at baseline ( 40 on a linear 0 to 100 vas pain scale ) , making the inclusion criteria similar to that used in fms trials of the other fda - approved agents . also contrary to the gendreau et al study,35 subjects with a current major depressive episode as determined by the mini were excluded from participation , but similar exclusions included severe psychiatric illness ; active suicidality ; abuse of alcohol , benzodiazepines or other substances ; a history of non - compliant behavior ; active cardiovascular , pulmonary , hepatic , renal , gastrointestinal , or autoimmune disease ; current systemic infection ; active cancer ; unstable endocrine disease ; severe sleep apnea ; prostate enlargement or other male genitourinary disorder ; pregnancy or breastfeeding . as in the gendreau et al study , patients were required to discontinue any centrally - acting medications that might interfere with the evaluation of pain or other symptoms associated with fms . however , contrary to the gendreau et al study and all other fms trials of indicated agents , patients were allowed up to 60 mg / day of hydrocodone as a rescue analgesic therapy provided it was not used during the 2-week data - collection period preceding the primary efficacy evaluation or during the 48-hour period immediately before study visits . this provision for opioid use during the study is surprising , as treatment trials used to obtain fda approval of the other indicated fms medications only allowed for acetaminophen as a rescue analgesic . also , narcotic use in treating fms is actively discouraged to prevent the development of rebound pain that can lead to dependence . after a 2-week baseline period , subjects were randomized to receive milnacipran or placebo tablets twice daily with food . doses were escalated over the course of 2 to 3 weeks to 50 mg twice daily ( 100 mg / day ) or 100 mg twice daily ( 200 mg / day ) and continued through 15 weeks total . the primary endpoints in this study were different from those used in prior fms studies and evaluated composite response rates to investigate the ability of milnacipran to manage global fms symptoms as well as fms pain . fms global composite responders were defined as subjects who simultaneously experienced clinically meaningful improvement in pain ( 30% or greater improvement in average morning vas pain e - diary scores for final 2 weeks ) , global status ( much or very much improved on the endpoint pgic scale ) , and physical function ( improvement of 6 points on the sf-36 physical component summary scale from baseline to week 15 ) . fms pain composite responders were defined as those who had improvement in pain and global status as defined above . it is important to realize these composite response outcomes are more difficult to achieve than the primary outcomes that were studied in the trials used to obtain fda approval of the other indicated fms medications which looked solely at reduction in pain . secondary end points included time - weighted averages of the individual components of the composite responder analyses as well as improvement in sleep quality , cognitive function and fatigue . for the primary endpoints , missing data were handled using baseline - observation - carried forward ( bocf ) methodology . bocf is a much more conservative methodology than the more commonly used last - observation - carried forward ( locf ) method , and for this reason bocf is now being required instead of locf by the fda for approval trials . however , the study authors also reported locf and observed cases ( oc ) analyses . oc analysis only includes data from trial subjects who fully completed the trial with no missing values and is thought to better reflect the treatment response that can be expected clinically in patients who tolerate the drug . both primary endpoints were met , with a significantly greater proportion of subjects in the milnacipran treatment groups achieving fms global ( 15% 100 mg / day , 14% 200 mg / day versus 9% placebo ) and pain ( 23% 100 mg / day , 25% 200 mg / day versus 17% placebo ) composite response outcome rates compared to those receiving placebo using bocf . while the responder percentages from bocf analysis were small , the more clinically relevant oc analyses showed about one - quarter of milnacipran treated subjects achieved fms global composite response rates ( 25% 100 mg / day , 26% 200 mg / day versus 13% for placebo ) and nearly half had a fms pain composite response ( 39% 100 mg / day , 46% 200 mg / day versus 25% for placebo ) , rates similar to our clinical experience . the authors also noted that analgesic response to milnacipran began as early as one week after starting treatment . this time was during the dose - escalation phase , and this observation is consistent with our clinical experience of many patients responding to lower than indicated milnacipran doses . a published extension study indicates the efficacy of milnacipran in improving fms symptoms may be sustainable for up to 1 year.45 in analysis of secondary endpoints , both milnacipran doses were associated with significant improvements in fatigue but only the 200 mg / day dose improved cognitive dysfunction . this is somewhat surprising as other dual reuptake inhibitors including duloxetine have been associated with insomnia , presumably due to their noradrenergic activity . since the majority of fms patients have nonrestorative sleep , it is reassuring that milnacipran treatment does not appear to worsen sleep quality . study 4 : the most recently published milnacipran study was a fixed - dose , multicenter ( 59 centers ) , randomized , placebo - control trial . study 4 was very similar to study 3 but lasted twice as long ( 27 weeks ) to show the benefits of milnacipran treatment are maintained over time.33 the strategy of performing a 3-month trial to demonstrate efficacy followed by a six month trial to show durability has now been used to gain fda approval of all three indicated fms medications . inclusion and exclusion criteria were similar to those used in study 3 , except that patients were required to have baseline vas pain scale scores 50 on a linear 0100 scale ( slightly higher than the typical 40 scores required in study 3 and other pain studies ) . while milnacipran dosing was identical to that used in study 3 , study 4 randomized fewer subjects ( 888 ) and distributed them unequally between treatment groups using a 2:1:1 ratio between the 200 mg / day , 100 mg / day , and placebo , respectively . primary outcomes were the same as those in study 3 , with composite responder rates used to demonstrate improvement in fms global symptoms ( simultaneous improvement in pain [ 30% or greater improvement in average morning vas pain e - diary scores for final 2 weeks ] , global status ( much or very much improved on the endpoint pgic scale ) , and physical function ( improvement of 6 points on the sf-36 physical component summary scale from baseline to week 27 ) ] and improvement in fms pain ( simultaneous improvement in vas pain e - diary and pgic scores as defined above ) . secondary end points were also identical to those used in study 3 and included time - weighted averages of the individual components of the composite responder analyses as well as improvement in sleep quality , cognitive function and fatigue . in contrast to study 3 where strict bocf analysis was used , a modified bocf methodology was utilized for imputing missing data in study 4 , with bocf used for patients who discontinued the trial before week 15 and locf used for patients who discontinued after week 15 . however , as before , the study authors also reported locf and oc data analyses . secondary outcomes were identical to those used in study 3 and included changes in fatigue , sleep quality and cognitive function . in contrast to study 3 , study 4 did not meet its primary endpoints at 27 weeks . while a higher percentage of milnacipran treated fms subjects achieved the predetermined fms global composite response outcome compared to placebo ( 18.3% for 100 mg / day and 18.1% for 200 mg / day versus 13% for placebo ) , the differences were not statistically significant . the fms pain composite outcome was met for the 200 mg / day milnacipran group , as significantly more of these subjects reached this endpoint compared with placebo ( 25.6% versus 18.4% , p = 0.034 ) . while the 100 mg / day group had a responder rate that was higher than the rate in the 200 mg / day group ( 26.8% versus 25.9% , respectively ) , this difference failed to reach statistical significance in comparison to placebo , likely caused by lower statistical power due to the lower number of subjects in the 100 mg / day versus 200 mg / day group . using the more clinically relevant oc comparison that limits analysis to patients who completed the trial , the 200 mg / day milnacipran treatment group met both primary outcomes , but the 100 mg / day group only met the fms pain composite response outcome . in secondary endpoint analyses using locf analysis , milnacipran at 200 mg / day significantly reduced fatigue compared to placebo at both 15- and 27-week time points . while the 100 mg / day dose improved fatigue scores at 15 weeks , statistical significance compared to placebo was lost at 27 weeks likely due to the lower number of patients in the 100 mg / day versus 200 mg / day group and a higher placebo response rate observed at the last visit . patients treated with milnacipran at 200 mg / day had significant improvement in cognition compared to placebo at both 15 and 27 week endpoints . while the 100 mg / day dose also improved cognition , the differences were not statistically significant . as in study 3 , there were no statistically significant effects on sleep quality for the milnacipran treatment groups compared to placebo . forty - two percent of randomized patients prematurely discontinued the study , which is a rate higher than that seen in the other milnacipran studies . however , this was a longer study and similar high discontinuation rates have been seen in other 6 month fms studies.4649 also , the rates of serious teaes did not differ across treatment groups . despite these caveats , higher discontinuation rates due to teaes were seen in the milnacipran versus placebo arms ( 27% for 200 mg / day and 19.6% for 100 mg / day versus 10.3% for placebo ) . gastrointestinal - related complaints were the most common teaes , with nausea being particularly common in all three groups ( 40.1% 200 mg / day , 32.6% 100 mg / day and 21.1% placebo ) . higher rates of constipation and vomiting were also seen in active treatment groups , as were increased rates of headache , hyperhidrosis , hot flush , heart rate increase and palpitation . these teaes are typical for patients taking norepinephrine active medications , and , on average , there were no clinically relevant changes in laboratory values , heart rate or blood pressure seen in the trials . we have found that starting with a low dose of medication , stopping at the dose patients find effective , and taking milnacipran with food can limit the occurrence of these side effects in the majority of fms patients . with three fda - approved medications for fms management , deciding which medication to use first in individual fms patients can be challenging since all three indicated medications have shown similar efficacy in improving fms pain . we recommend milnacipran as the first - line medication for fms patients who rate fatigue and/or fibrofog as the symptoms that are most limiting for them . this recommendation is based on our clinical experience and the fact that milnacipran is the only fda approved fms treatment shown to improve symptoms of fatigue and cognitive dysfunction in phase 3 clinical trials.33,36 also , our clinical experience shows that many patients who have previously failed to respond to either of the other two indicated medications can have an excellent therapeutic response to milnacipran . despite side effects such as headache , gastrointestinal complaints , hyperhidrosis , hypertension or palpitations that can limit use in fms patients , gradual uptitration of milnacipran starting with 12.5 mg however , it is important to note that the vast majority of subjects in the milnacipran trials and in our clinical practice are caucasian women , making generalization of these recommendations to other patient populations problematic . new fms trials in minorities and men are needed to determine whether these recommendations are valid for all fms patients . we have purposely limited the scope of our discussion of milnacipran to its role in managing fms since that is the only fda approved indication in the us . however , milnacipran has demonstrated clinical efficacy in treating major depressive disorder ( mdd ) and has been used for this indication in europe and japan for a decade.50 in the treatment of mdd , milnacipran shows similar efficacy to the tricyclic antidepressants but with better tolerability.51 the relative efficacy of milnacipran in managing mdd compared to other antidepressant classes remains unknown.52 while not indicated in the us , we have found milnacipran to be a reasonable treatment alternative for managing fms patients with coexisting mdd who fail duloxetine therapy . the fibro mnemonic can be used to recall fms symptoms including fatigue , fibrofog , insomnia , blues , rigidity , and ow ! for pain and work disability . effective fms treatment requires an individualized program of pharmacologic and nonpharmacologic treatments ( including graduated aerobic and resistance exercise ) that target problematic fibro symptoms . serotonin and norepinephrine play important roles in central pain processing , and fms patients have reduced levels of norepinephrine and serotonin in their cerebrospinal fluid . milnacipran is a balanced nsri with demonstrated efficacy in managing global fms symptoms and pain at doses of 100 and 200 mg divided twice daily . milnacipran is associated with numerous side effects including gastrointestinal complaints , headache , hyper - hidrosis , hypertension and palpitations . milnacipran may be particularly beneficial for fms patients with significant symptoms of fatigue , fibrofog and/or depression . milnacipran use has been linked to elevations in liver function tests and severe liver injury . we recommend regular lab monitoring and avoiding milnacipran use in patients with chronic liver disease or concomitantly with hepatotoxic substances . unlike duloxetine , milnacipran can be used in patients with severe renal disease at 50% of the indicated dose .	fibromyalgia syndrome ( fms ) is a widespread pain condition associated with fatigue , cognitive dysfunction , sleep disturbance , depression , anxiety , and stiffness . milnacipran is one of three medications currently approved by the food and drug administration in the united states for the management of adult fms patients . this review is the second in a three - part series reviewing each of the approved fms drugs and serves as a primer on the use of milnacipran in fms treatment including information on pharmacology , pharmacokinetics , safety and tolerability . milnacipran is a mixed serotonin and norepinephrine reuptake inhibitor thought to improve fms symptoms by increasing neurotransmitter levels in descending central nervous system inhibitory pathways . milnacipran has proven efficacy in managing global fms symptoms and pain as well as improving symptoms of fatigue and cognitive dysfunction without affecting sleep . due to its antidepressant activity , milnacipran can also be beneficial to fms patients with coexisting depression . however , side effects can limit milnacipran tolerability in fms patients due to its association with headache , nausea , tachycardia , hyper- and hypotension , and increased risk for bleeding and suicidality in at - risk patients . tolerability can be maximized by starting at low dose and slowly up - titrating if needed . as with all medications used in fms management , milnacipran works best when used as part of an individualized treatment regimen that includes resistance and aerobic exercise , patient education and behavioral therapies .
You are an expert at summarizing long articles. Proceed to summarize the following text: lung and breast cancers are the most common to metastasize to the head and neck region , followed by renal cell carcinoma ( rcc ) . at this site , very few cases of tongue lesion leading to diagnosis of a renal primary have been reported in the literature . this paper reports a case of a 63-year gentleman , chronic smoker , presented with progressively increasing swelling over dorsal surface of tongue with difficulty in swallowing for last 3 months . on examination a 3 3 cm superficial , mobile , pedunculated , proliferative growth was seen in midline of dorsal surface of tongue [ figure 1 ] . in view of histopathology , computed tomography ( ct ) scan of neck , chest and abdomen was done which revealed a 5 6 5 cm heterogeneously enhancing lesion in mid - region extending to upper pole of right kidney with multiple bilateral sub - centimeter lung nodules [ figure 2 ] . fluoride positron emission tomography - ct done as a part of metastatic work - up did not reveal any other site of metastases . image showing tongue metastases computed tomography scan showing right renal mass patient underwent right cytoreductive nephrectomy along with wide excision of tongue metastases . histopathology of kidney mass confirmed the diagnosis of conventional clear cell carcinoma with fuhrman 's grade one ( pt1bn0 ) . patient was started post - operatively on sunitinib in view of residual metastatic lung lesions . rcc predominantly occurs in males ( 1.5:1 ) with peak incidence in the sixth decade . approximately 30% of newly diagnosed rcc patients present with synchronous metastatic disease , and an additional 25 - 30% of patients with clinically localized disease eventually develop metastases . metastases in the head neck region from rcc are seen in 8 - 15% of patients with metastatic disease . usually , it is a manifestation of widespread metastases in a known case of rcc , but at times such lesions can also lead to detection of a silent rcc . primary localizing sites in the head neck region include paranasal sinuses , larynx , jaws , temporal bones , thyroid gland and parotid glands . lingual metastases represent only about 1% incidence of metastases to oral cavity and arise most commonly from melanomas , breast , gastrointestinal tract , lung and rarely from kidney . from 1973 to 2011 , 33 cases of a lingual metastasis of a primary kidney tumor were published in the english - language literature . until today , only 7 patients have presented primarily with tongue metastases leading to the diagnosis of rcc [ table 1 ] . previous reports of metastasis of tongue it has been postulated that in the absence of lung or liver lesions , the disease can spread through batson 's venous plexus or thoracic duct . most tongue metastases involve the base due to its high vascularity and low mobility . however , our patient had a dorsal midline lesion in tongue . like primary tongue lesions , lingual metastases can be ulcerated or polypoid and tumor cells can infiltrate the muscle without involving the overlying mucosa . tomita et al . in their review of 18 cases of rcc with lingual metastases , considered this site to be associated with poor prognosis : the rarity of this site of metastases can create a diagnostic dilemma in patients with clear cell histology . malignant clear cell tumors of the head and neck arise as primary from the salivary , thyroid , or parathyroid glands and as secondaries from lung , female genital tract and kidney . cytoreductive nephrectomy is justified in patients with metastatic disease to improve quality of life or local symptoms . prospective trials have demonstrated that , in patients with synchronous metastatic disease , cytoreductive nephrectomy and cytokine therapy give a distinct survival advantage over immunotherapy alone . several retrospective studies have suggested that patients undergoing complete resection of limited metastatic foci may experience long disease free intervals , with median 5 year survival rate of 35 - 50% in some reports . in a report by kyan and kato , surgical resection of lingual metastases followed by administration of interferon - alpha and interleukin - ii had a disease free interval of 2 years . many prospective trials have shown a prolongation of progression free survival and higher response rates with targeted molecular therapy in metastatic disease . tongue is a rare site for rcc metastases and in very few patients it has led to detection of an otherwise silent rcc . renal evaluation for primary should be considered in differential diagnosis of tongue lesion with clear cell histology . patients can achieve long disease free survival with combination of surgical excision and molecular therapy .	renal cell carcinoma ( rcc ) has the propensity to metastasize to any organ in the body . tongue metastases from rcc is very rare with most of them being metachronous in nature . we present a rare case where patient presented with a tongue lesion leading to a diagnosis of rcc . surgery is the preferred modality of treatment for tongue metastases .
You are an expert at summarizing long articles. Proceed to summarize the following text: within moments of birth , a human baby enters into a state of quiet alertness that lasts for hours . the newborn rests quietly with eyes open , orienting to the faces , voices , and scents of humans . some hardwiring in the baby s brain makes this bonding possible : babies are born with a preference for the high - frequency sounds of the human voice and for the general shape of the human face . this learning begins before birth as the baby becomes familiar with the mother s voice and odors . the newborn continues rapidly to learn more voices and smells , responding more intensely . throughout the animal kingdom , once a newborn knows its mother it will do its best to remain with her . animals that are mobile soon after birth , like horses or goslings , follow their mothers everywhere . it seems inconceivable that any child , puppy , baby monkey , or chick would develop an enduring bond with an abusive caregiver . puzzling out the explanation requires a shift in the ways brain and behavior have been traditionally viewed . for much of the twentieth century , psychology followed a behaviorist model : an animal will repeat a behavior that triggers a reward , and will not repeat a behavior that s punished . following this logic , a baby would do its best to avoid an abusive caregiver , or at least show signs of distress in that person s presence . but the research of harry harlow at the university of wisconsin , madison , beginning in the 1960s , showed just the opposite . harlow raised infant monkeys with a surrogate mother ( a wire tube wrapped in cloth with a plastic monkey head on top ) . in one experiment , baby monkeys clinging to the surrogate would hold on even when subjected to an unpleasant puff of air . similar results show up in studies of other species , including chicks and rats and in many heart - wrenching headlines of abused children , battered wives , and kidnapping victims who stand by their tormentors . scientists suspecting a brain - based explanation were hampered not only by the behavioral hypothesis but also by prevailing views of brain development . throughout much of the 20 century , researchers understandably assumed that the brain became more sophisticated with age that the young brain was , in fact , immature . the infant brain is actually perfectly developed to accomplish the tasks appropriate to the survival needs of infancy . some of the unique functions of the infant brain help to explain why a child will bond with whatever caregiver is available . because of ethical concerns , it s difficult to scan the brains of most children ; it is certainly unethical to upset an abused child even further by presenting traumatic events during a brain scan . scanning also presents logistical problems in children , since the child needs to remain perfectly still during the scan . though rats learn about their mothers entirely through smell seeing and hearing do not emerge until the rat pup is around two weeks old rats are similar to children in that they have a biologically predetermined attachment system in the brain but must learn to identify and remember the caregiver . attachment learning in rats to question what it is about an infant s brain that supports attachment , even when the caregiver is abusive . the neurotransmitter is released in massive amounts at birth and during bonding with the mother . it is also released when a young animal is in pain . this dual role for norepinephrine set us on the path to explore the fundamental , and puzzling , question of why a child can not learn to break the attachment to an abusive caregiver . after all , when humans and all other mammals experience pain , they learn to avoid anything that warns of the pain so that they can prevent it next time . so in an abusive relationship , why does nt the pain activate the brain s fear and avoidance circuits ? simply and amazingly , the brain area responsible for fear and avoidance learning is not turned on by pain in pups not old enough to leave the nest . even more amazingly , in older pups that live in the nest but also explore outside the nest , this brain area shuts off only when the pups are with the mother . we worked first with young rats still nesting with their mothers , when they are blind and deaf and must depend upon the mother s odor for attachment . first , we paired a novel odor with a painful experience , such as a mother rat handling pups roughly , a tail pinch , or a slightly painful electric shock . that odor became a new maternal odor that controlled the pups approach to the mother and enhanced social behavior and feeding with the mother . older pups that could leave the nest reacted very differently : they learned to avoid an odor paired with pain . but to our surprise , if the mother was with these older pups during the learning , they were attracted to the pain - associated odor , and examination revealed no activation of stress networks in the brain . that is , the mother s presence can act as a biochemical switch , determining whether rat pups will develop an aversion or an attraction to a mildly painful event.2,3 my colleagues and i were perplexed by these results . it took time for us to change our thinking and view the infant brain as a brain designed for infant survival , rather than as an incomplete adult brain . we were completely surprised by the paradoxical result that pain would lead an infant to approach , even seek , a newly learned odor . we were even more astonished by the ability of maternal presence to reinstate this paradoxical learning in older pups . the key to the young rat pups behavior is an almond - shaped structure known as the amygdala , a part of the brain involved in emotionally charged memories especially fearful ones . the presence of the mother during a painful event is enough to suppress activity in the amygdala of a rat pup . the mechanism proved to be surprisingly simple : pups still living in the nest , or older pups in the mother s presence , did not release the stress hormone corticosterone . the infant amygdala is uniquely dependent upon increases in corticosterone to learn and express fear . in a later study , we showed that the neurotransmitter dopamine is also released when the rat is learning to avoid the odor . though dopamine is often considered a reward chemical , when increased in the amygdala it helps to form fear - related memories . in this study , young rats still in the mother s nest showed a large decrease in dopamine in the amygdala , indicating a mechanism to further block pups from learning to fear.4 the benefits of this maternal off switch seem obvious . nearness to the mother offers comfort and courage in what might otherwise be a frightening situation , serving to strengthen the bond between mother and infant , and to remind an older child where its haven is in times of danger . when a child gets an injection , the presence of a parent can help the child cope with the pain . later on , when someone we care about is there to comfort us in a stressful situation , our stress hormones and fear are greatly reduced . the switch is flipped , however , when the intrepid older rat pup is out exploring . then , the threatening event sparks the fear response , propelling the youngster away from danger and , preferably , back into the nest while searing an indelible memory into the brain . but when the parent and the nest are themselves sources of danger , the suppression of fear circuits in the amygdala unfortunately still works . the fear , avoidance , and even memories associated with pain are extinguished explaining why an abused child , under normal circumstances , the ability to remember danger is so vital to survival that memories based on fear can not be erased . they can be overwritten through extinction , an active - learning process in which a new memory supplants the older one ( like teaching a rat that a shock will no longer follow the sound that used to precede it ) . but extinction is shaky and impermanent , and the underlying fear can re - emerge at any time . these perineuronal nets are made of cartilage - like tissue known as proteoglycans . like miniature chain - link fences , they are thought to protect important memories by blocking the re - modeling , or plasticity , that might otherwise dismantle the memory in favor of new information . andreas luthi of the friedrich miescher institute for biomedical research in switzerland and cyril herry of inserm in france showed that in adult mice whose fear of a shock has been reprogrammed through extinction , treatment with an enzyme that degrades the nets will bring the memory back in full force ; the mice showed the same freezing behavior in response to a warning odor that they did before extinction training took place.5 but younger pups , less than three weeks old , can lose a fearful memory completely . like our work with norepinephrine circuits , this research also illustrates a concrete mechanism that prevents young children from forming memories based on fear even when it might be in their best interest to do so . the animal research explains an infant s biological drive to stay close to a source of food and safety . but we know that even when basic needs food , clothing , shelter , medical care are adequately met , children are still devastated by the loss of a parent . children in orphanages whose basic needs are met ( who receive perfunctory care , in medical parlance ) often fail to grow and do not reach developmental milestones at a normal pace . later in life , these children are more likely to have profound mental problems , including difficulty maintaining social relationships . the early work of harlow and colleagues attests to the importance of attachment figures . in an experiment well known to every first - year psychology student , a baby monkey will choose a warm , soft , cuddly artificial mother with no food over a wire one with a bottle attached . more recently , the importance of a loving caregiver early in life has been underlined by studies of teenage romanian orphans left by impoverished parents to be raised in overcrowded , understaffed , improperly run state institutions . even when adopted and cared for by loving families , some of these children remain detached and have emotional and cognitive impairment . some encouraging findings , however , show a group of resilient children who overcame these early - life disadvantages ; they were adopted before age two and placed in loving families that gave enormous time and energy to their rescue . what specifically does a loving caregiver provide , without which even a healthy , well - fed child will languish ? rats are devoted mothers and spend copious time licking , snuggling , and crouching over their young . myron hofer of columbia university has shown that the intense sensory stimulation a rat pup receives regulates the behavioral , physiological , and neural development of the brain.6 this line of research has shown that when rat pups are separated from their mother , mimicking her actions by stroking can increase levels of growth hormones in the infants brains ; extra warmth ( normally provided by the mother s body heat ) increases levels of norepinephrine ; and giving the pups a whiff of their mother s scent ramps up their level of activity . once basic needs of the infant are met through social interactions , presenting an environment that is interesting but age appropriate appears to optimize behavioral and brain development . a recent study in rats by akaysha tang and bruce mcewen showed that infant rats that experienced just three minutes of novelty each day exhibited enhanced learning and more controlled stress response in adulthood.7 this fact may come as a surprise , since most of us think of genes as something we are born with . of course , the genetic material we receive from our parents is important , and lays the foundation for who we are and what we become . but how our parents treat us as we grow can greatly influence the way the genetic material is read and used . think of our genetic material as a vast library of information . the way this information is accessed , and what ends up being done with it , depend in great part on our early - life experiences . an epigenetic change occurs during the biochemical process of turning a gene s code into whatever protein that gene codes for in scientific parlance , the process of gene expression . the concept of epigenetics helps explain why so many effects of child abuse do not become apparent until adolescence or adulthood . it may seem obvious that a mother s handling of her child will influence the way that child treats her own children . the transmission of this trait from mother to daughter and beyond may result from epigenetic alterations to genes involving estrogen sensitivity , according to research from frances champagne of columbia university and michael meaney of mcgill university . these researchers showed that the amount of care a newborn female rat receives in the form of licking and grooming will determine how much licking and grooming she will do for her own pups and they for theirs.8 in 2006 this team found that a mother s licking and grooming activity can modify the expression of a gene involved in several aspects of reproduction , resulting in differing levels of a particular estrogen receptor in adulthood . consequently , a rat s sensitivity to her own estrogen levels can change , leading to changes in the way a mother handles her pups for at least two generations . tania roth , david sweat , and colleagues at the university of alabama , birmingham , found that when rat pups were raised by stressed mothers who behaved abusively , the pups and their pups showed changes in the gene producing a substance known as brain - derived neurotrophic factor ( bdnf).9 the job of growth factors is to nourish and protect brain cells ; bdnf in particular helps to stimulate the birth of new neurons ( a process known as neurogenesis ) . the gene alterations in the abused pups showed up in the hippocampus and the prefrontal cortex . since bdnf and neurogenesis in the hippocampus help to support learning and memory , and both help ward off stress and depression , this animal finding points to one route through which abused children can suffer from emotional and cognitive problems throughout life . some recent research hints at epigenetic changes in people who were abused as children . in 2009 , meaney and colleagues examined brain tissue obtained postmortem from a group of suicide victims , some of whom had been abused early in life and some of whom had not . the brains of the abuse victims showed several differences . in the hippocampus , receptors for the stress hormones known as glucocorticoids were fewer in number , and the genes encoding the receptors had alterations in the promoter glucocorticoid receptors are plentiful in the hippocampus , where they play several roles in memory , emotion , and the stress response including helping to consolidate fear - related memories . some conditions , like major depression and post - traumatic stress disorder , involve a loss of the neurons containing these receptors ; both conditions can involve memory loss as well . this pattern of epigenetic change did not show up in the brains of suicide victims who were not abused , or in control subjects who had died of natural causes.10 the differences imply that the abuse caused the changes in the hippocampus not that people who commit suicide have an underlying brain anomaly making them more likely to take their lives . technological advances , coupled with our new understanding of the uniqueness of the developing brain , have direct implications for child rearing . assuming that basic nutritional and health needs are met , what do we need to do to optimize brain development ? the developing brain overwhelmingly requires a loving , consistent , and patient caregiver who socializes with the infant , punctuated with age appropriate novelty and learning . this organizes brain circuits and gene expression for cognition and emotion to yield a cognitively and emotionally well - balanced child and adult . what is detrimental to brain development ? neglect or emotional or physical abuse that produces either heightened or prolonged activation of the stress system results in later - life difficulties . while we are less sure how these early - life experiences change the brain , we do know that the brain responds by changing its structure , gene expression , and function . , we are social creatures , and our social interactions in early life design the brain to greatly influence the person we become .	editor s note : why do abused children attach and remain attached to abusive parents ? in this article , dr . regina sullivan explains how her research with rat pups has led to greater understanding of the infant brain , and how negative early experiences can cause long - term genetic , brain , behavioral , and hormonal changes that can affect not only the abuse victim but also the victim s descendants .
You are an expert at summarizing long articles. Proceed to summarize the following text: the epic - interact is a large prospective type 2 diabetes case - cohort study ( 16 ) nested within the epic study ( 17 ) with more than half a million adult participants recruited in the 1990s from the following 10 european countries : denmark , france , germany , greece , italy , the netherlands , norway , spain , sweden , and the united kingdom . with the exception of greece and norway , all epic countries participated in the epic - interact study ( n = 455,680 ) . after the exclusion of individuals without stored blood ( n = 109,680 ) or with prevalent diabetes at baseline ( 5,821 ) , 340,234 participants with 3.99 million person - years of follow - up were included in this study . all participants gave written informed consent , and the study was approved by the local ethics committee in the participating countries and the internal review board of the international agency for research on cancer . a pragmatic , high - sensitivity approach for case ascertainment was used in order to identify all potential incident type 2 diabetes cases and to exclude all individuals with prevalent diabetes ( 16 ) , using at least two multiple sources of evidence including self - report and linkage to primary care registers , secondary care registers , medication registers , and hospital admissions and mortality data . cases in denmark and sweden were not ascertained by self - report , but were identified via local and national diabetes and pharmaceutical registers , and hence were considered as verified . follow - up was censored either on 31 december 2007 , the date of type 2 diabetes diagnosis , or the date of death , whichever occurred first . in total , 12,403 verified incident type 2 diabetic cases were identified . a random subcohort of 16,835 individuals was selected from the 340,234 participants with available stored blood samples , stratified by center . after the exclusion of 681 individuals without information on diabetes status , 16,154 subcohort individuals were included , of whom 778 individuals developed incident type 2 diabetes during follow - up . of the 27,779 participants ( 12,403 case subjects , of whom 778 were within the subcohort of 16,154 participants ) in the epic - interact study , we excluded 619 participants within the lowest and the highest 1% of the distribution of the ratio of reported energy intake ( determined from the questionnaire ) to estimate energy requirements ( calculated from age , sex , body weight , and height ) . in addition , we excluded 1,072 participants with missing information on nutritional intake or other covariates used in the statistical analysis . this resulted in a final sample of 26,088 participants for inclusion in the current analysis with 11,559 case subjects and a subcohort of 15,258 participants , including 729 case subjects in the subcohort . habitual diet during the 12 months prior to recruitment was recorded using country - specific validated food frequency questionnaires or diet histories ( 17,18 ) . most centers adopted a self - administered questionnaire of 98 to 266 food items . in spain and ragusa ( italy ) questionnaires in france , italy , spain , the netherlands , and germany were quantitative , estimating individual average portion size systematically . those in denmark , naples ( italy ) , and ume ( sweden ) were semiquantitative , with the same standard portion assigned to all subjects . in malm ( sweden ) and total energy and nutrient intakes were estimated using the standardized epic nutrient database ( 19 ) . estimated flavonoid and lignan intake was derived from foods included in the dietary questionnaires through a comprehensive food composition database on flavonoids and lignans , as we have previously described ( 20,21 ) . department of agriculture databases ( 22 ) , phenol - explorer ( 23 ) and the u.k . this database compiles composition data on lignans and the six flavonoid subclasses ( supplementary table 1 ) . furthermore , our flavonoid food composition database was expanded by using retention factors when no analytical data were provided by cooked food . the retention factors applied to all flavonoid classes , except isoflavones , were 0.70 , 0.35 , and 0.25 , respectively , after frying , cooking in a microwave oven , and boiling ( 25 ) . these retention factors were not applied to isoflavones and lignans because their cooking losses are usually minimal . our database was also expanded by calculating the flavonoid content of recipes , estimating missing values based on similar foods ( by botanical family and plant part ) , obtaining consumption data for food group items , and using botanical data for logical zeros ( when negligible amounts of flavonoids or lignans would be present in a food type , e.g. , anthocyanidins in plant foods without red , blue or purple color ) . in nature , flavonoids and lignans are usually found as glycosides , mainly with glucose or rhamnose moieties , but other sugars may also be involved . therefore , data on flavonoids and lignans are expressed as aglycone equivalents , after conversion of the flavonoid glycosides into aglycone contents using their respective molecular weights . the final database contains 1,877 food items , including raw foods , cooked foods , and recipes , and 10% of values for these food items are missing . a lifestyle questionnaire was used to collect information about sociodemographic characteristics , smoking status , and medical history ( 17 ) . occupational and leisure - time physical activity was assessed by questionnaire and classified according to the cambridge physical activity index ( 26 ) . a history of previous illness included hypertension , hyperlipidemia , previous cancers , and/or cardiovascular diseases ( angina , stroke , and myocardial infarction ) . information on family history of type 2 diabetes in a first - degree relative was collected for all participants except for individuals in italy , spain , germany , and oxford ( u.k . ) . height , weight , and waist circumference were measured by trained health professionals using standardized protocols , except in oxford ( u.k . ) and france , where self - reported measurements were obtained , and ume ( sweden ) , where waist circumference was not recorded ( 16 ) . blood samples were collected at baseline , and hemoglobin a1c ( hba1c ) was measured using high - performance liquid chromatography ( diamat automated glycated hemoglobin analyzer ; bio - rad laboratories ltd . , hemel hempstead , u.k . ) . dietary questionnaire - derived means , sds , medians , and 5th and 95th percentiles of total intake and intakes of subclasses of flavonoids and lignans were calculated . total flavonoid intake by country was also visualized in a box - and - whisker plot . baseline characteristics and dietary intakes in the subcohort were summarized by quintiles of total flavonoid intake using means and sds or frequencies . prentice - weighted cox regression models accounting for the case - cohort design ( 27 ) were used to estimate the associations between flavonoid and lignan intakes and type 2 diabetes of each epic country . total intake and intakes of subclasses of flavonoids and lignans were categorized using sub - cohort - wide quintiles . tests for linear trend were performed by assigning the medians of each quintile as scores . intakes were also analyzed continuously , after a log2 transformation that indicates a doubling in flavonoid and lignan intakes . age was used as the underlying time scale , with entry time defined as the participant s age at baseline , and exit time as the participant s age at diagnosis of diabetes , censoring , or death ( whichever came first ) . all analyses were stratified by center to control for center effects such as follow - up procedures and questionnaire design . model 1 included age ( as underlying time scale ) , sex , and total energy intake ( kilocalories per day ) . model 2 was additionally adjusted for the following potential lifestyle confounders : educational level ( none , primary school , technical / professional , secondary school , higher education ) ; physical activity ( inactive , moderately inactive , moderately active , and active ) ; smoking status ( never , former , and current ) ; bmi ( kilograms per square meter ) ; and alcohol intake ( grams per day ) . model 3 was additionally adjusted for the following potential dietary confounders : intakes of red meat , processed meat , sugar - sweetened soft drinks , and coffee ( grams per day ) . model 4 was additionally adjusted for the following potential mediators : intakes of fiber ( grams per day ) , vitamin c ( milligrams per day ) , and magnesium ( milligrams per day ) . hrs and 95% cis were estimated within each country and then combined by using random - effects meta - analysis . between - country heterogeneity was assessed using the i statistic . effect modification by sex , baseline bmi category ( bmi < 25 , 25 to < 30 , and 30 kg / m ) , and smoking status ( never , current , former smokers ) was assessed by modeling interaction terms , in model 4 , between these variables and total flavonoid intake , and conducting stratified analyses . moreover , the proportional hazards assumption was assessed by testing the interaction between flavonoid intake and age ( < 60 and 60 years of age ) , and for all exposures there was no evidence against the assumption . sensitivity analyses were conducted excluding 975 diabetes case subjects in whom type 2 diabetes had been diagnosed within the first 2 years of recruitment . in a second sensitivity analysis , model 4 was additionally adjusted for hypertension and hyperlipidemia , after the exclusion of 1,971 participants with cancer and/or cardiovascular diseases at recruitment , because participants in these subgroups may have modified their diets . in a third sensitivity analysis , model 4 was additionally adjusted for history of diabetes in a first - degree relative ( with the exclusion of 12,977 participants with missing data ) , an important risk factor of type 2 diabetes ( 28 ) ; finally , model 4 was additionally adjusted for waist circumference ( exclusion of 1,824 participants without this data ) , another independent risk factor strongly associated with type 2 diabetes ( 29 ) . in a further sensitivity analysis , non - case subjects from the subcohort were excluded if they had an hba1c level 6.5% ( 48 mmol / mol ) , as this cutoff can be used as a diagnostic criterion for type 2 diabetes ( as per the american diabetes association and the world health organization ) . all statistical analyses were performed using stata / se 12.0 ( statacorp , college station , tx ) . all p values were based on two - sided tests , and statistical significance was set at p < 0.05 . the epic - interact is a large prospective type 2 diabetes case - cohort study ( 16 ) nested within the epic study ( 17 ) with more than half a million adult participants recruited in the 1990s from the following 10 european countries : denmark , france , germany , greece , italy , the netherlands , norway , spain , sweden , and the united kingdom . with the exception of greece and norway , all epic countries participated in the epic - interact study ( n = 455,680 ) . after the exclusion of individuals without stored blood ( n = 109,680 ) or with prevalent diabetes at baseline ( 5,821 ) , 340,234 participants with 3.99 million person - years of follow - up were included in this study . all participants gave written informed consent , and the study was approved by the local ethics committee in the participating countries and the internal review board of the international agency for research on cancer . a pragmatic , high - sensitivity approach for case ascertainment was used in order to identify all potential incident type 2 diabetes cases and to exclude all individuals with prevalent diabetes ( 16 ) , using at least two multiple sources of evidence including self - report and linkage to primary care registers , secondary care registers , medication registers , and hospital admissions and mortality data . cases in denmark and sweden were not ascertained by self - report , but were identified via local and national diabetes and pharmaceutical registers , and hence were considered as verified . follow - up was censored either on 31 december 2007 , the date of type 2 diabetes diagnosis , or the date of death , whichever occurred first . in total , a random subcohort of 16,835 individuals was selected from the 340,234 participants with available stored blood samples , stratified by center . after the exclusion of 681 individuals without information on diabetes status , 16,154 subcohort individuals were included , of whom 778 individuals developed incident type 2 diabetes during follow - up . of the 27,779 participants ( 12,403 case subjects , of whom 778 were within the subcohort of 16,154 participants ) in the epic - interact study , we excluded 619 participants within the lowest and the highest 1% of the distribution of the ratio of reported energy intake ( determined from the questionnaire ) to estimate energy requirements ( calculated from age , sex , body weight , and height ) . in addition , we excluded 1,072 participants with missing information on nutritional intake or other covariates used in the statistical analysis . this resulted in a final sample of 26,088 participants for inclusion in the current analysis with 11,559 case subjects and a subcohort of 15,258 participants , including 729 case subjects in the subcohort . habitual diet during the 12 months prior to recruitment was recorded using country - specific validated food frequency questionnaires or diet histories ( 17,18 ) . most centers adopted a self - administered questionnaire of 98 to 266 food items . in spain and ragusa ( italy ) questionnaires in france , italy , spain , the netherlands , and germany were quantitative , estimating individual average portion size systematically . those in denmark , naples ( italy ) , and ume ( sweden ) were semiquantitative , with the same standard portion assigned to all subjects . in malm ( sweden ) and the u.k . total energy and nutrient intakes were estimated using the standardized epic nutrient database ( 19 ) . estimated flavonoid and lignan intake was derived from foods included in the dietary questionnaires through a comprehensive food composition database on flavonoids and lignans , as we have previously described ( 20,21 ) . department of agriculture databases ( 22 ) , phenol - explorer ( 23 ) and the u.k . this database compiles composition data on lignans and the six flavonoid subclasses ( supplementary table 1 ) . furthermore , our flavonoid food composition database was expanded by using retention factors when no analytical data were provided by cooked food . the retention factors applied to all flavonoid classes , except isoflavones , were 0.70 , 0.35 , and 0.25 , respectively , after frying , cooking in a microwave oven , and boiling ( 25 ) . these retention factors were not applied to isoflavones and lignans because their cooking losses are usually minimal . our database was also expanded by calculating the flavonoid content of recipes , estimating missing values based on similar foods ( by botanical family and plant part ) , obtaining consumption data for food group items , and using botanical data for logical zeros ( when negligible amounts of flavonoids or lignans would be present in a food type , e.g. , anthocyanidins in plant foods without red , blue or purple color ) . in nature , flavonoids and lignans are usually found as glycosides , mainly with glucose or rhamnose moieties , but other sugars may also be involved . therefore , data on flavonoids and lignans are expressed as aglycone equivalents , after conversion of the flavonoid glycosides into aglycone contents using their respective molecular weights . the final database contains 1,877 food items , including raw foods , cooked foods , and recipes , and 10% of values for these food items are missing . a lifestyle questionnaire was used to collect information about sociodemographic characteristics , smoking status , and medical history ( 17 ) . occupational and leisure - time physical activity was assessed by questionnaire and classified according to the cambridge physical activity index ( 26 ) . a history of previous illness included hypertension , hyperlipidemia , previous cancers , and/or cardiovascular diseases ( angina , stroke , and myocardial infarction ) . information on family history of type 2 diabetes in a first - degree relative was collected for all participants except for individuals in italy , spain , germany , and oxford ( u.k . ) . height , weight , and waist circumference were measured by trained health professionals using standardized protocols , except in oxford ( u.k . ) and france , where self - reported measurements were obtained , and ume ( sweden ) , where waist circumference was not recorded ( 16 ) . blood samples were collected at baseline , and hemoglobin a1c ( hba1c ) was measured using high - performance liquid chromatography ( diamat automated glycated hemoglobin analyzer ; bio - rad laboratories ltd . , hemel hempstead , u.k . ) . dietary questionnaire - derived means , sds , medians , and 5th and 95th percentiles of total intake and intakes of subclasses of flavonoids and lignans were calculated . total flavonoid intake by country was also visualized in a box - and - whisker plot . baseline characteristics and dietary intakes in the subcohort were summarized by quintiles of total flavonoid intake using means and sds or frequencies . prentice - weighted cox regression models accounting for the case - cohort design ( 27 ) were used to estimate the associations between flavonoid and lignan intakes and type 2 diabetes of each epic country . total intake and intakes of subclasses of flavonoids and lignans tests for linear trend were performed by assigning the medians of each quintile as scores . intakes were also analyzed continuously , after a log2 transformation that indicates a doubling in flavonoid and lignan intakes . age was used as the underlying time scale , with entry time defined as the participant s age at baseline , and exit time as the participant s age at diagnosis of diabetes , censoring , or death ( whichever came first ) . all analyses were stratified by center to control for center effects such as follow - up procedures and questionnaire design . model 1 included age ( as underlying time scale ) , sex , and total energy intake ( kilocalories per day ) . model 2 was additionally adjusted for the following potential lifestyle confounders : educational level ( none , primary school , technical / professional , secondary school , higher education ) ; physical activity ( inactive , moderately inactive , moderately active , and active ) ; smoking status ( never , former , and current ) ; bmi ( kilograms per square meter ) ; and alcohol intake ( grams per day ) . model 3 was additionally adjusted for the following potential dietary confounders : intakes of red meat , processed meat , sugar - sweetened soft drinks , and coffee ( grams per day ) . model 4 was additionally adjusted for the following potential mediators : intakes of fiber ( grams per day ) , vitamin c ( milligrams per day ) , and magnesium ( milligrams per day ) . hrs and 95% cis were estimated within each country and then combined by using random - effects meta - analysis . between - country heterogeneity was assessed using the i statistic . effect modification by sex , baseline bmi category ( bmi < 25 , 25 to < 30 , and 30 kg / m ) , and smoking status ( never , current , former smokers ) was assessed by modeling interaction terms , in model 4 , between these variables and total flavonoid intake , and conducting stratified analyses . moreover , the proportional hazards assumption was assessed by testing the interaction between flavonoid intake and age ( < 60 and 60 years of age ) , and for all exposures there was no evidence against the assumption . sensitivity analyses were conducted excluding 975 diabetes case subjects in whom type 2 diabetes had been diagnosed within the first 2 years of recruitment . in a second sensitivity analysis , model 4 was additionally adjusted for hypertension and hyperlipidemia , after the exclusion of 1,971 participants with cancer and/or cardiovascular diseases at recruitment , because participants in these subgroups may have modified their diets . in a third sensitivity analysis , model 4 was additionally adjusted for history of diabetes in a first - degree relative ( with the exclusion of 12,977 participants with missing data ) , an important risk factor of type 2 diabetes ( 28 ) ; finally , model 4 was additionally adjusted for waist circumference ( exclusion of 1,824 participants without this data ) , another independent risk factor strongly associated with type 2 diabetes ( 29 ) . in a further sensitivity analysis , non - case subjects from the subcohort were excluded if they had an hba1c level 6.5% ( 48 mmol / mol ) , as this cutoff can be used as a diagnostic criterion for type 2 diabetes ( as per the american diabetes association and the world health organization ) . all statistical analyses were performed using stata / se 12.0 ( statacorp , college station , tx ) . all p values were based on two - sided tests , and statistical significance was set at p < 0.05 . table 1 shows the mean ( sd ) and median and percentiles ( 5th and 95th ) of both total intake and intakes of subclasses of dietary flavonoids and lignans . as indicated by the large differences between means and medians , flavanols were the most important contributor ( 80% ) to total flavonoid intake ( proanthocyanidins 44% , flavan-3-ols monomers 35% , theaflavins 1% ) , followed by anthocyanidins ( 6.3% ) , flavanones ( 6.2% ) , and flavonols ( 6.0% ) . total flavonoid intake varied markedly across countries , with median intakes ranging from 201.7 mg / day in sweden to 850.6 mg / day in the u.k . total flavonoid intake and intake of some flavonoid subclasses ( flavanols and flavonols ) were highly correlated ( r > 0.8 ) , whereas other flavonoid subclasses ( such as anthocyanidins , flavanones , flavones , and isoflavones ) had low to moderate correlation ( r = between 0.1 and 0.4 ) . the main food sources of total flavonoid intake were fruits ( 36.4% ) , tea ( 33.1% ) , wine ( 8.6% ) , chocolate products ( 4.2% ) , fruit juices ( 3.9% ) , beer ( 2.5% ) , vegetables ( 2.3% ) , and legumes ( 2.3% ) ( table 1 ) . dietary intake of flavonoids and lignans in the epic - interact subcohort ( n = 15,258 ) baseline characteristics of the subcohort according to quintiles of total flavonoid intake are shown in table 2 . participants in the highest quintile of total flavonoid intakes were likely to be older and to have the lowest bmi and waist circumference compared with those participants in the lowest quintile . with increasing total intake of flavonoids , participants tended to have a more health - conscious lifestyle pattern with greater educational level and physical activity ; lower tobacco consumption ; a higher intake of fruits , vegetables , fiber , vitamin c , and magnesium ; and a lower consumption of processed meat . however , participants in the top quintile reported greater alcohol and red meat intake and lower coffee intake . baseline characteristics and dietary intakes of the epic - interact subcohort according to quintiles of total flavonoid intake the pooled hrs ( 95% cis ) for type 2 diabetes by quintiles of total intake and intakes of subclasses of flavonoids and lignans are shown in table 3 . significant inverse associations were observed in model 1 ( stratified by center and adjusted for age [ as underlying time - scale ] , sex , and total energy ) for total intakes of flavonoids , flavanols ( including flavan-3-ol monomers , proanthocyanidins , and theaflavins ) , anthocyanidins , flavonols , flavones , and lignans . after further adjustment for potential confounders ( models 2 and 3 ) , all associations were attenuated but were still statistically significant for flavan-3-ol monomers and flavonols . when fiber , vitamin c , and magnesium intakes were additionally included in the multivariable models ( model 4 ) , similar risk estimates were observed between the intake of all flavonoid subclasses and lignans , and the incidence of type 2 diabetes as in model 3 , showing significant inverse associations with intakes of flavanols ( hr for highest vs. lowest quintile 0.82 [ 95% ci 0.680.99 ] ; p for trend 0.012 ) ; flavan-3-ol monomers ( hr 0.73 [ 95% ci 0.570.93 ] ; p for trend 0.029 ) ; and flavonols ( hr 0.81 [ 95% ci 0.690.95 ] ; p for trend 0.020 ) . a significant trend was also detected for total flavonoids ( hr 0.90 [ 95% ci 0.771.04 ] ; p for trend 0.040 ) . a borderline significant trend was seen for theaflavins ( hr 0.83 [ 95% ci 0.691.01 ] ; p for trend 0.084 ) . no significant association was observed with lignans ( hr 0.88 [ 95% ci 0.721.07 ] ; p for trend 0.119 ) . association between flavonoid and lignan intakes and type 2 diabetes : epic - interact study in multivariable analyses ( model 4 ) , similar associations of type 2 diabetes were observed when dietary flavonoid and lignan exposures were assessed as continuous variables after a log2 transformation ( fig . 1 and supplementary fig . 2 ) . no statistically significant heterogeneity between countries was detected for the associations of total intake and intakes of subclasses of flavonoids and lignans with type 2 diabetes , except for flavanones ( i = 52.8% , p = 0.038 ) and flavones ( i = 53.3% , p = 0.036 ) ( supplementary fig . no interactions were found with sex ( p for interaction = 0.609 ) , bmi ( p = 0.680 ) , or smoking status ( p = 0.526 ) for total flavonoid intake . hrs ( and 95% cis ) for incident type 2 diabetes for a doubling of total flavonoid ( a ) and lignan ( b ) intakes across countries in the interact study . the pooled hr is based on a random - effects meta - analysis using prentice - weighted cox regression analysis with age as the underlying time scale ( model 4 ; see statistical analysis section ) ; stratified by center ; and adjusted for sex , educational level , smoking status , physical activity levels , bmi , total energy , and intakes of alcohol , red meat , processed meat , sugar - sweetened soft drinks , coffee , fiber , vitamin c , and magnesium . in sensitivity analyses ( supplementary table 2 ) , similar results were observed after the exclusion of diabetes case subjects in whom type 2 diabetes had been diagnosed within the first 2 years of follow - up or participants with prevalent cardiovascular diseases . when family history of diabetes was added in model 4 , after the exclusion of 84 non - case subjects from the subcohort with an hba1c level 6.5% ( 48 mmol / mol ) at baseline , the results were almost identical . in this large european case - cohort study , an inverse trend between dietary total flavonoid intake and incidence of type 2 diabetes was observed . flavanols , including flavan-3-ol monomers and flavonols , were the flavonoid subclasses significantly related to a lower hazard of type 2 diabetes . to date , there are only two large u.s . cohort studies that have evaluated the association between the total flavonoid intake and incident type 2 diabetes , each using a different update of the u.s . department of agriculture database on flavonoids ( 22 ) . only the study using the database release 2.1 ( year 2007 ) observed a consistent inverse association between intake of anthocyanidins and type 2 diabetes risk ( 8,11 ) . this is in line with the crude , but not the multivariable adjusted , findings in our study , based on the database version from 2007 . this inconsistency could be due to the different dietary intakes between studies ; in our study , the median anthocyanidin intake in the first quintile ( 7.1 mg / day ) was similar to that in the third quintile ( 8.1 mg / day ) in the u.s . study , the hrs were almost identical for the third ( hr 0.87 [ 95% ci 0.800.94 ] ) , fourth ( hr 0.88 [ 95% ci 0.830.94 ] ) , and fifth quintiles ( hr 0.85 [ 95% ci 0.800.91 ] ) compared with the first quintile ( 8) . this suggests that the lower risk of type 2 diabetes due to intake of anthocyanidins might reach a plateau at a certain intake level . two other prospective studies have assessed the relationships between the intake of some flavonoid subclasses and the risk of the development of type 2 diabetes ( 9,10 ) . the u.s . study reported no association with intake of either flavonols or flavones ( 9 ) ; however , the finnish study reported inverse significant trends for two individual flavonols ( 10 ) , as in our study . these differences in the results for intakes of flavonols and flavanols between european and u.s . studies could be a result of european countries having approximately twice the intake compared with the u.s . ( 8,21 ) . in both asian studies , inverse associations with isoflavone intakes were reported ( 12,13 ) , but not in western studies ( 8,11 ) . asian countries still have the highest isoflavone intakes worldwide ( 10-fold higher than in european countries ) ( 20,30 ) , which may explain the differences observed in association with type 2 diabetes between asian and western countries . in our study , there was no association between lignan intake and risk of type 2 diabetes , although in a u.s . study , lignan levels were significantly associated with a lower fasting insulin level ( 31 ) . indeed , in two recent experimental studies lignans have been associated with an improvement of glucose homeostasis by increasing glucose disposal rates and enhancing hepatic insulin sensitivity ( 14 ) and an inhibition of -amylase activity ( 15 ) . the main food sources of flavonoids were fruits and vegetables , tea , and wine . these foods ( 2,32,33 ) , as well as the mediterranean diet , a dietary pattern based on flavonoid - rich foods ( e.g. , fruits and vegetables , olive oil , and moderate wine consumption ) ( 3 ) were associated with a reduced risk of type 2 diabetes in the epic - interact study . studies , where anthocyanidin - rich foods ( blueberries and apples / pears ) ( 8) and wine consumption ( 11 ) , a rich source of anthocyanidins and flavanols , were inversely associated with type 2 diabetes risk . notably , after adjustment for potential compounds co - occurring in flavonoid - rich foods , such as fiber , vitamin c , magnesium , and alcohol , associations between flavonoids and the risk of type 2 diabetes were still statistically significant in the current study , suggesting that it is unlikely that these compounds confound or mediate the association between intake of flavonoids and type 2 diabetes risk . the potential mechanisms underlying these inverse associations between flavonoids and type 2 diabetes risk may include the modulation of the postprandial glucose levels by reducing the activity of digestive enzymes ( e.g. , -amylase and -glucosidase ) ( 34 ) and decreasing the active transport of glucose across intestinal brush border membrane , inhibiting sodium glut2 ( 35 ) . furthermore , some flavonoid - rich extracts improved hyperglycemia and insulin sensitivity in type 2 diabetic mice via activation of amp - activated protein kinase and accompanied by an upregulation of glut4 ( 36 ) . in vitro , flavonoids also had a protective effect on pancreatic -cells by reducing the inducible form of nitric oxide synthase gene expression mediated through the suppression of nuclear factor-b and c - jun nh2-terminal kinase signaling pathways ( 37,38 ) . other antioxidant , anti - inflammatory , and antiangiogenic activities of flavonoids may also contribute to their potential protective effect against type 2 diabetes ( 5 ) . strengths of the current study include the multicenter design and the large sample size at recruitment , from which a large number of verified incident cases of type 2 diabetes accrued during 3.99 million person - years of follow - up . this study also includes a wide variation in flavonoid and lignan intakes among participants in eight european countries . furthermore , we were able to control for a number of plausible confounders and factors that may mask the etiological pathway of the association between flavonoid and lignan intake and type 2 diabetes . in all sensitivity analyses , the associations were almost identical , denoting the robustness of our results . limitations of the current study included the use of a single baseline assessment of diet and other lifestyle variables . in addition , our results may be influenced by measurement errors of the dietary questionnaires that may have attenuated our findings , although country - specific validated questionnaires for some flavonoid - rich foods , such as fruits , vegetables , tea , and wine ( 17,18 ) , were used . furthermore , flavonoid and lignan intakes are likely to be underestimated since the flavonoid database was incomplete ( although an extensive common database was used ) ( 20,21 ) and herb / plant supplement intakes were omitted in these analyses ( up to 5% in denmark , the highest consumer country ) ( 39 ) . nutritional biomarkers offer an alternative and objective method for estimating dietary intake and provide more accurate measures than self - reported questionnaires . to date , there are only a few validated biomarkers of flavonoid and lignan intakes , so further research in this field is warranted ( 40 ) . however , we were unable able to evaluate the association between the intakes of other polyphenols , such as phenolic acids and stilbenes , and type 2 diabetes because data on these are not yet available in the epic cohort . moreover , the association of dietary intakes of flavonoids and lignans with type 2 diabetes risk might be susceptible to confounding since high flavonoid and lignan intake reflects a healthier lifestyle . in our models , we have adjusted for other determinants of healthy lifestyle ; however , possible residual confounding can not be excluded . in conclusion , this large case - cohort study conducted in eight european countries supports a role for dietary intake of flavonoids in the prevention of type 2 diabetes in men and women . high total intakes of flavonoids , flavanols , flavan-3-ol monomers , and flavonols were associated with a 10 , 18 , 27 , and 19% lower risk , respectively , of type 2 diabetes . these results highlight the potential protective effect of eating a diet rich in flavonoids ( a dietary pattern based on plant - based foods ) on type 2 diabetes risk .	objectiveto study the association between dietary flavonoid and lignan intakes , and the risk of development of type 2 diabetes among european populations.research design and methodsthe european prospective investigation into cancer and nutrition - interact case - cohort study included 12,403 incident type 2 diabetes cases and a stratified subcohort of 16,154 participants from among 340,234 participants with 3.99 million person - years of follow - up in eight european countries . at baseline , country - specific validated dietary questionnaires were used . a flavonoid and lignan food composition database was developed from the phenol - explorer , the u.k . food standards agency , and the u.s . department of agriculture databases . hazard ratios ( hrs ) from country - specific prentice - weighted cox regression models were pooled using random - effects meta-analysis.resultsin multivariable models , a trend for an inverse association between total flavonoid intake and type 2 diabetes was observed ( hr for the highest vs. the lowest quintile , 0.90 [ 95% ci 0.771.04 ] ; p valuetrend = 0.040 ) , but not with lignans ( hr 0.88 [ 95% ci 0.721.07 ] ; p valuetrend = 0.119 ) . among flavonoid subclasses , flavonols ( hr 0.81 [ 95% ci 0.690.95 ] ; p valuetrend = 0.020 ) and flavanols ( hr 0.82 [ 95% ci 0.680.99 ] ; p valuetrend = 0.012 ) , including flavan-3-ol monomers ( hr 0.73 [ 95% ci 0.570.93 ] ; p valuetrend = 0.029 ) , were associated with a significantly reduced hazard of diabetes.conclusionsprospective findings in this large european cohort demonstrate inverse associations between flavonoids , particularly flavanols and flavonols , and incident type 2 diabetes . this suggests a potential protective role of eating a diet rich in flavonoids , a dietary pattern based on plant - based foods , in the prevention of type 2 diabetes .
You are an expert at summarizing long articles. Proceed to summarize the following text: glycogen storage disease type ii ( gsdii ) , also known as pompe s disease or acid maltase deficiency , is a rare , autosomal recessive , progressive neuromuscular disease caused by the deficiency of lysosomal acid -glucosidase ( gaa ) or acid maltase . the enzyme catalyzes the hydrolysis of -1,4 and -1,6 links of glycogen and its deficiency leads to intra - lysosomal accumulation of glycogen . in patients with the classic infantile form , the deposition of glycogen in the heart , skeletal , and respiratory muscles causes severe cardiomyopathy , hypotonia , and respiratory failure , typically leading to death within the first year of age . in late - onset gsdii patients glycogen deposition is confined mainly to skeletal and respiratory muscles , causing progressive limb - girdle myopathy and respiratory insufficiency . a considerable number of patients become wheelchair dependent and may require assisted ventilation later in life . an inverse correlation is usually observed between the amount of residual gaa activity and disease severity , and in general the symptoms do not emerge until the gaa activity remains above 30% of average normal activity . until recently , no effective therapy for gsdii patients was available , even if physical activity alone or in parallel with an high - protein and low - carbohydrate dietary regime has been demonstrated to improve quality of life and motor function . enzyme replacement therapy ( ert ) with recombinant human -glucosidase became available in 2000 , and currently a number of studies have been published on the efficacy and safety of ert in gsdii disease . clinical studies in infants have shown that ert led to improvement in skeletal and cardiac muscle function and to increased survival in many patients . few studies on the efficacy of this treatment in older children and adults have been published so far [ 69 ] . overall these studies demonstrated that ert is associated , over a 12- to 36-month period , with a stabilization of pulmonary function and with an improved exercise tolerance , as estimated by the 6-min walking test ( 6mwt ) , which has been demonstrated to be an appropriate outcome measure . however , besides being intrinsically imprecise , the 6mwt does not provide specific information on the function of the different organs and systems involved in exercise , or the mechanism of exercise limitation . insights into these issues , together with a more precise quantification of the exercise intolerance , could derive from standard cardiopulmonary exercise testing , associated with measurements of pulmonary o2 uptake , cardiac and skeletal muscle functions carried out during incremental exercise up to voluntary exhaustion . in the present study we report a 12-month follow - up of four late - onset gsdii patients who underwent ert . in particular , data related to cardiovascular and metabolic responses to exercise and respiratory function are provided . all measurements were non - invasive , so they could be easily repeated as a function of time . four late - onset gsdii patients ( 2 males and 2 females , 45 6 years ) were investigated . ert with recombinant human -glucosidase ( myozymegenzyme corporation , cambridge , mass ) was administered intravenously at a dose of 20 mg / kg every 2 weeks . before treatment ( before ) and after 12 months of ert ( after ) the patients were evaluated in our laboratory . the subject were fully informed of any risk and discomfort associated with the experiments before giving their written informed consent to participate to the study , which was approved by the local institutional ethics committees . all procedures were in accordance with the recommendations found in declaration of helsinki ( 2000 ) of the world medical association . pulmonary function variables , forced vital capacity ( fvc ) and forced expiratory volume in 1 s ( fev1 ) , were measured with the patients in sitting position by a spirometer . exercise tolerance was assessed on an electronically braked cycle ergometer ( corival , lode , the netherlands ) . an incremental exercise was performed : after sitting for a few minutes at rest the patient performed at 1530 w for 5 min , and thereafter the workload was increased by 510 w ( according to the subject s estimated level of physical fitness ) every minute until voluntary exhaustion was reached . for cardiovascular , gas exchange , and muscle oxygenation variables mean values were calculated during the last 30 s of each load . the highest values reached by the patient were taken as peak values.pulmonary ventilation ( ve , expressed in btps ) , o2 uptake ( vo2 ) , and co2 output ( vco2 ) , both expressed in stpd , were determined breath - by - breath by a metabolic cart ( sensormedics vmax29c ; the netherlands ) . expiratory flow measurements were performed by a mass flow sensor ( hot wire anemometer ) , calibrated before each experiment by a 3-l syringe at three different flow rates . tidal volume and ve were calculated by integration of the flow tracings recorded at the mouth of the subject . vo2 and vco2 were determined by continuously monitoring partial pressures of oxygen ( po2 ) and carbon dioxide ( pco2 ) at the mouth throughout the respiratory cycle and from established mass balance equations , after alignment of the expiratory volume and expiratory gases tracings and a / d conversion . calibration of o2 and co2 analyzers was performed before each experiment by utilizing gas mixtures of known composition . gas exchange ratio ( r ) was calculated as vco2/vo2 . heart rate ( hr ) was monitored from the electrocardiogram ; stroke volume ( sv ) was estimated beat - by - beat by impedance cardiography ( physio flow , manatec , paris , france ) . the accuracy of this device has been previously evaluated during incremental exercise in healthy subjects against the direct fick method . oxygenation changes in a superficial portion of the vastus lateralis muscle were evaluated by near - infrared spectroscopy ( nirs ) . a portable nir single - distance continuous - wave photometer ( heo-100 , omron , japan ) , the instrument provides separate measurements of changes in deoxygenated hb and mb concentrations , as well as of oxygenated hb and mb concentrations , expressed in arbitrary units . further details on the method can be found in a previous article by our group . for a detailed discussion regarding advantages and limitations of nirs measurements in skeletal muscle , concentration changes of deoxygenated hb + mb ( [deoxy(hb + mb ) ] ) , with respect to an initial value arbitrarily set equal to zero , were calculated and expressed in arbitrary units . [deoxy(hb + mb ) ] was taken as an oxygenation index , since this variable is relatively insensitive to changes in blood volume . [deoxy(hb + mb ) ] data were expressed as a percentage of the values determined after the exercise by obtaining a maximal deoxygenation of the muscle , by pressure cuff inflation ( at about 300 mmhg ) carried out at the inguinal crease of the thigh ( subject in the sitting position on the cycloergometer ) , for a few minutes until [deoxy(hb + mb ) ] increase reached a plateau . results were expressed as mean values standard deviation ( x sd ) . the statistical significance of differences between means was checked by paired students t - test . no side effects of therapy were observed . we did not use validated scales to assess quality of life , however , the patients reported a progressive subjective improvement of daily performance , and particularly less fatigability both at work and during activities of daily living . after 9 months of treatment one of the patients spontaneously started to perform 23 times per week short sessions ( about 15 min ) of exercise on a stationary bicycle . 1 . in before fvc and fev1 were , on the average , 73% and 74% of the predicted normal value , respectively . for both variables , either expressed in l or as a percentage of the predicted value , ert was associated with a slight improvement , which , however , did not reach statistical significance , presumably as a consequence of the low number of patients ( see also below ) . the patients tolerated the exercise testing relatively well and none of them complained of significant discomfort , pain or delayed onset muscle soreness . peak values of work rate , vo2 , co and ( [deoxy(hb + mb ) ] are shown in fig . 2 ( also for [deoxy(hb + mb ) ] peak the data were not available for one of the patients ) . vo2peak increased by 10% from before ( 1.01 0.42 l / min , or 17.2 4.4 ml / kg / min ) to after ( 1.14 0.36 l / min , or 19.7 3.5 ml / kg / min ) . on the average vo2peak values obtained in after correspond to 5 mets , or 5 times the resting energy expenditure ( 1 met = 3 . ml o2 kg min ) , and to 59% of the sex- and age - predicted peak value . the increase in vo2peak was associated with a clear tendency for an increase in copeak , from 12.3 5.3 to 14.8 4.5 l / min . both hrpeak ( which increased from 134 16 beats / min to 145 25 beats / min ) and peak stroke volume ( from 91 29 to 101 21 ml ) contributed to the increased copeak . for copeak , hrpeak and svpeak the increase , in after vs. before , the same can be said for [deoxy(hb + mb ) ] peak , taken as an estimate of skeletal muscle fractional o2 extraction ( for details see ) . the major finding of the present study is that in patients with glycogen storage disease type ii enzyme replacement therapy is associated , over a 12-month period , with a stabilization of pulmonary function and with a mild improvement of exercise tolerance . two widely accessible and simple parameters , fvc and fev1 , were used to monitor respiratory function . a nonsignificant trend in fvc and fev1 improvement these findings are consistent with those recently reported in larger ert trials on late - onset gsdii patients treated for 12 , 18 and 36 months . the stabilization or the slight improvement of pulmonary function observed in our study , as well as in the previously mentioned ones , is in contrast to the progressive deterioration that characterizes the natural course of the disease ( annual decline of almost 23% in the percentage of predicted fvc ) . significantly higher values of vo2peak after were associated with ( and presumably were responsible for ) a significant improvement of exercise tolerance , as shown by the significantly higher peak work rate values . the increase in vo2peak was associated with an improvement of copeak and [deoxy(hb + mb ) ] peak . in two of the patients [deoxy(hb + mb ) ] peak values were markedly lower than the values observed by our group in healthy subjects , but higher than those obtained on patients with mitochondrial myopathies or myophosphorylase deficiency . + mb ) ] peak values were substantially unchanged compared with those determined at rest ; in this patient , characterized by the lowest work rate and vo2peak values , the variable was unaffected by ert . as nicely discussed by poole et al . , it would be an oversimplification to interpret skeletal muscle fractional o2 extraction simply as a result of muscle factors . the enhanced peak fractional o2 extraction described with training in two of our patients may indeed be the result of a combination of factors such as increased bulk blood flow and o2 delivery ; enhanced vasodilation and capillary recruitment ; improved intramuscular matching of o2 delivery and o2 utilization ; enhanced peripheral o2 diffusion ; improved endothelial function ; reduced levels of inflammatory , catabolic and pro - apoptotic mediators and oxidative stress ; increased mitochondrial volume density and activity of oxidative enzymes ; enhanced oxidative phosphorylation , etc . all these factors , which could be at least in part related to a decreased disease - induced damage in muscle tissue , could explain ( and be a result of ) the increased exercise tolerance . in late - onset gsdii patients skeletal muscle pathology is extremely heterogeneous , ranging from substantially unaffected fibers to a complete destruction of contractile machinery . the pathogenic mechanisms of muscle damage are still under debate , but autophagy is increasingly identified as a pivotal contributor to muscle destruction and mitochondrial abnormalities have been repeatedly found . regarding the response to ert , clinical trials have indicated that ert is effective in glycogen clearance in cardiac muscle , whereas a reversal of the damage in skeletal muscle has not always been achieved , and highly variable responses between patients should be expected . unfortunately , muscle biopsies were not performed in the present study , thus we have no data of the degree of muscle damage in the patients in before and in after . as discussed above , the subjective improvement observed by one of the patients during the first few months of ert induced her to spontaneously adopt a home - based light exercise training protocol . thus , it can not be excluded that the beneficial effects described in the present study after ert can be attributed , at least in part , to the increased level of physical activity . recently , evidence has been provided that ert and exercise training could have additive positive effects on these patients exercise tolerance and , ultimately , on their quality of life . for some of the variables determined in the present study ( spirometry data , copeak , [deoxy(hb + mb ) ] peak ) the observed increases did not reach statistical significance . this could be attributed to the low number of patients , which does not allow us to exclude the possibility of a type 2 error . thus , the findings need to be validated on a larger group of patients and with a longer follow - up period . in conclusion , our findings showed that in late - onset gsdii patients ert is associated with a mild improvement of pulmonary function and exercise tolerance over a 12 month period . the improved exercise tolerance seems associated with improvements both in cardiovascular and in skeletal muscle functions . the findings need to be validated on a larger group of patients and with a longer follow - up period . in addition , the results highlight the role that cardiopulmonary exercise testing , with simultaneous non - invasive measurements of pulmonary o2 uptake , cardiac output and skeletal muscle oxygenation , can play in the assessment and follow - up of late onset gsdii patients .	enzyme replacement therapy ( ert ) has recently became available for patients with glycogen storage disease type ii . previous studies have demonstrated clinical efficacy of enzyme replacement therapy , however , data on physiological variables related to exercise tolerance are scarce . four glycogen storage disease type ii late - onset patients ( 45 6 years ) performed an incremental exercise on a cycle ergometer , up to voluntary exhaustion , before ( before ) and after 12 months of ert ( after ) . peak workload , oxygen uptake , heart rate , cardiac output ( by impedance cardiography ) and vastus lateralis oxygenation indices ( by continuous - wave near - infrared spectroscopy , nirs ) were determined . peak workload and oxygen uptake values significantly increased during ert ( 54 30 vs. 63 31 watt , and 17.2 4.4 vs. 19.7 3.5 ml / kg / min , respectively , in before vs. after ) . on the other hand , for both peak cardiac output ( 12.3 5.3 vs. 14.8 4.5 l / min ) and the nirs - determined peak skeletal muscle fractional o2 extraction , expressed as a percentage of the maximal values during a transient limb ischemia ( 30 39% vs. 38 28% ) , the observed increases were not statistically significant . our findings suggest that in glycogen storage disease type ii patients enzyme replacement therapy is associated with a mild improvement of exercise tolerance . the findings need to be validated during a longer follow - up on a larger group of patients .
You are an expert at summarizing long articles. Proceed to summarize the following text: preswallow pharyngeal bolus presence is viewed on videofluoroscopic swallow study ( vfss ) or fiberoptic endoscopic examination of swallowing ( fees ) amongst many patients presenting with oropharyngeal dysphagia . there are two main causes for this presentation : poor oral bolus containment with premature bolus spillage and/or a delayed pharyngeal swallow trigger . poor oral bolus containment results in passive or ineffective movement of a liquid or viscous bolus from the oral cavity into the pharynx prior to pharyngeal swallow onset . by definition this occurs whilst the oral preparatory or oral stage of swallow is still underway , that is , before or during lingual propulsion . separately , a delay in the pharyngeal swallow trigger is defined by a failure of a coordinated and timely pharyngeal response following a purposeful transfer of the bolus into the pharynx . poor oral containment leading to premature bolus spillage can occur in isolation or in combination with a delayed pharyngeal swallow trigger [ 13 ] . high resolution manometry with impedance ( hrmi ) with automated pressure flow analysis ( pfa ) is a new method to diagnostically interpret pharyngeal and ues function . pressure sensors detect activity of swallow musculature whilst impedance electrodes provide measures which indicate bolus flow . pfa derives a range of swallow metrics that indicate bolus flow timing , intrabolus pressure , contractile vigour , bolus presence , and ues luminal diameter , making it possible to measure and describe the function of different mechanical components of pharyngeal swallowing . a global swallow risk index ( sri ) generated from pfa metrics as a means to amplify dysfunction has shown to correlate with the presence of aspiration and/or postswallow residue as seen on videofluoroscopy [ 4 , 5 ] . following on from this previous work , the purpose of this study was to use hrmi in combination with automated pfa to objectively describe pressure - flow patterns in the pharynx and ues in the event of preswallow pharyngeal bolus presence as seen on videofluoroscopy . we hypothesised that specific pfa metrics would be altered in patients with preswallow pharyngeal bolus presence compared to patients without preswallow pharyngeal bolus presence and controls . the aim of this study was to identify the altered pfa metrics which may provide a means to describe functional changes in the pharynx in the event of preswallow pharyngeal bolus presence . we analysed vfss investigations performed in 40 adult patients with broad dysphagia ( 24 males , mean age 46 yrs , and age range 2395 yrs ) and 8 adult controls ( 3 males , mean age 38 yrs , and age range 2447 yrs ) . at the time of initial investigation , all subjects were enrolled in study protocols that were approved by the research ethics committee , university hospital leuven , belgium . in patients with dysphagia , underlying diseases / conditions were identified through a review of medical records . eighteen patients had a neurological history ( 10 patients were post stroke , 2 had parkinson 's disease , 1 huntington 's disease , 1 multiple sclerosis , 2 dementia , 1 spina bifida and 1 post - neurosurgery ) . of the remaining four patients 1 was post - cervical surgery , 1 wegener disease , 1 postseptic shock , and 1 diabetes . this database of patients with broad dysphagia has previously been reported on [ 49 ] , and the diverse clinical presentations and dysphagia severity have been purposeful in order to concept - test pfa metrics in relation to radiological measures from vfss . previously this has included aspiration status , postswallow residue , ues opening , and in this case preswallow pharyngeal bolus presence . studies were performed in the radiology department , university hospital leuven , with a 3.2 mm diameter solid state high resolution manometry and impedance catheter incorporating 25 1 cm - spaced pressure sensors and 12 adjoining impedance segments , each of 2 cm ( unisensor ag catheter , attikon switzerland ) . subjects were intubated after topical anaesthesia ( lignocaine spray ) and the catheter was positioned with sensors straddling the entire pharyngoesophageal segment ( velopharynx to proximal esophagus ) . pressure and impedance data were acquired at 20 hz ( solar gi acquisition system , mms , netherlands ) with the subject sitting upright . most subjects were tested with at least 5 boluses in the lateral view : liquid ( x3 ) , semisolid ( x1 ) , and solid boluses ( x1 ) . a standard liquid contrast material ( micropaqueh ) was given as liquid bolus and used with thickener ( thick & easy ) for semisolid boluses . a low osmotic hydrosoluble iodium compound ( ultravisth ) the bingham viscosity of the liquid barium ( micropaqueh ) was 0.22 pascal seconds ( pas ) , 4.50 pas for the semisolid bolus . all controls were given boluses of 10 ml volume while patients were given either 5 ml or 10 ml volumes as determined on clinical grounds by the attending specialist . solid boluses consisted of a 4 cm piece of bread soaked in the appropriate radiological marker which was chewed and swallowed . all swallows were prompted and the first swallow following bolus administration was marked for analysis . continuous videofluoroscopy sequences ( 25 frames / sec ) of swallows were analysed by a speech pathologist ( author lara ferris ) who was not present during acquisition and was blinded to study functional measures , patient history , and clinical reports . each primary bolus swallow was reviewed to determine movement of the bolus and the level reached from the oral cavity into the distal pharynx prior to the onset of the pharyngeal swallow , defined by onset of rapid laryngeal excursion [ 1 , 10 , 11 ] . the levels reached were defined as follows : still in the oral cavity , base of tongue , valleculae , or pyriform sinuses . based on the results for all the analysed swallows patients were classified as follows : group 1 : patients who never demonstrated bolus movement to the pyriform sinuses prior to swallow onset.group 2 : patients who demonstrated bolus movement to the pyriform sinuses at least once prior to swallow onset . group 1 : patients who never demonstrated bolus movement to the pyriform sinuses prior to swallow onset . group 2 : patients who demonstrated bolus movement to the pyriform sinuses at least once prior to swallow onset . note : amongst controls , at swallow onset bolus movement was observed in the mouth or at the base of tongue for all swallows . pharyngeal pfa was performed using automated impedance manometry software ( aimplot ) to calculate pfa metrics and two global indices which have been previously validated against vfss , namely , the sri , indicative of global dysfunction , and the integrated ratio of nadir impedance to impedance ( izn / z ) , indicative of postswallow residue . the calculations used to derive pfa metrics have been previously described [ 49 , 12 ] . in brief , pressure impedance recordings are displayed as pressure topography plots with embedded impedance recordings which show bolus flow movements , the pharyngeal stripping wave , and relaxation and movement of the ues pressure zone ( figure 1(b ) ) . on selection of specific landmarks on the pressure topography space - time plot ( with embedded impedance recordings ) , specific regions of interest ( roi ) are mapped . the landmarks identified are ( 1 ) time of onset of pharyngeal swallow , ( 2 ) position of the ues proximal margin postswallow , and ( 3 ) position of the velopharynx during the swallow ( numbered 13 in figure 1(b ) ) . there are three roi encompassing ( 1 ) the pharynx , ( 2 ) distal pharynx , and ( 3 ) ues . within each of the roi specifically , identification of peak pressure defines the maximum contraction in space and time , the nadir impedance ( nadimp ) defines the centre of the swallowed bolus in space and time ( figures 1(b ) and 1(c ) ) , and the pressure at nadir impedance ( pnadimp , figure 1(c ) ) defines pharyngeal intrabolus pressure . the time interval from nadir impedance to peak pressure ( tnadimp to peakp , figure 1(c ) ) measures the time from bolus passage to pharyngeal contraction ; the flow interval ( fi ) correlates with pharyngeal bolus transit time . the ues nadir impedance ( ues nadimp ) is measured as a correlate of ues opening diameter and the ues intrabolus pressure ( ues ibp ) is measured using the established method of ghosh et al . the swallow risk index ( sri ) was empirically derived and designed to amplify difference in swallow metrics previously shown to be altered in relation to swallow dysfunction and aspiration risk [ 4 , 5 ] . these validation studies using concurrent vfss showed that the sri for liquid and viscous swallows is significantly higher in patients demonstrating penetration - aspiration compared to patients with no penetration or aspiration [ 4 , 5 ] . therefore the sri quantifies the overall level of swallowing dysfunction potentially predisposing to aspiration risk [ 4 , 5 ] . a postswallow residue score was designed using the integrated ratio of nadir impedance to impedance ( izn / z ratio ) which relates postswallow impedance to the impedance during bolus passage . data gathered from multiple swallows were averaged for each subject . only liquid boluses ( 5 or 10 mls ) were assessed . data distribution across the study cohort was nonparametric and therefore medians ( interquartile range ) are presented . mann - whitney u tests were used to compare controls and patients grouped on vfss assessment with bonferroni correction for multiple comparisons , p < 0.017 . amongst controls a total of 24 liquid swallows were analysed and 102 swallows were analysed from 40 broad dysphagia patients . controls showed bolus in the mouth or at the base of tongue at the time of swallow onset in all cases . 20 of 40 patients showed bolus to the pyriform sinuses at least once and this was the most frequent bolus position at swallow onset in 75% of this patient group . as shown in figure 2 using mann - whitney u tests , in this cohort of broad dysphagia patients the sri , a global measure of swallowing dysfunction , was higher in relation to evidence of bolus to the pyriform sinuses prior to the pharyngeal swallow onset compared to controls ( p < 0.001 ) , and a trend was observed compared to patients who did not spill to the pyriform sinuses ( p = 0.02 ) . the tnadimp - peakp ( poor ability for the bolus to be propelled ahead of the pharyngeal stripping wave ) was shorter in both patient groups ( group 1 p < 0.002 and group 2 p < 0.001 ) compared to controls ; the flow interval ( suggesting extended pharyngeal bolus dwell time ) was longer in both patient groups ( group 1 p < 0.001 and group 2 p < 0.002 ) compared to controls ; and the izn / z ( postswallow residue metric ) was significantly higher in both patient groups ( group 1 p < 0.000 and group 2 p two individual metrics in this data differentiate the patient groups : group 1 : patients without preswallow bolus presence to the pyriform sinuses prior to swallow onset and group 2 : patients with preswallow bolus presence to the pyriform sinuses prior to swallow onset . pharyngeal mean impedance ( correlating with reduced pharyngeal distension ) was significantly higher ( p < 0.002 ) in group 2 compared to group 1 and controls ( p < 0.001 ) and the ues mean impedance ( correlating with reduced ues opening ) was higher ( p < 0.000 ) in group 2 compared to group 1 and controls ( p < 0.001 ) . collectively this pattern of pressure - flow differences is consistent with impairment of the mechanisms that drive bolus propulsion , pharyngeal distension , and relaxation and opening of the ues in accordance with laryngeal excursion . as has been shown previously , a higher ues impedance recording correlates with reduced ues opening diameter and therefore , in the case of preswallow pharyngeal bolus presence to the pyriform sinuses , an explanation for the increase in pharyngeal and ues mean impedance : bolus movement to the pyriform sinuses before swallow onset leads to a greater loss of bolus volume of the remaining propelled bolus . a smaller bolus volume for propulsion results in reduced distension of the pharynx and ues during the swallow . vfss and fees are currently the most widely used instrumental swallow assessments ; however differential diagnosis of oropharyngeal dysphagia can be difficult based on visualisation alone . the use of pharyngeal manometry for the assessment of dysphagia has its limitations and has been described by nativ - zeltzer et al . in 2012 ; however the integration of concurrent high resolution manometry with impedance allows for dynamic swallow function assessment , and its potential to assist in the evaluation of swallow function is beginning to emerge , as has been demonstrated in this and a number of other recent studies [ 49 , 12 ] . within the adult population , there is discussion in the literature regarding normal variability for the presence of the bolus lower in the pharynx prior to swallow onset [ 1519 ] . however , the spillage or propulsion of all or part of the bolus to the pyriform sinuses prior to pharyngeal swallow trigger is a pathological event [ 13 , 20 ] suggesting markedly altered mechanics of bolus transport through the pharynx . the clinical relevance in detecting preswallow pharyngeal bolus presence lies in the fact that recognising the causes for bolus presence in the pharynx prior to pharyngeal swallow onset is important for treatment of oropharyngeal dysphagia . our findings demonstrate how specific pfa metrics are altered in relation to a general pathological observation of preswallow pharyngeal bolus presence in dysphagia patients . this pilot study presents specific pressure flow analysis metrics ( using integrated high resolution manometry and impedance ) which are significantly associated with a known pathological presentation of oropharyngeal dysphagia , that is , preswallow pharyngeal bolus presence to the pyriform sinuses . these results provide reason to further explore the potential differences in pressure flow analysis metrics that may distinguish the causes for preswallow pharyngeal bolus presence , that is , poor oral containment and/or delayed pharyngeal trigger pressure flow analysis with aimplot software deriving metrics and a global measure of dysfunction , the swallow risk index , have the potential to guide diagnosis and treatment of patients with oropharyngeal dysphagia and may be used to track patient changes in swallow function over time .	objectives . preswallow pharyngeal bolus presence is evident in patients with oropharyngeal dysphagia . pressure flow analysis ( pfa ) using high resolution manometry with impedance ( hrmi ) with aimplot software is a method for objective interpretation of pharyngeal and upper esophageal sphincter ( ues ) pressures and bolus flow patterns during swallowing . this study aimed to observe alterations in pfa metrics in the event of preswallow pharyngeal bolus presence as seen on videofluoroscopy ( vfss ) . methods . swallows from 40 broad dysphagia patients and 8 controls were recorded with a hrmi catheter during simultaneous vfss . evidence of bolus presence and level reached prior to pharyngeal swallow onset was recorded . aimplot software derived automated pfa functional metrics . results . patients with bolus movement to the pyriform sinuses had a higher sri , indicating greater swallow dysfunction . amongst individual metrics , tnadimp to peakp was shorter and flow interval longer in patient groups compared to controls . a higher pharyngeal mean impedance and ues mean impedance differentiated the two patient groups . conclusions . this pilot study identifies specific altered pfa metrics in patients demonstrating preswallow pharyngeal bolus presence to the pyriform sinuses . pfa metrics may be used to guide diagnosis and treatment of patients with oropharyngeal dysphagia and track changes in swallow function over time .
You are an expert at summarizing long articles. Proceed to summarize the following text: the effects of various treatments on outcome have been studied extensively both in psychotherapy and substance abuse treatment [ 1 , 2 ] . the investigation of between - therapist variation in outcome has been infrequent , although it has proved an important factor in both disciplines [ 3 , 4 ] . other little - studied factors are the gender and gender roles of the therapist and their impact on treatment effectiveness . it refers to the set of attitudes and behaviors socially expected of the members of a particular gender . according to bem 's theory , a traditionally gender - typed person is highly attuned to the cultural definitions of gender - appropriate behavior and uses such definitions as the ideal standard against which her or his own behavior is to be evaluated . it means that a person is both masculine and feminine ; these traits are not mutually exclusive . research on psychotherapy has indicated that therapy effectiveness may be predicted on the basis of gendered factors . in the substance abuse field , there is evidence that therapist 's attitudes toward clients vary according to the client 's gender . dejong et al . demonstrated that therapists were more confrontational and critical with male clients , while female clients received more empathy and support . the male clients were seen by the therapists as threatening , in which case the attitudes became confrontational , while female clients were seen as submissive , which led to empathetic attitudes . the therapist 's own gender had no impact on the attitudes toward clients in the study by dejong et al . by contrast , findings on mental health professionals have indicated that males generate more stereotypical attitudes toward clients than do females [ 10 , 11 ] . these findings corroborate a study by bernstein and lecomte reporting that psychotherapist 's gender is an important factor for attitudes toward clients , males being more stereotypical than females . a small study by saarnio et al . showed that the clients of male therapists dropped out of inpatient substance abuse treatment significantly more frequently than did the clients of female therapists ( 20 versus 10% ) . unfortunately , a more detailed examination of the findings was not possible because no in - session data were collected . however , one possible explanation is that female therapists were more adept at avoiding alliance ruptures that easily lead to dropping out . this explanation is supported by a recent finnish study which found that female therapists in substance abuse treatment were significantly more empathetic and friendly toward clients than were their male colleagues . moreover , avoidance of excessive directiveness was considered more important by female therapists than by males . miller et al . demonstrated that therapist empathy explained as much as 67% of variance in treatment outcome ; in other words , the more empathetic the therapist , the better the outcome . miller et al . showed experimentally that the therapist 's style affects the client 's drinking after treatment . from among two experimental groups , the therapists in one group were instructed to work in a directive - confrontational style and the therapists in the other group in a client - centered style . the therapists in the second group followed the principles of the motivational interviewing ( mi ) in which emphasis is placed on a positive attitude toward the client , especially empathy . the directive - confrontational style caused significantly more opposition than did the client - centered style . the treatment results can be summed up as follows : the more the therapist confronted , the more the client drank one year after the treatment . mi can be hypothesized to be feminine rather than masculine in nature as in it avoidance of confrontation and excessive directiveness play important roles . it would be interesting to find out whether there are between - therapist differences in attitudes toward mi due to gender or gender roles . the purpose of the present study was to investigate the impact of therapist 's gender and gender roles on attitudes toward clients with different genders and sexual orientations . these were formulated as three questions : are there differences in masculinity , femininity , or androgyny between male and female therapists?are there between - therapist differences due to gender or gender roles in attitudes toward clients?are gender or gender roles connected with attitudes toward mi ? are there differences in masculinity , femininity , or androgyny between male and female therapists ? are there between - therapist differences due to gender or gender roles in attitudes toward clients ? the subjects ( n = 246 ) were finnish substance abuse therapists employed by the a - clinic foundation . the a - clinic foundation has treated clients with various addictions since 1955 , and today it provides about 40% of the substance abuse treatment in finland . an electronic questionnaire was sent via the internet to counselors , social workers , nurses , physicians , psychologists , and team leaders ( n = 546 ) . regardless of job title , they all had the same task , therapy with clients . out of those participating in the study , 29.7% were men ( n = 73 ) and 70.3% women ( n = 173 ) . the gender distribution was similar to that of the total personnel of the a - clinic foundation ( 22.2% men and 77.8% women ) . the age of the subjects varied between 24 and 63 years ( m = 44.0 ; sd = 10.0 ) , while the women ( m = 42.8 ; sd = 9.9 ) were significantly ( t244 = 3.3 ; p = 0.001 ) younger than the men ( m = 47.2 ; sd = 9.5 ) . on average , the female therapists had a higher level of professional education than the male therapists , even though the males had acquired university degrees more often than their female colleagues ( table 1 ) . among therapists in team leader position , male therapists reported more often being homosexual than did female therapists . according to a population - based finnish study , the proportion of individuals with gay , lesbian , or bisexual identities has been estimated to be 2.5% of males and 1.9% of females in the year 2007 . the question on mi was formulated as follows : in motivational interviewing one avoids directly telling the client what to do . how important do you consider this principle to be in substance abuse treatment ? the subjects were requested to use a five - point scale ( 1 = not so important5 = very important ) . the different gender roles of the subjects , which in this study included masculinity , femininity , and androgyny , were measured by the bem sex - role inventory ( bsri ) . for this purpose , a short version of the bsri consisting of 30 items i am assertive , was rated on a seven - point scale ( 1 = never or almost never true7 = almost or almost always true ) . both masculine and a high positive score indicates femininity and a high negative score indicates masculinity ; the closer the score is to zero , the more androgynous the person is . the alpha reliabilities of masculinity and femininity in the present study were of the same level as in the original study : 0.78 ( 0.86 ) and 0.81 ( 0.81 ) . despite having been developed over 30 years ago , the bsri continues to be extensively used in both research and clinical work . however , recent studies have reanalyzed and questioned the bsri 's factor structure and validity [ 23 , 24 ] . the attitudes of the therapists toward clients were measured with a vignette task , in which the subject had to associate adjectives with three different fictional client cases . the cases differed from each other only as regards the client 's gender and sexual orientation . the second vignette had to do with a heterosexual female , and the third vignette concerned a homosexual male . the vignette rating made use of 50 adjectives extracted from the adjective check list ( acl ) , which is commonly used to measure personality traits . , the acl has a wide range of uses and applications in both research and clinical work . the test , t - test , correlations , and analyses of variance ( anova , repeated measures , and manova ) were used . effect sizes were calculated with cohen 's d , defined as the difference between the means of female and male therapists , divided by the pooled standard deviation of these groups . first , the results on gender roles measured by the bsri were compared between male and female therapists . the raw scores were not converted to t - scores because they were based on normative data over 30 years old . the male therapists received a slightly higher mean score for masculinity ( m = 4.9 ; sd = 0.6 ) than the female therapists ( m = 4.8 ; sd = 0.6 ) . according to the t - test , the difference was not statistically significant ( t244 = 0.7 ; p = 0.5 ) . as for femininity , the means between males ( m = 5.2 ; sd = 0.6 ) and females ( m = 5.5 ; sd = 0.6 ) differed significantly ( t244 = 2.6 ; p = 0.01 ) from each other . however , effect size was small ( d = 0.3 ) . as regards the androgynous gender role , the male ( m = 0.3 ; sd = 0.8 ) and female therapists ( m = 0.6 ; sd = 0.8 ) differed significantly ( t244 = 2.5 ; p = 0.01 ) from each other . the mean score of female therapists deviated more from zero than that of men , so the male therapists were more androgynous than the women . in the next section of the questionnaire , the therapists had to select six adjectives that best described the client in each of the three vignettes . gender roles were not significantly connected with the ratings . instead , gender as such was a significant factor : female therapists had a more positive attitude than the males toward all cases ( figure 1 ) . a repeated analysis of variance indicated that the difference between the columns was significant ( f1,244 = 19.8 ; p = 0.000 ) . on the basis of the t - test , the difference between genders was greatest in attitudes toward the homosexual male client ( t244 = 5.4 ; p = 0.000 ) and smallest in attitudes toward the heterosexual female client ( t244 = 3.1 ; p = 0.002 ) . the genders also differed significantly ( t244 = 4.0 ; p = 0.000 ) from each other in their attitudes toward the heterosexual male client . effect sizes for vignettes were medium to large ( d = 0.8 ; 0.6 ; 1.1 ) . when controlling for background variables described in table 1 , the one - way analysis of variance revealed three variables on the basis of which the therapists differed significantly from each other as regards their attitudes toward the vignettes : professional education , job title , and technical eclecticism . when these variables and gender were used as independent variables and the combined score of the vignettes as a dependent variable in manova , only technical eclecticism ( f3,235 = 3.1 ; p = 0.03 ) and gender ( f3,235 = 7.4 ; p = 0.000 ) remained significant . in addition , anova indicated that the therapist 's marital status and length of therapy training interacted significantly with gender on the attitudes toward the vignettes ( table 2 ) . single men had more positive attitudes toward all vignettes than men in pair relationships . for women the inverse was true : female therapists in a pair relationship had a more positive attitude than single women toward all cases . as for lengthy therapy training , the attitudes of the male therapists who had completed lengthy training were more positive , while among the female therapists such training weakened the positive client images . the study also focused on the connections between gender , gender roles , and attitude toward mi . femininity had a significant positive correlation ( r = 0.18 ; p < 0.01 ) with attitude toward mi : as the therapist 's femininity increased , so did the preference for mi . mi was slightly more important for female therapists ( m = 4.0 ; sd = 0.7 ) than in males ( m = 3.8 ; sd = 0.7 ) . however , they did not differ significantly ( t244 = 1.3 ; p = 0.2 ) from each other . effect size was small ( d = 0.2 ) . when controlled for , mi usage per se the present study investigated the impact of therapist 's gender and gender roles on attitudes toward clients . the female therapists were significantly more feminine than the male therapists . however , masculinity was at the same level in both genders . the degree of androgyny was higher among the male therapists than among the females . female therapists were significantly more positive than male therapists in their attitudes toward all the cases in the vignette task . this finding indirectly supports saarnio 's conclusion : on the grounds of personality and interpersonal functioning , female therapists were keener on working according to motivational interviewing that therapist 's gender has no impact on attitudes toward clients . on the contrary , the vignette task result corroborates the findings for mental health professionals , indicating between - gender differences in attitudes toward clients [ 1012 ] . client 's gender was not a relevant factor for therapist 's attitudes : the vignette task scores were at the same level for both male and female cases . this finding also differs from the result of dejong et al . that depending on the client 's gender , substance abuse therapists will prefer different attitudinal stances . as to why the female therapists had the most positive attitudes toward the homosexual male client , no answer can be given on the basis of the present study . however , in an international comparison , there were population - level differences between the genders in attitudes toward homosexuals , particularly in the nordic countries . the topic is an important one , since some studies have indicated that homosexuals may have a higher risk of substance use disorders than heterosexuals . in addition , it has been demonstrated that homosexuals have difficulties in entering substance abuse services . we hypothesized mi to be feminine rather than masculine in nature as in it avoidance of confrontation and excessive directiveness play important roles . the results indicated that gender as such had no significant association with the therapist 's attitude toward mi , while a significant correlation was found between femininity and the attitude score : the more feminine therapist was , the more s / he preferred mi . to the best of our knowledge , there are no studies available on this subject . our findings showed that eclectic therapists were more positive toward clients when compared with single - method therapists . we also found that therapist 's marital status and length of therapy training interacted significantly with gender on attitudes toward the vignettes . however , these interactions were explorative in nature , and no parallel information was available . although the questionnaires sent via the internet had a high degree of anonymity , more responses might have been obtained if data had been gathered through personal contacts . second , while the bsri has been used extensively , there are questions and limitations as to how valid its underlying factors currently are [ 23 , 24 ] . third , the assessment of attitudes toward clients could partly have been confounded by socially desirable responding . tourangeau and yan concluded in their review that responses to questions on sexual orientation , in particular , are often based on social expectations . what the findings mean for practical substance abuse treatment remains open . in the future , an examination of this kind should be combined with the measurement of treatment processes and outcomes . are there differences between female and male therapists in the continuity and outcome of substance abuse treatment ? these are necessary steps before we can draw sound conclusions on gendered effects in this field .	the purpose of the present study was to investigate the impact of therapist 's gender and gender roles on attitudes toward clients . attitudes toward motivational interviewing were also a focus as mi can be hypothesized to be feminine rather than masculine in nature . the subjects ( n = 246 ) were finnish substance abuse therapists . their attitudes toward clients were measured using a vignette task . results indicated that female therapists were significantly more positive toward clients than were male therapists . although females were significantly more feminine than males , they saw themselves as masculine as the males did . the more feminine the therapist was , the more s / he preferred mi . in the future , an examination of this kind should be combined with measurement of treatment processes and outcomes .
You are an expert at summarizing long articles. Proceed to summarize the following text: the first patient was a 23-month - old girl who was referred on march 6 , 2006 , to a tertiary hospital for bilateral otitis media that had not responded to amoxicillin or later to cefprozil . blood cultures were negative , as were antigen detection tests for influenza a and b viruses , the respiratory syncytial virus , and adenoviruses . after 24 hours , the girl was discharged with a diagnosis of bilateral acute otitis media secondary to a viral infection . a nasopharyngeal aspirate collected at the time of admission was inoculated onto different continuous cell lines including human lung adenocarcinoma ( a-549 ) ; human rhabdosarcoma ( rd ) ; transformed human kidney ( 293 ) ; human colon adenocarcinoma ( ht-29 ) ; human laryngeal carcinoma ( hep-2 ) ; human foreskin fibroblast ; mink lung ; and vero , mdck , and rhesus monkey kidney ( llc - mk2 ) cells . cultures were incubated for 3 weeks at 37c in 5% co2 . a viral isolate ( can112051 - 06 ) with cytopathic effects ( round cells ) suggestive of a picornavirus was observed only in llc - mk2 cells after 6 days of incubation ( figure 1 ) . an immunofluorescent assay that used the pan - enterovirus blend kit ( light diagnostics , levingston , uk ) gave a moderate fluorescent signal . nucleic acid extracts from can112051 - 06 were further analyzed with a multiplex real - time reverse transcription pcr ( rt - pcr ) assay for common respiratory viruses ( influenza a and b viruses , human respiratory syncytial virus , and human metapneumovirus ) ( 11 ) as well as rt - pcr assays for enteroviruses and parechoviruses ( 12 ) ; results were negative . a ) cytopathic effects ( round cells ) observed 6 days after infection of rhesus monkey kidney ( llc - mk2 ) cells ( second passage ) with the can112051 - 06 saffold virus like cardiovirus strain . the supernatant from llc - mk2infected cells was treated with dnase and divided into 2 aliquots for dna and rna extractions by using the qiaamp blood mini kit and qiaamp viral rna extraction kits ( qiagen , mississauga , ontario , canada ) , respectively . nucleic acids were then used in the sequence - independent single - primer amplification method as described ( 13 ) . sequence determination of cloned amplicons followed by tblastx analysis showed similarity of can112051 - 06 sequences with the saf - v vp4 and 2c sequences ( data not shown ) . subsequent pcr amplifications and sequencing reactions that used primers selected from our clones and the complete saf - v genome sequence ( genbank accession no . ef165067 ) enabled us to determine the complete polyprotein encoding region of the can112051 - 06 isolate ( genbank accession no . this region was 6,879 nt long compared with 6,888 nt for the saf - v polyprotein sequence ; nucleotide identity between the 2 strains was 82.5% . the can112051 - 06 polyprotein comprised 2,293 aa compared with 2,296 aa for the saf - v polyprotein ; amino acid identity between the 2 strains was 91.2% . deletions of 1 aa in the vp2 and 2 in the vp1 proteins were found in can112051 - 06 with regard to the prototype saf - v strain . as expected , the 8 aa flanking these sites were conserved ; 6 sites were identical in the 2 strains , whereas the remaining sites had 1- or 2-aa differences ( table 1 ) . the resulting 12 proteins of can112051 - 06 and saf - v had 76.1%100% aa identities ( table 2 ) . in addition to the l peptide , some cardioviruses , in particular theiler s murine encephalomyelitis virus strains that are associated with persistent infections , contain an alternate open reading frame ( orf ) , the so - called l * ( 14 ) . as for the prototype saf - v strain , the can112051 - 06 putative l * orf is unlikely to encode a protein because it has an acg ( instead of atg ) start codon ( data not shown ) . in addition , contrasting with the saf - v l * , which contained 57 aa , the can112051 - 06 l * sequence contained only 34 aa . comparison of the l * sequence of can112051 - 06 with the first 34 aa of the saf - v l * sequence showed 60.6% identity ( data not shown ) . four small loops are exposed on the virion surface of cardioviruses ; 2 are part of the vp2 ef loop structure , and 2 are part of the vp1 cd loop structure . the ef loop structure of can112051 - 06 , which contained 55 aa ( residues 274 - 328 of the polyprotein ) , had 61.8% aa identity with that of saf - v ( figure 2 , panel a ) . similarly , the cd loop structure of can112051 - 06 , which contained 40 aa ( residues 712 - 751 of the polyprotein ) , had 67.5% aa identity with the saf - v counterpart ( figure 2 , panel b ) . comparison of amino acid sequences of the a ) ef loop structure ( part of the vp2 protein ) and b ) the cd loop structure ( part of the vp1 protein ) between can112051 - 06 and other cardioviruses including saffold virus ( saf - v ) , theiler s murine encephalomyelitis virus ( tmev ) , theiler - like virus , encephalomyocarditis virus ( emcv ) , and mengovirus . other respiratory samples with picornavirus - like cytopathic effects on llc - mk2 cells and weakly immunofluorescent signals according to the pan - enterovirus blend kit were screened for cardiovirus saf - v by using a specific rt - pcr assay targeting a 2a2c encoding region ( 1,407 nt , 469 aa ) . with use of this strategy , 2 more cases were noted in september 2006 : 1 in a 19-month - old child hospitalized for suspected bacteremia and a cold and 1 in a 4-year - old child hospitalized for right lung pneumonia and otitis media . the 2a-2c aa sequences of these additional isolates were identical and shared 96.6% and 97.2% aa identities with the corresponding regions of can112051 - 06 and the prototype saf - v , respectively . our findings suggest a pathogenic role for saf - v - like viruses in humans . although the polyprotein sequences of the can112051 - 06 strain and the original us strain were related , the ef and cd loop structures varied substantially ( 61.8% and 67.5% aa identities , respectively ) . for comparison , the ef and cd loop structure sequences of emcv and mengovirus ( 2 members of the emcv species ) have 95.2% and 95.1% aa identities , respectively . the difference between time of isolation of saf - v ( 1981 ) and the can112051 - 06 strain ( 2006 ) is unlikely to be responsible for such a high level of sequence variation . we previously showed that the amino acid sequences of the vp0-vp1 capsid region of canadian human parechovirus 1 strains isolated from 1985 through 2004 had 89.2% to 97.5% identities ( 12 ) . because the ef and cd loop structures are exposed on the viral surface of cardioviruses and thus constitute an important site for recognition by neutralizing antibodies ( 15 ) , can112051 - 06 and the original saf - v might represent different serotypes , although further serologic studies are needed to confirm this hypothesis . the implication of the weak immunofluorescent signal seen in cardiovirus - infected cells stained with an enterovirus antibody is uncertain because of the considerable difference between the capsid proteins of cardioviruses and enteroviruses , which constitute 2 separate picornavirus genera . in contrast to the initial recovery of this virus from a stool sample ( 10 ) , our 3 strains were recovered from nasopharyngeal aspirate samples of children with fever and some other respiratory signs . the cardioviruses were the only pathogens identified in these samples . whether saf - v and the related canadian strains described in this study should be classified as a new human cardiovirus species or as a new clade within the theilovirus species remains to be determined	in canada , cardiovirus isolates related to saffold virus were detected in nasopharyngeal aspirates from 3 children with respiratory symptoms . polyprotein sequence of the can112051 - 06 isolate had 91.2% aa identity with saffold virus ; however , ef and cd loops of the viral surface varied substantially .
You are an expert at summarizing long articles. Proceed to summarize the following text: nevertheless , the longevity and integrity of composite restorations depend on multiple factors , such as polymerization shrinkage and hydrolysis of the hybrid layer , which may lead to postoperative sensitivity , secondary caries formation and , as a result , failure of the restoration . recently , many investigators have focused their research on the subject of durability of the bond between dental adhesive systems and dentin . it is well known that in composite restorations the hybrid layer gradually degenerates , as a result of the hydrolytic degradation of collagen fibers , even when bacteria and their toxins are not present . matrix metalloproteinases ( mmps ) are a group of 23 mammalian enzymes capable of degrading all extracellular matrix components . human dentin matrix contains at least mmp-2 and mmp-9 ( gelatinolytic activity ) , mmp-8 ( collagenolytic activity ) , and enamelysin mmp-20 . the incomplete infiltration of collagen fibers by the dental adhesive leads to the exposure of the fibers and to the collagenolytic activity of mmps , which may be activated by low ph values during the etching of dentin . as a result , the hybrid layer degenerates , the bond strength gradually deteriorates , and the composite restoration ends in failure . chlorhexidine ( chx ) is a cationic bisbiguanide , widely known as an antimicrobial agent in oral health . moreover , it has been reported that chx is able to inhibit the mmps collagenolytic activity , improving the longevity of the bond between adhesives and dentin . in fact , gendron et al . found that the minimum concentrations that are adequate for this inhibition are 0.001% for mmp-2 , 0.02% for mmp-8 , and 0.002% for mmp-9 . therefore , the aim of this study was to systematically review and discuss the results of previous studies regarding the effect of chx on the bond strength of dental adhesive systems in composite resin restorations . the hypothesis tested was that there is no difference between the bond strength of dental adhesives and dentin when chx treatment is applied . the electronic databases that were searched to identify manuscripts for inclusion were the medline via pubmed and google search engine . the search strategies were computer search of the database and review of reference lists of the related articles . no publication year limit was used , and the last search was made in november 2015 . search words / terms were as follows : ( digluconate chlorhexidine * ) and ( dentin * or adhesive system * or bond strength * ) . in terms of reviewing reference lists , the references of all included articles were manually searched for further relevant studies that could fulfill the inclusion criteria . the inclusion criteria were as follows : in vitro or in vivo studies that evaluated the effect of chx application on the bond strength of composite restorations during the adhesive procedure after storing of the adhesive interface for at least 6 months ( any type of storage medium ) . studies that did not evaluate the immediate and the aged bond strength were also excluded from evaluation . exclusion criteria of the study the 144 studies titles found were checked for duplicity , and 102 studies were finally included . the abstracts and if necessary the full texts of 102 studies were then assessed , depending on the exclusion and inclusion criteria , to identify 17 relevant studies ; 85 studies were excluded . the study design of the selected studies and the percentage of bond strength reduction during the experimental periods are shown in table 2 . the oldest report was published in 2007 and the most recent in 2015 . bond strength reduction after chx treatments varied among the studies , ranging 0 - 84.9% . in most of the studies , exceptions were reported by zhou et al . , who found that the reduction of bond strength after treatment with 0.05% chx was 28.8% compared to 18.7% of the control group after 12 months of storage , and by de munck et al . , who demonstrated a bond strength reduction of 68.7% after pretreatment with 0.05% chx compared to 49.7% of the control group after 12 months of storage . in addition , sabatini reported that there was no reduction of bond strength after storage for 6 months : neither in chx treated specimens nor in control specimens . data from the studies included in the present systematic review in three studies , chx was incorporated into the adhesive system , while in other studies chx was applied as a pretreatment solution . fourteen studies evaluated bond strength using microtensile bond strength ( tbs ) test , one study using tensile bond strength ( tbs ) test , one study using microshear bond strength ( sbs ) test , and one study using shear bond strength ( sbs ) test . furthermore , in seven studies the type of storage medium was distilled water , in seven other studies was artificial saliva , in one study was deionized water , and in one study was 0.9% sodium fluoride ( naf ) and 0.02% sodium azide solution . in one study , the evaluation of bond strength of the specimens was carried out after 6 months of storage in seven studies , after 9 months in one study , after 12 months in four studies , after 6 and 12 months in four studies , and after 18 months in one study . fifteen studies investigated the effect of 2% chx on bond strength between dentin and dental adhesive ; four studies the effect of 0.2% chx ; two studies the effect of 0.5% chx ; two studies the effect of 0.05% chx ; and one study the effects of 0.002% , 0.004% , 0.02% , 0.1% , 1% , and 4% chx . there are many factors that influence the bond strength of a restorative material to the dentinal substrate . mechanical stresses by chewing forces , changes of temperature and ph , water sorption , resin shrinkage , and enzymatic action affect bond integrity to different extents . moreover , the type and composition of the composite resin and the adhesive system as well as the dentinal substrate are of great importance . previous scientific reports have demonstrated that application of chx for 60 s immediately after etching with three - step and two - step etch - and - rinse dental adhesives results in preservation of bond strength . additionally , stanislawczuk et al . found that even when chx is contained in self - etch adhesives or in the etchant agent , a better stability of hybrid layer may be observed . it has been found that resin - dentin bonds often fail 30 - 40% in 6 - 12 months . as it was previously mentioned , dentin is known to contain mmps , which are activated by weak acids released by caries - producing bacteria , and acid - etchants used in adhesive systems . the clinical application of 2% chx for 60 s to the etched dentin after rinsing off the acid and before applying the dentin - bonding primer and resin minimizes significant in vivo degradation of bond strength from mmps for at least 14 months . therefore , many clinicians currently apply 2% chx for 60 s to acid - etched dentin during resin bonding in an attempt to increase the durability of resin - dentin bonds by inhibiting endogenous mmps in the dentin matrix . this method is the only one proven clinically , is easy to adopt , and will likely be the first to gain wider acceptance . some researchers who previously investigated the effect of chx on the bond strength of dental adhesive systems on dentin have reported that the chx pretreatment did not affect the sbs of dental adhesives . nevertheless , other studies have reported that chx acting as mmps inhibitor decelerates the rate of resin - dentin bond degradation . furthermore , campos et al . reported that application of 2% chx was deleterious to bond strength and should be avoided prior to self - etch adhesive systems . this is in agreement with previous reports investigating the effect of 0.05% chx solution on the bond strength of adhesive systems with dentin . these contrary results of the previous studies may be attributed to different experimental methods , different design of the experiments , and the different materials investigated . chang and shin evaluated the influence of chx with different application methods on bond strength to dentin . the application methods used in this study were : chx + rinsing + etching;chx + etching;etching + chx + rinsing ; andetching + chx . chx + rinsing + etching ; etching + chx + rinsing ; and considering the amount of reduction in bond strength after thermocycling , this study showed improved dentin bond strength with chx when used after etching . other studies have postulated that the incorporation of chx into the adhesive or the etchant in order to avoid a chx treatment step can preserve bond strength to dentin . another factor that may influence the effectiveness of chx in bond strength between adhesives and dentin is the type of the applied chx solution . ali et al . demonstrated that pretreatment of the dentin with ethanol - based chx had a negative effect on the bonding of the tested single - step self - etch adhesive ; however , water - based chx showed bond stability under intrapulpal pressure simulation . moreover , ekambaram et al . found that the incorporation of 2% chx into ethanol - wet bonding preserved bond strength to both sound and caries - affected dentin , and reduced interfacial nanoleakage after 12 months , while the incorporation of 2% chx to water - wet bonding preserved bond strength only to sound dentin after aging , who evaluated the effect of chx and/or ethanol application on the bond strength of an etch - and - rinse , hydrophobic adhesive system under either in vitro aging or in situ cariogenic challenge , showed that chx , ethanol , or chx + ethanol did not affect the tbs after 6 months . it has been reported that the association between the concentration of chx and the bond strength is not linear . chx has been shown to directly inhibit mmp-2 , -8 , and -9 activities at extremely low concentrations ( 0.0001 - 0.02% ) . however , with low chx concentrations ( 0.2 - 0.45% ) , there is no interaction with synthetic hydroxyapatites , while with higher concentrations ( 1 - 4.5% ) , chx was progressively retained with time . on the other side , loguercio et al . found that the use of 0.002% chx for 15 s seems to be sufficient to preserve resin - dentin interfaces over a 6-month period . in addition , breschi et al . and lin et al . reported that treating acid - etched dentin with 0.2% chx reslted in the formation of hybrid layers that are as stable as those formed with 2% chx . claimed that the addition of chx in concentrations until 0.2% in the simplified etch - and - rinse adhesive systems may be an alternative to increase the long - term stability of resin - dentin interfaces , without jeopardizing the adhesives evaluated mechanical properties . moreover , zhou et al . , who investigated the incorporation of chx into a two - step self - etch adhesive in different concentrations , found that chx can preserve the dentin bond as long as the concentration is higher than or equal to 0.1% . in another research study , it has been demonstrated that the addition of 2% chx or , especially , 5% chx in experimental adhesives produced significant reductions in both the tbs and the fourier transform infrared spectroscopy ( ft - ir ) percent conversion . specifically , komori et al . evaluated the effect of 2% chx on the long - term bond strengths of two etch - and - rinse adhesive systems to normal and caries - affected dentin and found that chx significantly lowered the loss of bond strength after 6 months in normal dentin specimens , but did not alter the bond strength of caries - affected dentin specimens . in another , similar study , it has been postulated that 2% chx has an interaction effect on the preservation of bond durability to both sound and caries - affected dentin after 12 months . on the contrary , other studies reported that application of a self - etch adhesive on chx - treated sound dentin or artificially caries - affected dentin did not change the bond strengths . generally , the presence of carious dentin decreases bond strengths and the higher the level of caries progression , the lower the bond strengths of adhesives to carious dentin . moreover , francisconi et al . , who focused on the effect of 2% chx on the bond strength of adhesive systems to sound and artificially eroded dentin , found that although there was a significantly lower tbs in artificially eroded dentin compared to sound dentin , the influence of chx on bond strength conservation was not found to be persistent . in another investigation , manfro et al . assessed the effect of 0.5% chx and 2% chx on bond strength to primary tooth dentin and reported that chx pretreatment may prevent the degradation of adhesive interface in primary teeth . there are many studies that have investigated the sbs of etch - and - rinse and self - etch adhesive systems . some of them found that there were no statistically significant differences between etch - and - rinse and self - etch adhesives , but other reports have claimed that etch - and - rinse adhesives exhibit higher sbs than self - etch adhesive systems due to the better hybridization or infiltration of resin within the exposed collagen fibrils of the dentinal surface . a recent study has investigated the effect of 2% chx on the tbs of an etch - and - rinse and a self - etch adhesive after 9 months of aging , and the authors concluded that chx prevented the decrease in tbs upon aging of the etch - and - rinse but not of the self - etch adhesive . this finding is supported by nishitani et al . , who concluded that self - etch adhesives may activate mmps and increase their adverse activity , contributing to the degradation of the dentin bond over time . the presence of solvents in resins is absolutely necessary in the chemical composition of adhesives so that they can bond to dentin adequately . water is a poor solvent for organic compounds , such as monomers , which are rather hydrophobic , while acetone and ethanol can combine hydrophobic and hydrophilic components . additionally , the absence of solvents in the chemical compositions of some new dental adhesives results in a thicker adhesive layer . some authors have assumed that this increases hydrophilicity and that as a result the adhesive behaves as a semipermeable membrane , allowing more fluids to pass through , leading to lower bond strength . it has been found that solvent - free self - etch adhesives , when used for bonding composite resins to dentin , underperform as compared to conventional adhesives . intermediary strong ( ph ~1.5 ) self - etch dental adhesives have a hybrid layer of about 1 - 2 m , with some hydroxyapatite preserved at the bottom part . ultramild ( ph > 2.5 ) self - etch adhesives do not eliminate the smear layer , and interact with the smear layer - covered dentin only up to a few hundredths of nm . previous reports have shown that mild self - etch adhesives create lower dentin bonds than the other adhesives . in many studies , scanning electron microscopy ( sem ) analysis has been used to reveal the failure mode distribution of the specimens demonstrating adhesive failures . failure is considered to be a ) adhesive , if it occurred at the dentin - adhesive interface ; b ) cohesive , if it occurred in the material or in the substrate ; and c ) mixed , if it involved both the interface and the material . quantitative sem analysis improves the accuracy of the failure mode distribution analysis and allows for discrimination between the failures occurring in the top and the bottom of the hybrid layer to understand the mechanisms leading to the reduction in dentin bond strengths with time . found significantly lower percent failure mode in the hybrid layer , especially in the bottom part , after 6 months with chx pretreatment and indicated that the higher bond strengths observed in this group reflected the preservation of hybrid layer collagenous matrix , especially in the bottom zone , where partially exposed collagen fibrils are most prone to initial enzymatic degradation . researchers who previously investigated the effect of chx on the bond strength of dental adhesive systems with dentin have reported contrary results . although there is evidence that chx is able to inhibit mmps collagenolytic action , it is not clear if this ability has a clinical significance in composite restorations . due to the many factors that influence the bond strength of a composite material to the dentinal substrate , further investigations , in particular clinical studies would be necessary to clarify the effect of chx on the longevity of dentin bonds .	aim : this study aimed to systematically review the literature for the effect of digluconate chlorhexidine ( chx ) on bond strength between dental adhesive systems and dentin of composite restorations.materials and methods : the electronic databases that were searched to identify manuscripts for inclusion were medline via pubmed and google search engine . the search strategies were computer search of the database and review of reference lists of the related articles . search words / terms were as follows : ( digluconate chlorhexidine * ) and ( dentin * or adhesive system * or bond strength*).results : bond strength reduction after chx treatments varied among the studies , ranging 0 - 84.9% . in most of the studies , pretreatment chx exhibited lower bond strength reduction than the control experimental groups . researchers who previously investigated the effect of chx on the bond strength of dental adhesive systems on dentin have reported contrary results , which may be attributed to different experimental methods , different designs of the experiments , and different materials investigated.conclusions:further investigations , in particular clinical studies , would be necessary to clarify the effect of chx on the longevity of dentin bonds .
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Proceed to summarize the following text: various bone - substitute materials are used to promote bone reconstruction in oral and maxillofacial areas . they have osteoconductivity properties , as they provide a scaffold for cell adhesion and proliferation and allow bone growth on their surfaces . deproteinized bovine bone matrix ( dbbm ) and biphasic calcium phosphate ( bcp ) are two calcium phosphate - based bone substitutes with different resorption patterns , widely used in the management of periodontal and peri - implant bone defects as well as of bone augmentation procedures [ 14 ] . it is a bovine bone that undergoes a low heat ( 300c ) chemical extraction process by which all organic components are removed , but it maintains the natural architecture of bone and results in cap crystals characterized as hydroxyapatite with morphological and structural properties similar to that of human bone hydroxyapatite . in vivo , it is resorbed very slowly or not at all . tested extensively in vitro and in vivo , this material has demonstrated osteoconductive properties that improve bone regeneration . bcps are synthetic bone substitutes with different ratios of hydroxyapatite ha and beta - form of tricalcium phosphate ( -tcp ) depending on the preparation and synthesis procedure . variations of bcp regarding ha / tcp ratio showed different patterns in biodegradation and osteoconduction [ 8 , 9 ] . some clinical studies comparing bcp ( 60/40 ha / tcp ratio ) and dbbm regenerative potential have reported conflicting results [ 24 , 10 ] while preclinical studies using bcp in comparison with dbbm in standardized critical - size defects are not consistent [ 9 , 1113 ] . to promote bone regeneration , the aforementioned bone substitutes are supplemented with autologous platelet concentrates such as platelet - rich fibrin ( prf ) , platelet - rich plasma ( prp ) and platelet lysate ( pl ) [ 14 , 15 ] . activated platelets release several chemical compounds including various growth factors and cytokines that are involved in wound healing , angiogenesis , and bone regeneration processes . it is hypothesized , therefore , that a platelet concentrate delivered at the site of a bone defect may increase the local concentration of bioactive compounds , such as growth factors , and subsequently affect functions of bone - healing related cells . despite the attractive rationale and the in vitro observed beneficial effects of platelet concentrates , in vivo preclinical [ 1723 ] and clinical [ 14 , 24 ] studies , which evaluated the efficiency of particulate bone substitutes supplemented with platelet concentrates in the treatment of intrabony defects , have provided promising but not consistent results . with the exception of the biological environment of the defect , differences in key parameters that include implanted material behavior , mode of preparation of the platelet - concentrates , and/or individual variability [ 18 , 26 ] could explain these discrepancies . in particular , most of these studies are focused on the effects of platelets concentrates in the form of prp , a gel obtained by treating concentrated platelets with thrombin . in this case , the observed results on bone healing might be due to formation of a fibrin clot / gel , which held material particles in situ , and/or to the effects of the compounds released by the activated platelets . alternatively , platelet concentrates are used also in the platelet lysate form ( pl ) . showed that pl improves the migration and/or proliferation of bone healing related cells [ 2830 ] and is used for the clinical grade expansion of hmsc . clinical studies suggested that it promotes oral lesions healing and could be potentially useful in bone reconstructive surgery [ 32 , 33 ] . however , the effect of pl in conjunction with particulate bone substitutes on bone healing has not been determined . it is hypothesized that the mixture of bioactive molecules in the pl added to a bone substitute promotes bone healing and its effect depends on the implanted material . the objective of the present study was to compare healing around particulate dbbm and bcp and to evaluate the benefit of apl use on the potential of these bone graft substitutes to promote bone repair . to investigate these aspects , dbbm and bcp were associated with autologous pl and implanted in critical - size femoral defects , in rabbits . the materials used in the present study were as follows : particles ( 5001000 microns in diameter ) of biphasic calcium phosphate ( bcp ; boneceramic ; straumann , agbasel , switzerland ) , a composite of 60% hydroxyapatite ( 100% crystalline ) , and 40% -tricalcium phosphate , with 90% porosity and interconnected pores . this material is used as an alloplastic bone graft substitute in dental and orthopedic clinical practice;particles ( 2501000 m in diameter ) of deproteinized bovine bone dbbm ( bio - oss ; geistlich , switzerland ) , obtained from excised bone through a multistage purification process . particles ( 5001000 microns in diameter ) of biphasic calcium phosphate ( bcp ; boneceramic ; straumann , agbasel , switzerland ) , a composite of 60% hydroxyapatite ( 100% crystalline ) , and 40% -tricalcium phosphate , with 90% porosity and interconnected pores . this material is used as an alloplastic bone graft substitute in dental and orthopedic clinical practice ; particles ( 2501000 m in diameter ) of deproteinized bovine bone dbbm ( bio - oss ; geistlich , switzerland ) , obtained from excised bone through a multistage purification process . . twelve four - month - old male new zealand rabbits ( segav , saint - mars d'egrenne , france ) with a mean weight of 3.5 kg were used in the present in vivo study . animal procedures were approved by the local ethics committee ( comit 4 , le - de - france , paris , france ) . the animals were housed individually in metal hutches in an appropriate environment ( ambient temperature of 21c and 50% air humidity ) that met the requirements of the european guidelines for care and use of laboratory animals ( directive du conseil 24.11.1986 , 86/609/cee ) . artificial light conditions were used in the animal facility to maintain a normal day / night biological rhythm in the animals . the animals drank water and ate commercial food concentrates ( pietrement , sainte colombe france ) ad libitum . rabbit apl was prepared one week before surgery and stored according to previously established methods . briefly , g21 infusion sets ( ago microperfusore , artsana , como , italy ) were used to obtain 10 ml blood from the intermediate branch of the caudal auricular vein from each rabbit . the blood samples were collected in venoject tubes ( centravet , plancoet , france ) containing acd - a : citric acid - dextrose - formula a ( b braun medical s.a . , boulogne , france ) ( 1 : 3 acd - a ( v / v)/blood ) and gently centrifuged at 150 g and 20c for 10 min . aliquots ( 5 ml ) of the platelet - rich supernatant plasma were mixed with 0.6 ml acd - a and centrifuged at 1500 g and 20c for 10 min to obtain platelet pellets . the supernatant platelet - poor plasma ( ppp ) was removed and stored at 80c . the packed platelets were suspended in isotonic saline to obtain a platelet suspension of 10 platelets / ml . each apl was obtained using a series of five cycles of freezing - thawing of the respective autologous platelet suspensions followed by centrifugation at 10 g and 4c for 1 hour to remove platelet membranes and other cell related debris and then stored at 80c . the biological activity of apl , with different concentrations of suspended platelets ( 0.010.3 10 cells / ml ) , was also assessed in vitro by testing its mitogenic effect . preconfluent foetal rat calvaria cells were incubated with rabbit apl used in the present study or not ( controls ) for 24 h. the cells were then removed by trypsinisation and counted using established techniques . platelet lysate enhanced , respectively , the proliferation of foetal rat calvaria cells by about 100% to 200% . the rabbits tested in the present study were randomly assigned to one of two groups , each composed of six animals . the femoral defects in the first group of rabbits were randomly filled either with 250 mm dbbm mixed with 250 l of physiological saline ( control ) or with dbbm mixed with 250 l apl ( with a platelet concentration of approximately 10 platelets/l ; lysate from 2.5 10 platelets ) . the femoral defects in the second group of rabbits were filled either with 250 mm bcp mixed with 250 l of physiological saline ( control ) or with bcp mixed with 250 l apl . prior to surgery , the rabbits were anesthetized using an intramuscular injection of 0.5 mg / kg diazepam ( valium ; roche ; basel , switzerland ) , 0.25 mg / kg metedomidine hydrochloride ( domitor , virbac ; france ) , and 100 mg / kg ketamine hydrochloride ( ketalar 500 , pfizer ; france ) . the anesthetized animals were prepared for aseptic surgery , shaved , and disinfected ; both lower limb sites were draped using standard sterile procedures . the surgical technique used was adapted from the one described by soffer et al . . briefly , a longitudinal skin incision was made to expose the distal lateral aspect of each femoral condyle . a circular cortical window was formed using a 6 mm wide internal diameter trephine . a cylindrical critical - size defect of 10 mm deep and 5.5 mm wide was then created in the lateral condyle in a stepwise fashion using color - coded drills , ( diffusion international , paris , france ) . these cavities were thoroughly rinsed with isotonic saline to remove bone debris and then , using a specially designed injector and applying a light pressure , they were filled with dbbm or bcp mixed with either physiological saline or apl . the aforementioned fillers were prepared and used under sterile conditions at the time of animal surgery . each soft tissue wound was closed in two successive layers soft tissue and skin , respectively , using resorbable vicryl 5/0 sutures , and the surgical site was disinfected . all animals were given intramuscular injections of 0.2 mg / kg metoxicam ( metakam boehringer ingelheim vetmedica ; gmbh germany ) to relieve pain during the postoperative 24 h period . prophylactic antibacterial treatment , consisting of 25 mg / kg animal weight sulfadimethoxine trimethoprim ( copylap biov ; france ) , was also administrated for 5 days after surgery by qualified animal - care staff . after surgery , all operated animals were monitored until they were able to walk on their own . the rabbits were sacrificed using an overdose of pentobarbital 6 weeks after implantation . at that time , the femoral condyles were removed and cleared of the surrounding soft tissue . all excised bone tissue specimens were prepared for histological analysis as described in the histology section . each excised bone specimen was cleared of soft tissue , fixed in 10% phosphate - buffered formalin , rinsed in water , dehydrated in ethanol , cleared in xylene , and embedded in plastic resin ( technovit 7200 vcl , kulzer & co. gmbh , wehreim , germany ) . each section was then ground down to a thickness of 100 m using the exact grinding system ( exact aparatebau gmbh norderstedt , germany ) and polished down to 80 m . all tissue specimens were then stained using giemsa - paragon for histomorphometric analysis . each section was examined under a light microscope ( olympus bx 60 , olympus corporation , tokyo , japan ) connected to a digital camera ( olympus , e330 ) . new bone formation was quantified using image tool 3.0 software ( uthsca , sanantonio , tx , usa ) . the spatial calibration of the histological sections was done by drawing manually the surface of the newly formed bone and the residual bone substitutes . the total surface was obtained by adding all the delimited areas measurements ( in mm ) . the distribution of newly formed bone and residual implanted material was also determined by classifying the defect area into an inner zone ( ic ) of 2.5 mm and an outer zone ( oc ) , including the remaining of the 5.5 mm defect ( figure 1 ) . two - way analysis of variance between groups was conducted to determine the effect of bone substitutes with apl on new bone formation and on the resorption of these materials . variables were tested for normal distribution and equality of variance using the kolmogorov smirnov and levene tests , respectively . comparison of the data between the different groups tested was performed using the student 's t - test . all statistical analyses were performed using commercially available software programs ( spss for windows version 16.0 ) . all rabbits recovered within 2 - 3 hours after surgery . in all cases , tissue healing was uneventful . rabbits were systematically checked in order to detect any problems during the experimental period and they were weighed every two weeks . no postsurgical complications , infections , animal behavior , or general health changes were observed after surgery and for the duration of the study . rabbits presented a normal weight gain of 103,8 g. after 6 weeks of implantation , histological analysis of specimens excised from all animals tested showed no inflammatory reactions . new bone formation was evident in close contact to both the bcp and dbbm particles that have been used to fill the femoral defects in the present study , even in the central part of the defect ( figures 2(a ) and 3(a ) ) . in the defects filled with dbbm , the dbbm particles were distributed over the entire grafted area of the femoral defects and were surrounded and bridged by newly formed bone ( figure 2(b ) ) . . a thin layer of osteoid and osteoblast lining - cells was observed in some areas around dbbm particles ( figures 2(b ) and 2(c ) ) . multinucleated giant cells or howship lacunae , pits , or resorptive trails were not seen on the dbbm surface regardless of the presence or absence of apl . the dbbm thus exhibited osteoconductive properties but not ongoing material resorption . in the defects filled with bcp , the material particles were distributed over the entire defect area and they were surrounded by newly formed bone ( figure 3(a ) ) both in the presence and absence of apl addition . some particles were covered by a layer of newly formed bone ( figure 3(b ) ) . some lining osteoblasts , as well as multinuclear phagocytic cells , were observed at the surface of the bcp particles ( figures 3(c ) and 3(d ) ) . in some areas , examination at high magnification ( 40 ) revealed that the most newly formed bone had woven bone characteristics . new bone formation was more prominent in the outer circle ( oc ) , bridging with the edges of the defects , than in the inner circle ( ic ) . at 6 weeks after implantation , the bone areas in defects filled with bcp were similar to those observed in defects filled with bcp+apl ( 6.69 1.53 mm and 6.57 1.90 mm , respectively ; figure 4 ) . the amount of bone formed in defects filled with either dbbm or with dbbm+apl was also similar ( 4.49 1.19 mm and 4.28 1.42 mm , resp . ) . the addition of apl did not promote new bone formation above the extent obtained with the two bone substitutes tested alone . in contrast , the bone areas in defects filled with bcp were significantly ( p < 0.0001 ) higher than those observed when dbbm alone was used as the filler . specifically , the mean difference in the bone amount in defects filled with either bcp or dbbm was 2.29 mm ( confidence interval ( ci ) 1.233.36 ) in the absence of apl and 2.19 mm ( confidence interval ( ci ) 1.133.25 ) in the presence of apl . for the two substitute materials as well as for all of the experimental conditions tested in the present study , the bone surface areas in the outer zone ( oc ) were significantly higher than those observed in the inner zone ( ic ) of the defects ( for bcp : p < 0.0001 in the presence and absence of apl ; for dbbm : p < 0.037 and p < 0.001 in the presence and absence of apl ; figure 5 ) . distribution of newly formed bone throughout the respective original defect was not affected by the presence or absence of apl . the bone surface area at the inner as well as at the outer zones of the defects was significantly ( p < 0.001 ) higher when the defects were filled with bcp than when they were filled with dbbm ( figure 5 ) . at 6 weeks after implantation , the surface areas of the residual implanted material in defects filled with bcp was similar to those in defects filled with bcp+apl formulations ( 9.43 1.89 mm and 9.25 1.61 mm , respectively ; figure 6 ) . in addition , the surface areas of the residual material in defects filled with dbbm was similar to those filled with dbbm+apl formulations ( 14.61 1.64 mm and 14.36 1.93 mm , resp . ) . the amount of implanted material in defects filled with bcp was significantly ( p < 0.0001 ) lower ( specifically , close to 65% ) compared to that observed with dbbm regardless of the presence of apl . analysis of the distribution of the nonresorbed implant material throughout the defect provided evidence that , in the defects filled with bcp , the amount of residual material was significantly ( p < 0.0001 ) higher in the outer zone than in the inner zone of the defect ( mean difference 1.04 mm ; confidence interval ( ci ) : 0.700.84 ; figure 7 ) . similar results were observed in the defects filled with bcp+apl formulations ( mean difference 0.95 mm ; p < 0.0001 ; confidence interval ( ci ) : 1.110.79 ) . in contrast , the amount of residual material present in the defects filled with dbbm in either the presence or the absence of apl was similar in the inner and the outer zones ( mean difference 0.06 mm ; p < 0.43 ; confidence interval ( ci ) : 0.090.21 and 0.07 mm ; p < 0.368 ; confidence interval ( ci ) : 0.080.22 with and without apl , resp . ) . this study compared the osteoconductive and resorption behavior of bcp and dbbm alone or associated with apl , in critical - size rabbit femoral defects . in particular , the femoral site is an interesting site for the evaluation of the biofunctionality of biomaterials because of the equilibrium of the osteogenesis and the biodegradation activities . in order to achieve standardization in the evaluation of bone substitutes , a critical - size defect is defined as the smallest intraosseous wound that does not heal by bone formation during the lifetime of the animal [ 36 , 37 ] . according to international standards ( iso 10993 - 6 ) , bone substitute materials must be evaluated in bone defects of at least 2 mm diameter and 6 mm in length for rabbit femurs . dbbm and bcp are the most widely used grafting materials in oral , maxillofacial , and implant surgery [ 1 , 7 ] and they are frequently used associated with platelet concentrates [ 14 , 24 ] . however , there are few experimental studies in the literature focusing on the evaluation of their regenerative potential using standardized intrabony defects within the same study and , to our knowledge , none has used critical - size femoral defects to compare the regenerative properties of these bone substitutes . results obtained in this study showed the presence of newly formed bone in close contact with the particles of dbbm and bcp as reported in previous preclinical [ 9 , 12 , 13 ] and clinical studies [ 24 , 10 ] . regarding the distribution of newly formed bone , between the inner and outer zones , new bone formation was more prominent in the oc than in the ic , which shows a slight tendency of augmented bone formation in the outer zone of the defect . other histomorphometric studies have suggested that the majority of the bone formation starts from the borders of the defect , as a centripetal bone colonization . interestingly , under the experimental conditions of the present study , significantly ( p < 0.001 ) more bone formation was observed in the bcp group ( close to 70% ) after 6 weeks implantation time in the presence or absence of additional apl ( figure 4 ) . a similar tendency was described in standardized intrabony defects when bcp was compared to pure hydroxyapatite in the mandible of minipigs at 4 and 8 weeks after implantation . in contrast to the results of the present study , bcp and dbbm rendered similar amount of newly formed bone in noncritical size intrabony defects in the mandible of minipigs at 4 and 8 weeks after implantation , in the rabbit calvaria as well as around implant placed in circumferential defects in dogs mandibles at 8 weeks after implantation . the reasons for these differences are not clear ; however , the anatomical and dimensional characteristics of the defects sites ( femur in the present study versus calvaria ) or mandible and the duration of the study ( 6 weeks in the present study versus 4 , 8 , and 16 weeks in others ) [ 11 , 12 ] ) may be contributory factors . the efficiency of bcp could be related to its partial dissolution and the enhanced bioactivity of soluble tcp . further studies with different healing intervals are necessary to clarify the differences between dbbm and bcp osteoconduction efficiency as well as the characteristics of the newly formed bone in their presence . the most striking difference between the two bone substitutes tested in the present study was their resorption behavior . for the same volume of implanted material , the amount of bcp residual particles in the defect was significantly ( p < 0.001 ) lower ( specifically , close to 65% ) than that observed in defects filled with dbbm at 6 weeks after implantation . in contrast , a limited resorption was observed in mandible bone defects in minipigs and no bcp resorption in calvaria bone defects in rabbits at 4 and even at 16 weeks after implantation . this disparity may be explained by the differences in the anatomical site of the defect ; calvaria is a site where blood supply and bone marrow are poor . however , the results of this work are in accordance with previous studies that reported also approximately 50% resorption of bcp ( 60/40 ha / tcp ratio ) in osseous defects in rabbits within one year after implantation . the resorption of implanted bcp particles provided space and may explain the higher new bone formation observed with this material . comparison of the distribution of the nonresorbed material throughout the defect showed that , in the defects filled with bcp , the amount of the residual material was significantly ( p < 0.001 ) higher in the outer zone than in the inner zone of the defect . this finding may be correlated to the greater bone formation observed around bcp particles in continuity to the surrounding bone . regarding the dbbm resorption , residual material in defects filled with dbbm was significantly ( p < 0.001 ) higher than that observed in defects filled with bcp , indicating that dbbm resorption is very slow . moreover , under the experimental conditions of the present study , multinucleated cells were not observed on the dbbm particles in line with previous findings . since the use of high local concentrations of platelet - derived soluble compounds with bone substitute material remains an attractive strategy for improving bone repair given its autologous origin , minimally invasive nature , and cost - effective ratio , but that it has not provided consistent results , the present study was motivated to explore bone healing in defects filled with either dbbm or bcp in the presence of apl . the results obtained at 6 weeks after implantation in the rabbit femoral defects showed that osteoconduction of bcp particles was more pronounced than in the case of dbbm , and the addition of pl did not affect those outcomes . a direct comparison between the results of the present study and published reports on platelet - derived bioactive components is difficult because of the differences in the platelet preparation ( prp , prf , and apl ) and/or in the study design . nevertheless , the findings of the present study corroborate those of other animal studies , which also showed that prp in conjunction with dbbm in critical - size rabbit calvaria and frontal defect of goat [ 17 , 20 ] does not improve dbbm osteoconductivity . as regards bcp , prp in combination with bcp ( ratio 60/40 ) was tested only in rat calvaria defects [ 19 , 23 ] and no positive effect of prp was established . however , recently it has been described that pl coating of bcp increases in vitro chemoattraction and adhesion of mscs and improves in vivo neovascularization and new bone formation . moreover , it has been suggested that platelet concentration plays a role in the effect of platelet concentrates in bone healing . in the present study , apl with a platelet concentration of 10 platelets/l ( 3.5 to 5x fold of baseline ) was used . with a similar concentration , previous studies have shown biological effects using rabbit pl or rabbit prp apl with a concentration of 10 platelets resulting in higher resorption of calcium carbonate ceramic in rabbit femoral defects . prp with platelet concentration ranging from 9.5 10 to 1.7 10 platelets/l showed improved peri - implant bone regeneration in rabbits . below this range the effect is suboptimal ; beyond this range , there may be a parodoxical inhibitory effect . the time period during which platelet growth factors remain active and the bioavailability of platelet components in relation to dbbm and/or bcp dissolution in osseous defect could explain the absence of platelet concentrates effect on tissue repair . these hypotheses were not explored in this work , and further investigations may be needed to optimize the use of platelet concentrates . it is not clear how far the results of this rabbit study can be extrapolated to different species . nevertheless , previous studies reported biological effects using autologous rabbit as well as miniature pig , dog , and human pl . moreover , the quantitative histomorphometric measurements performed in this study demonstrated that the addition of apl did not affect the resorption of either dbbm or bcp . however , previous studies reported that platelet concentrates increase the resorption of carbonate phosphate and carbonated apatite calcium phosphate particles probably by triggering anti - inflammatory reaction and/or biological responses in the early events of healing . in contrast to dbbm , that did not or slowly resorbed , and bcp that resorbed mildly , calcium carbonate is a material with high and fast resorption . the discrepancy observed in the resorption pattern of these materials , related to the material surface properties and the ph of the biological environment , could influence the adsorption and the release of pl adsorbed proteins and growth factors . in summary , this study demonstrated the osteoconductive properties of these two biomaterials in critical - size osseous defects and showed in a quantitative way that compared to dbbm , bcp was resorbed and promoted a greater bone formation while apl addition had no impact on those outcomes . results obtained in the present investigation are , therefore , relevant to the contribution of bcp and dbbm particles , used either alone or in conjunction with autologous bioactive compounds released from platelet concentrates to promote intrabony defects repair .	bone substitutes alone or supplemented with platelet - derived concentrates are widely used to promote bone regeneration but their potency remains controversial . the aim of this study was , therefore , to compare the regenerative potential of preparations containing autologous platelet lysate ( apl ) and particles of either deproteinized bovine bone mineral ( dbbm ) or biphasic calcium phosphate ( bcp ) , two bone substitutes with different resorption patterns . rabbit apl was prepared by freeze - thawing a platelet suspension . critical - size defects in rabbit femoral condyle were filled with dbbm or dbbm+apl and bcp or bcp+apl . rabbits were sacrificed after six weeks and newly formed bone and residual implanted material were evaluated using nondemineralized histology and histomorphometry . new bone was observed around particles of all fillers tested . in the defects filled with bcp , the newly formed bone area was greater ( 70% ; p < 0.001 ) while the residual material area was lower ( 60% ; p < 0.001 ) than that observed in those filled with dbbm . new bone and residual material area of defects filled with either apl+dbbm or apl+bcp were similar to those observed in those filled with the material alone . in summary , osteoconductivity and resorption of bcp were greater than those of dbbm , while apl associated with either dbbm or bcp did not have an additional benefit .
You are an expert at summarizing long articles. Proceed to summarize the following text: ileosigmoid knot ( also known as compound volvulus ) is an unusual and rare cause of intestinal obstruction . in this condition , loops of ileum wrap around the base of a redundant sigmoid colon to form a knot . the condition progresses rapidly to gangrene , so early diagnosis and operative treatment are vital . we describe the ct scan features of this rare condition , the awareness of which can help achieve a preoperative diagnosis . a 22-year - old woman presented with a 3-day history of progressive abdominal pain associated with one episode of vomiting . she had no other concurrent disease and no significant past medical or surgical history . on examination the abdominal radiograph revealed a large gas - lled loop of the bowel in the right mid and lower quadrants [ figure 1 ] . abdominal radiograph reveals a large gas - filled loop of the bowel in the right mid and lower quadrants contrast - enhanced ct scan of the abdomen revealed a markedly dilated loop of sigmoid colon with loss of haustral pattern , and a thin wall in the right lower abdomen ; the whirl sign was present , created by the twisted mesentery and bowel , and the afferent and efferent limbs of the sigmoid colon had the appearance of a beak [ figure 2a c ] . ileal loops were also wrapped around the central whirl , and these too had thin , nonenhancing walls as well as afferent and efferent limbs with the appearance of a beak [ figure 2b d ] . the cecum and the distal descending colon were deviated medially , with their medial borders having a pointed appearance [ figure 2a , b ] . stretched superior mesenteric vessels converged toward the whirl . in view of the history and the ct scan findings , a diagnosis of ileosigmoid knot with gangrene of the involved sigmoid colon and ileal loops was made . axial ct scan of the abdomen ( a ) shows a dilated loop of the sigmoid colon with a thinned - out wall ( thin arrow ) . there is medial deviation of the cecum with a pointed medial border ( thick arrow ) . ( b ) caudal ct scan section shows the whirl sign ( within the circle ) created by the twisted mesentery and bowel ( branches arising from the superior mesenteric artery are present in the whirl ) . there is medial deviation of the distal descending colon with a pointed medial border ( thick arrow ) . fluid - filled ileal loops with thin non - enhancing walls are seen ( dotted arrow ) . ( c ) a more caudal ct scan section shows efferent limbs of the sigmoid colon ( thick arrow ) and the ileal loop ( dotted arrow ) with appearances of a beak . a further caudal ct scan ( d ) shows fluid - filled ileal loops in the pelvis with thin non - enhancing walls ( solid arrow ) and free fluid present in the pelvic cavity ( dotted arrow ) the patient was immediately operated . laparotomy showed a gangrenous sigmoid volvulus with gangrenous distal ileal loops twisted around it [ figures 3 and 4 ] and approximately 300 ml of hemorrhagic fluid in the peritoneal cavity . the gangrenous loops were resected and ileo - ascending colon and colo - colic anastomoses were done , along with a proximal defunctioning ileostomy . the postoperative course was uneventful and the patient was discharged a week after the surgery . intraoperative photographs show a distended sigmoid colon ( solid arrow ) and gangrenous ileal loops ( dashed arrow ) wrapped around each other forming an ileosigmoid knot diagrammatic illustration of the ileosigmoid knot . the long arrow points to the knot while the short arrows point to the medial displacement of the descending colon and cecum overall a rare entity , ileosigmoid knot is more common in asia , africa and the middle east than in the west . alver classified it into four types based on the mechanism of formation of the knot . in type i ( the commonest ) , the ileum is the active component , wrapping itself around the sigmoid colon ( passive component ) to form the knot , while in type ii , it is the other way round . in type iii , the ileocecal segment acts as the active component , while in type iv ( undetermined type ) it is not possible to differentiate the two components from each other . types i and ii can be classified into subtypes a and b depending on whether the torsion is clockwise or counterclockwise , respectively . a new classification based on preoperative and operative criteria is also used . although the mechanism by which an ileosigmoid knot develops is still speculative , it is generally accepted that certain anatomical predisposing factors are present , including a hypermobile small intestine with an elongated mesentery and a redundant sigmoid colon with a long mesocolon and a short attachment at the base of the mesentery . the knotting leads to closed - loop obstruction and causes gangrene of both the ileal loops and the sigmoid colon within a few hours in most patients . plain abdominal radiographs may show the characteristic double closed - loop obstruction , with the sigmoid colon in the right upper quadrant and the small bowel loops in the left , but this is only an occasional finding . more often , the picture is that of either simple sigmoid volvulus or small bowel obstruction . ct scan reveals the classic whirl sign of volvulus created by the involved , twisted loops of the intestine and the mesocolon . the whirl is reported to be visible on a larger number of contiguous slices in an ileosigmoid knot as compared to sigmoid volvulus . ct scan may show medial deviation of the distal descending colon , with a pointed appearance of its medial border , which is a distinct feature of the ileosigmoid knot . this is presumed to be due to the traction effect causing the peritoneum of the left paracolic gutter to move toward the center of the knot and the mass effect of the distended ileal loops interposing between the descending and proximal sigmoid colon and the left body wall . similarly , the cecum may show medial deviation due to the tightly stretched terminal ileum between the sigmoid mesocolon and the cecum . stretched and elongated superior mesenteric ( along with inferior mesenteric ) vascular structures converging toward the sigmoid colon also indicate the diagnosis . a radial distribution of the intestine and mesenteric vascular structures on ct scan can also suggest the diagnosis . at surgery , a prolonged attempt to untwist the knot is not recommended , especially if the bowel has become gangrenous . the ileosigmoid knot is a rare but life - threatening cause of closed - loop intestinal obstruction . findings of simultaneous ileal and sigmoid ischemia with nonischemic colon interposed in between should prompt suspicion of this diagnosis . the presence of the whirl sign on ct scan medially deviated distal descending colon and cecum , with mesenteric vascular structures from the superior mesenteric vessels converging toward the sigmoid colon can help clinch the diagnosis .	the ileosigmoid knot is an uncommon but life - threatening cause of closed loop intestinal obstruction . its treatment is different from a simple volvulus in that it has to be operated upon immediately . preoperative ct scan diagnosis and prompt treatment can lead to a good outcome . findings of simultaneous ileal and sigmoid ischemia with non - ischemic colon interposed in between should , in an appropriate clinical setting , indicate this condition . the presence of the whirl sign , medially deviated distal descending colon and cecum , and mesenteric vascular structures from the superior mesenteric vessels that converge toward the sigmoid colon on ct scan help clinch the diagnosis .
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Proceed to summarize the following text: the agency for healthcare research and quality ( ahrq ) requested production of a methods guide for comparative effectiveness reviews that specifically addresses the unique challenges of preparing a systematic review of the use of a medical test . this chapter describes the considerations needed when selecting the outcomes that will be included in a systematic review of a medical test . we describe in this chapter the range of effects that medical tests have and how these outcomes from testing should be incorporated into a systematic review to make it maximally useful to those using the review . we define decision - relevant outcomes as the outcomes that result from a testing encounter that may affect the decision to use the test . we consider a broad range of outcomes to illustrate how these may affect the balance of the benefits and risks of the test . the outcomes to be discussed are those that are relevant to screening tests , diagnostic tests , and prognostic tests , although prognostic tests are also discussed in chapter 11 . we also address unique issues that might arise if the test in question is a genetic test ( although genetic tests are explored in more detail in chapter 10 ) . we include a framework for generating potential outcomes for inclusion , and discuss the role of stakeholders in choosing the outcomes for study . finally , we give examples of systematic reviews that either included a range of outcomes in the review or might have done so . investigators are tasked with choosing the outcomes to consider in a systematic review about a medical test . resource limitations require judicious selection from among all possible outcomes , which necessitates setting priorities for the outcomes to include . if reviewers do not explore the full range of outcomes at the outset of the project , the likelihood of excluding important outcomes is high ; the systematic review may miss outcomes relevant to the stakeholder(s ) . the balance of the benefits and harms from testing will be skewed by the absence of information about key outcomes . the consequence may be that recommendations based on the systematic review are inapt when the test is used in practice . additionally , for tests that offer modest clinical gains over another test , information on additional outcomes may be essential for making decisions with the results , like information about costs or convenience . however , we caution that if the initially broad range of outcomes is not carefully condensed , the quality of the review will be threatened by resource limitations . 1 ) figure 1balance of outcomes against resources . balance of outcomes against resources . the narrow review may be incomplete , and the broad review may be too superficial to provide meaningful insights . we recommend a two - step approach for choosing the outcomes for inclusion in a review about a medical test . the first step is to catalog outcomes methodically , and the second is to solicit input from the stakeholder(s ) . below is a description of a conceptual approach to identifying outcomes to ensure that relevant outcomes are not overlooked . the preceding chapter included description of frameworks for designing systematic reviews about medical tests that include consideration of picots ( i.e. , population , intervention , comparisons , outcomes , timing , and setting ) . here we present another framework specifically for thinking about the outcomes from using a test in a clinical setting . here , outcomes are separated into those attributable to the testing process and those attributable to knowledge of the test results . in general , outcomes attributable to the testing process are direct effects of the test ; outcomes attributable to the test results are more plentiful and include the patient s response to the test results and how the patient and clinician act upon the results . bossuyt and mccaffery described a useful framework for thinking about patient outcomes attributable to medical testing.1 they classified outcomes into three groups : ( 1 ) outcomes that result from clinical management based on the test results ; ( 2 ) the direct health effects of testing ; and ( 3 ) the patients emotional , social , cognitive , and behavioral responses to testing . we extend this model by including two additional elements to arrive at five types of outcomes : ( 4 ) the legal and ethical effects of testing , which may or may not be a consideration depending on the test under consideration ; and ( 5 ) the costs of testing . these five categories of outcomes can be associated with the testing process , or with the test result , or with both . we suggest that the relative importance of these outcomes may differ substantially depending on the intention of the test : screening , diagnosis , or prognosis ( table 1 ) . to illustrate , the adverse emotional effects , and the legal and ethical outcomes of testing might be more significant for medical tests used for screening than tests used for diagnosis , due to the high prevalence of false positive test results associated with many tests used for screening purposes . additionally , screening tests are conducted in individuals who are without symptoms of the disease of interest so any adverse or disruptive consequences of testing may be more pronounced . mammography is a useful example since the emotional reaction to a false positive test may be substantial . correspondingly , the potential legal consequences of a false negative test are substantial as a false negative test may lead to the filing of a malpractice suit . missed diagnoses , in particular breast cancer diagnoses , are a large category of radiology - related malpractice suits.2table 1outcomes that might be particularly consequential depending on type of medical testoutcomesscreening testdiagnostic testprognostic testclinical management+++++++direct health effects+++++emotional , social , cognitive , behavioral responses+++++++legal and ethical+++++++costs++++++ outcomes that might be particularly consequential depending on type of medical test systematic reviewers should remember as well that a normal test result , that is a test that has correctly excluded the presence of disease , may be as affecting as a test that has made a diagnosis , and inclusion of outcomes resulting from a negative test may be important in the review . the primary studies of the medical test may have assessed behaviors and consequences after a normal test result , which may include additional testing when a diagnosis is sought or a change in behavior in response to a normal test result ( e.g. , less attention to healthy lifestyle or possibly redoubled efforts at maintaining good health ) . the impact of testing on clinical management is a more important consideration when reviewing diagnostic testing and less important for screening tests where the clinical management may be quite removed from the screening step . a useful example of diagnostic testing is the use of computed tomography ( ct ) for detection of pulmonary embolism : a positive test will result in many months of anticoagulation therapy , an important clinical management consequence for the patient . therefore , systematic reviews will ideally include primary literature that tests the clinical consequences resulting from the use of ct in this setting ( rather than just the sensitivity and specificity and predictive values of the test ) . it is likely that the direct health effects of screening tests are less than in tests used for diagnosis and prognosis : screening tests are generally designed to be less invasive than tests used to make diagnoses in individuals suspected of having disease . an example is pap testing for cervical cancer screening there should be no direct health effects of this process . the range downstream activities that result from a test are also appropriate for consideration as inclusion as outcomes . these may be particularly prominent in imaging tests where there is a high likelihood of identifying unexpected findings that necessitate further evaluation ( e.g. , unexpected adrenal masses seen during abdominal imaging ) or imaging tests that identify unexpected findings that worry the patient ( e.g. , degenerative spine changes seen on chest imaging ) . in these situations , one might consider the emotional and cognitive outcomes of unexpected findings , or the monetary costs of the downstream evaluation of incidentally identified abnormalities . in addition to the direct costs of the test , one might consider the downstream costs triggered by the results of the testing that may include confirmatory testing following a positive result , treatment costs resulting from detecting disease , and costs for treatment of adverse effects of the testing ( direct harms of the test and downstream harms resulting from additional testing or treatment , or evaluation of incidental findings . ) other costs to consider might be the costs to society from direction of funds to testing and away from other services . as an example , one might include , in a systematic review of universal newborn screening , the impact of diverting funding away from other childhood programs ( such as vaccination ) . in addition to consideration of the consequences of testing , we suggest that reviewers also consider an additional axis ; namely , who experiences the outcome . the individual being tested is not the only one who can experience outcomes from the testing process . outcomes may be experienced by family members , particularly in the case of testing an index person for heritable conditions . outcomes may be experienced by the population away from which resources are diverted by a screening activity , e.g. , widespread newborn screening which diverts resources away from population - based smoking cessation activities . society as a whole may experience some outcomes , as when a test of an individual leads to a public health intervention , e.g. prophylactic antibiotics or quarantine after exposure to an infectious individual , or diversion of resources in order to pay for testing of other individuals . payers are affected if they need to pay for a treatment of a newly - diagnosed condition . 2 ) figure 2mapping outcomes to the testing process and to the test results . the range of outcomes that could be included in a systematic review of a test is wide . we encourage investigators doing systematic literature reviews to think through this range of outcomes , considering the testing process , the test results , the range of associated outcomes , and the parties that may experience the outcomes . these considerations may differ depending on the type of test under consideration , as we discuss below , and will differ importantly by the specific test and the question being addressed by the systematic review . because the range of outcomes that a reviewer might include is broad , expecting such reviews to include all possible outcomes is unrealistic . the ahrq methods guide recommends that stakeholders be involved at several steps in the systematic review process.3 we describe additional considerations regarding the role of the stakeholder in reviews of medical tests , as their inputs are particularly relevant to the choice of outcomes for inclusion . little to no empiric evidence exists regarding what outcomes are most essential for inclusion in a systematic review . if the systematic reviewers knew that some outcomes are universally valued by users of reports , these would be routinely included . it is likely , however , that the choice of outcomes depends largely on the needs of stakeholders and how they intend to use the review . clinicians and patients are frequently the primary users of the results of a systematic review and therefore are important stakeholders in the process . an understanding of what evidence the patient or clinician needs in order to make a decision about use of the test is vital in selecting outcomes for inclusion in a review . certainly the health effects of testing and the emotional or behavioral , social , or cognitive outcomes are directly relevant to the patient ; a comprehensive review must include outcomes that are important to patients and would influence their use of a test . to give an example of another stakeholder , the evaluation of genomic applications in practice and prevention ( egapp ) group of the centers for disease control and prevention ( cdc ) has sponsored several epc reports.4 - 6 egapp uses these reports to generate guidelines that the cdc issues about genetic testing . egapp s interests are broad ; it aims to maximize the effectiveness of genetic testing at a societal level . understandably , the outcomes that they consider to be relevant are broad and range from the analytic validity of the test to the impact of the testing process on family members . when the possible outcomes for inclusion are many , the investigators have a responsibility to work with the stakeholder to refine the questions carefully so that the task can be accomplished . other stakeholders , like professional societies such as the american college of physicians , may be most interested in evidence reports that can be used to generate recommendations or guidelines for practicing clinicians . therefore , as stakeholders , they may be more focused on how clinical outcomes vary as a result of medical testing , and perhaps less interested in outcomes that may be more relevant to payers , such as cost - shifting to accommodate costs of testing and downstream costs . not infrequently , the primary users of systematic reviews are federal agencies such as the center for medicare & medicaid services ( cms ) . this agency is responsible for decisions regarding coverage of their beneficiaries medical care , including medical tests . therefore , cms may specify that the outcome most relevant to their coverage decision is the analytic validity of the test , as it would not want to cover a test that inadequately identifies the condition of interest . the researchers doing comprehensive systematic reviews have a role in helping stakeholders to understand the breadth of outcomes . the researchers might assist stakeholders with mapping the range of outcomes depicted in figure 2 . this will allow the stakeholder to review the breadth of outcomes and characterize the outcomes as being more or less vital depending on the intended use of the review . we recommend a two - step approach for choosing the outcomes for inclusion in a review about a medical test . the first step is to catalog outcomes methodically , and below is a description of a conceptual approach to identifying outcomes to ensure that relevant outcomes are not overlooked . the preceding chapter included description of frameworks for designing systematic reviews about medical tests that include consideration of picots ( i.e. , population , intervention , comparisons , outcomes , timing , and setting ) . here we present another framework specifically for thinking about the outcomes from using a test in a clinical setting . here , outcomes are separated into those attributable to the testing process and those attributable to knowledge of the test results . in general , outcomes attributable to the testing process are direct effects of the test ; outcomes attributable to the test results are more plentiful and include the patient s response to the test results and how the patient and clinician act upon the results . bossuyt and mccaffery described a useful framework for thinking about patient outcomes attributable to medical testing.1 they classified outcomes into three groups : ( 1 ) outcomes that result from clinical management based on the test results ; ( 2 ) the direct health effects of testing ; and ( 3 ) the patients emotional , social , cognitive , and behavioral responses to testing . we extend this model by including two additional elements to arrive at five types of outcomes : ( 4 ) the legal and ethical effects of testing , which may or may not be a consideration depending on the test under consideration ; and ( 5 ) the costs of testing . these five categories of outcomes can be associated with the testing process , or with the test result , or with both . we suggest that the relative importance of these outcomes may differ substantially depending on the intention of the test : screening , diagnosis , or prognosis ( table 1 ) . to illustrate , the adverse emotional effects , and the legal and ethical outcomes of testing might be more significant for medical tests used for screening than tests used for diagnosis , due to the high prevalence of false positive test results associated with many tests used for screening purposes . additionally , screening tests are conducted in individuals who are without symptoms of the disease of interest so any adverse or disruptive consequences of testing may be more pronounced . mammography is a useful example since the emotional reaction to a false positive test may be substantial . correspondingly , the potential legal consequences of a false negative test are substantial as a false negative test may lead to the filing of a malpractice suit . missed diagnoses , in particular breast cancer diagnoses , are a large category of radiology - related malpractice suits.2table 1outcomes that might be particularly consequential depending on type of medical testoutcomesscreening testdiagnostic testprognostic testclinical management+++++++direct health effects+++++emotional , social , cognitive , behavioral responses+++++++legal and ethical+++++++costs++++++ outcomes that might be particularly consequential depending on type of medical test systematic reviewers should remember as well that a normal test result , that is a test that has correctly excluded the presence of disease , may be as affecting as a test that has made a diagnosis , and inclusion of outcomes resulting from a negative test may be important in the review . the primary studies of the medical test may have assessed behaviors and consequences after a normal test result , which may include additional testing when a diagnosis is sought or a change in behavior in response to a normal test result ( e.g. , less attention to healthy lifestyle or possibly redoubled efforts at maintaining good health ) . these are all appropriate outcomes for consideration for inclusion in a systematic review . the impact of testing on clinical management is a more important consideration when reviewing diagnostic testing and less important for screening tests where the clinical management may be quite removed from the screening step . a useful example of diagnostic testing is the use of computed tomography ( ct ) for detection of pulmonary embolism : a positive test will result in many months of anticoagulation therapy , an important clinical management consequence for the patient . therefore , systematic reviews will ideally include primary literature that tests the clinical consequences resulting from the use of ct in this setting ( rather than just the sensitivity and specificity and predictive values of the test ) . it is likely that the direct health effects of screening tests are less than in tests used for diagnosis and prognosis : screening tests are generally designed to be less invasive than tests used to make diagnoses in individuals suspected of having disease . an example is pap testing for cervical cancer screening there should be no direct health effects of this process . the range downstream activities that result from a test are also appropriate for consideration as inclusion as outcomes . these may be particularly prominent in imaging tests where there is a high likelihood of identifying unexpected findings that necessitate further evaluation ( e.g. , unexpected adrenal masses seen during abdominal imaging ) or imaging tests that identify unexpected findings that worry the patient ( e.g. , degenerative spine changes seen on chest imaging ) . in these situations , one might consider the emotional and cognitive outcomes of unexpected findings , or the monetary costs of the downstream evaluation of incidentally identified abnormalities . in addition to the direct costs of the test , one might consider the downstream costs triggered by the results of the testing that may include confirmatory testing following a positive result , treatment costs resulting from detecting disease , and costs for treatment of adverse effects of the testing ( direct harms of the test and downstream harms resulting from additional testing or treatment , or evaluation of incidental findings . ) other costs to consider might be the costs to society from direction of funds to testing and away from other services . as an example , one might include , in a systematic review of universal newborn screening , the impact of diverting funding away from other childhood programs ( such as vaccination ) . in addition to consideration of the consequences of testing , we suggest that reviewers also consider an additional axis ; namely , who experiences the outcome . the individual being tested is not the only one who can experience outcomes from the testing process . outcomes may be experienced by family members , particularly in the case of testing an index person for heritable conditions . outcomes may be experienced by the population away from which resources are diverted by a screening activity , e.g. , widespread newborn screening which diverts resources away from population - based smoking cessation activities . society as a whole may experience some outcomes , as when a test of an individual leads to a public health intervention , e.g. prophylactic antibiotics or quarantine after exposure to an infectious individual , or diversion of resources in order to pay for testing of other individuals . payers are affected if they need to pay for a treatment of a newly - diagnosed condition . 2 ) figure 2mapping outcomes to the testing process and to the test results . the range of outcomes that could be included in a systematic review of a test is wide . we encourage investigators doing systematic literature reviews to think through this range of outcomes , considering the testing process , the test results , the range of associated outcomes , and the parties that may experience the outcomes . these considerations may differ depending on the type of test under consideration , as we discuss below , and will differ importantly by the specific test and the question being addressed by the systematic review . because the range of outcomes that a reviewer might include is broad , expecting such reviews to include all possible outcomes is unrealistic . the ahrq methods guide recommends that stakeholders be involved at several steps in the systematic review process.3 we describe additional considerations regarding the role of the stakeholder in reviews of medical tests , as their inputs are particularly relevant to the choice of outcomes for inclusion . little to no empiric evidence exists regarding what outcomes are most essential for inclusion in a systematic review . if the systematic reviewers knew that some outcomes are universally valued by users of reports , these would be routinely included . it is likely , however , that the choice of outcomes depends largely on the needs of stakeholders and how they intend to use the review . clinicians and patients are frequently the primary users of the results of a systematic review and therefore are important stakeholders in the process . an understanding of what evidence the patient or clinician needs in order to make a decision about use of the test is vital in selecting outcomes for inclusion in a review . certainly the health effects of testing and the emotional or behavioral , social , or cognitive outcomes are directly relevant to the patient ; a comprehensive review must include outcomes that are important to patients and would influence their use of a test . to give an example of another stakeholder , the evaluation of genomic applications in practice and prevention ( egapp ) group of the centers for disease control and prevention ( cdc ) has sponsored several epc reports.4 - 6 egapp uses these reports to generate guidelines that the cdc issues about genetic testing . egapp s interests are broad ; it aims to maximize the effectiveness of genetic testing at a societal level . understandably , the outcomes that they consider to be relevant are broad and range from the analytic validity of the test to the impact of the testing process on family members . when the possible outcomes for inclusion are many , the investigators have a responsibility to work with the stakeholder to refine the questions carefully so that the task can be accomplished . other stakeholders , like professional societies such as the american college of physicians , may be most interested in evidence reports that can be used to generate recommendations or guidelines for practicing clinicians . therefore , as stakeholders , they may be more focused on how clinical outcomes vary as a result of medical testing , and perhaps less interested in outcomes that may be more relevant to payers , such as cost - shifting to accommodate costs of testing and downstream costs . not infrequently , the primary users of systematic reviews are federal agencies such as the center for medicare & medicaid services ( cms ) . this agency is responsible for decisions regarding coverage of their beneficiaries medical care , including medical tests . therefore , cms may specify that the outcome most relevant to their coverage decision is the analytic validity of the test , as it would not want to cover a test that inadequately identifies the condition of interest . the researchers doing comprehensive systematic reviews have a role in helping stakeholders to understand the breadth of outcomes . the researchers might assist stakeholders with mapping the range of outcomes depicted in figure 2 . this will allow the stakeholder to review the breadth of outcomes and characterize the outcomes as being more or less vital depending on the intended use of the review . the preceding chapter included description of frameworks for designing systematic reviews about medical tests that include consideration of picots ( i.e. , population , intervention , comparisons , outcomes , timing , and setting ) . here we present another framework specifically for thinking about the outcomes from using a test in a clinical setting . here , outcomes are separated into those attributable to the testing process and those attributable to knowledge of the test results . in general , outcomes attributable to the testing process are direct effects of the test ; outcomes attributable to the test results are more plentiful and include the patient s response to the test results and how the patient and clinician act upon the results . bossuyt and mccaffery described a useful framework for thinking about patient outcomes attributable to medical testing.1 they classified outcomes into three groups : ( 1 ) outcomes that result from clinical management based on the test results ; ( 2 ) the direct health effects of testing ; and ( 3 ) the patients emotional , social , cognitive , and behavioral responses to testing . we extend this model by including two additional elements to arrive at five types of outcomes : ( 4 ) the legal and ethical effects of testing , which may or may not be a consideration depending on the test under consideration ; and ( 5 ) the costs of testing . these five categories of outcomes can be associated with the testing process , or with the test result , or with both . we suggest that the relative importance of these outcomes may differ substantially depending on the intention of the test : screening , diagnosis , or prognosis ( table 1 ) . to illustrate , the adverse emotional effects , and the legal and ethical outcomes of testing might be more significant for medical tests used for screening than tests used for diagnosis , due to the high prevalence of false positive test results associated with many tests used for screening purposes . additionally , screening tests are conducted in individuals who are without symptoms of the disease of interest so any adverse or disruptive consequences of testing may be more pronounced . mammography is a useful example since the emotional reaction to a false positive test may be substantial . correspondingly , the potential legal consequences of a false negative test are substantial as a false negative test may lead to the filing of a malpractice suit . missed diagnoses , in particular breast cancer diagnoses , are a large category of radiology - related malpractice suits.2table 1outcomes that might be particularly consequential depending on type of medical testoutcomesscreening testdiagnostic testprognostic testclinical management+++++++direct health effects+++++emotional , social , cognitive , behavioral responses+++++++legal and ethical+++++++costs++++++ outcomes that might be particularly consequential depending on type of medical test systematic reviewers should remember as well that a normal test result , that is a test that has correctly excluded the presence of disease , may be as affecting as a test that has made a diagnosis , and inclusion of outcomes resulting from a negative test may be important in the review . the primary studies of the medical test may have assessed behaviors and consequences after a normal test result , which may include additional testing when a diagnosis is sought or a change in behavior in response to a normal test result ( e.g. , less attention to healthy lifestyle or possibly redoubled efforts at maintaining good health ) . the impact of testing on clinical management is a more important consideration when reviewing diagnostic testing and less important for screening tests where the clinical management may be quite removed from the screening step . a useful example of diagnostic testing is the use of computed tomography ( ct ) for detection of pulmonary embolism : a positive test will result in many months of anticoagulation therapy , an important clinical management consequence for the patient . therefore , systematic reviews will ideally include primary literature that tests the clinical consequences resulting from the use of ct in this setting ( rather than just the sensitivity and specificity and predictive values of the test ) . it is likely that the direct health effects of screening tests are less than in tests used for diagnosis and prognosis : screening tests are generally designed to be less invasive than tests used to make diagnoses in individuals suspected of having disease . an example is pap testing for cervical cancer screening there should be no direct health effects of this process . the range downstream activities that result from a test are also appropriate for consideration as inclusion as outcomes . these may be particularly prominent in imaging tests where there is a high likelihood of identifying unexpected findings that necessitate further evaluation ( e.g. , unexpected adrenal masses seen during abdominal imaging ) or imaging tests that identify unexpected findings that worry the patient ( e.g. , degenerative spine changes seen on chest imaging ) . in these situations , one might consider the emotional and cognitive outcomes of unexpected findings , or the monetary costs of the downstream evaluation of incidentally identified abnormalities . in addition to the direct costs of the test , one might consider the downstream costs triggered by the results of the testing that may include confirmatory testing following a positive result , treatment costs resulting from detecting disease , and costs for treatment of adverse effects of the testing ( direct harms of the test and downstream harms resulting from additional testing or treatment , or evaluation of incidental findings . ) other costs to consider might be the costs to society from direction of funds to testing and away from other services . as an example , one might include , in a systematic review of universal newborn screening , the impact of diverting funding away from other childhood programs ( such as vaccination ) . in addition to consideration of the consequences of testing , we suggest that reviewers also consider an additional axis ; namely , who experiences the outcome . the individual being tested is not the only one who can experience outcomes from the testing process . outcomes may be experienced by family members , particularly in the case of testing an index person for heritable conditions . outcomes may be experienced by the population away from which resources are diverted by a screening activity , e.g. , widespread newborn screening which diverts resources away from population - based smoking cessation activities . society as a whole may experience some outcomes , as when a test of an individual leads to a public health intervention , e.g. prophylactic antibiotics or quarantine after exposure to an infectious individual , or diversion of resources in order to pay for testing of other individuals . payers are affected if they need to pay for a treatment of a newly - diagnosed condition . 2 ) figure 2mapping outcomes to the testing process and to the test results . the range of outcomes that could be included in a systematic review of a test is wide . we encourage investigators doing systematic literature reviews to think through this range of outcomes , considering the testing process , the test results , the range of associated outcomes , and the parties that may experience the outcomes . these considerations may differ depending on the type of test under consideration , as we discuss below , and will differ importantly by the specific test and the question being addressed by the systematic review . because the range of outcomes that a reviewer might include is broad , expecting such reviews to include all possible outcomes is unrealistic . the ahrq methods guide recommends that stakeholders be involved at several steps in the systematic review process.3 we describe additional considerations regarding the role of the stakeholder in reviews of medical tests , as their inputs are particularly relevant to the choice of outcomes for inclusion . little to no empiric evidence exists regarding what outcomes are most essential for inclusion in a systematic review . if the systematic reviewers knew that some outcomes are universally valued by users of reports , these would be routinely included . it is likely , however , that the choice of outcomes depends largely on the needs of stakeholders and how they intend to use the review . clinicians and patients are frequently the primary users of the results of a systematic review and therefore are important stakeholders in the process . an understanding of what evidence the patient or clinician needs in order to make a decision about use of the test is vital in selecting outcomes for inclusion in a review . certainly the health effects of testing and the emotional or behavioral , social , or cognitive outcomes are directly relevant to the patient ; a comprehensive review must include outcomes that are important to patients and would influence their use of a test . to give an example of another stakeholder , the evaluation of genomic applications in practice and prevention ( egapp ) group of the centers for disease control and prevention ( cdc ) has sponsored several epc reports.4 - 6 egapp uses these reports to generate guidelines that the cdc issues about genetic testing . egapp s interests are broad ; it aims to maximize the effectiveness of genetic testing at a societal level . understandably , the outcomes that they consider to be relevant are broad and range from the analytic validity of the test to the impact of the testing process on family members . when the possible outcomes for inclusion are many , the investigators have a responsibility to work with the stakeholder to refine the questions carefully so that the task can be accomplished . other stakeholders , like professional societies such as the american college of physicians , may be most interested in evidence reports that can be used to generate recommendations or guidelines for practicing clinicians . therefore , as stakeholders , they may be more focused on how clinical outcomes vary as a result of medical testing , and perhaps less interested in outcomes that may be more relevant to payers , such as cost - shifting to accommodate costs of testing and downstream costs . not infrequently , the primary users of systematic reviews are federal agencies such as the center for medicare & medicaid services ( cms ) . this agency is responsible for decisions regarding coverage of their beneficiaries medical care , including medical tests . therefore , cms may specify that the outcome most relevant to their coverage decision is the analytic validity of the test , as it would not want to cover a test that inadequately identifies the condition of interest . the researchers doing comprehensive systematic reviews have a role in helping stakeholders to understand the breadth of outcomes . the researchers might assist stakeholders with mapping the range of outcomes depicted in figure 2 . this will allow the stakeholder to review the breadth of outcomes and characterize the outcomes as being more or less vital depending on the intended use of the review . to explain these points in more detail , we use three examples : one each of a screening test , a diagnostic test , and a prognostic test . in discussing these examples , we consider both outcomes that result from the process of testing and outcomes associated with the results of testing , and those that affect the tested individual and others . we conclude with a discussion of additional considerations when the test is a genetic test . screening tests are used to detect disease in asymptomatic individuals or individuals with unrecognized symptoms.7 screening tests should be able to separate individuals with the disease of interest from those without , and should be employed when there is a treatment available and where early treatment improves outcomes . the us preventive services task force ( uspstf ) develops recommendations for use of clinical preventive services in the united states . an epc is sometimes tasked with preparing the supporting review of the evidence.8,9 other stakeholders have interest in screening tests as well , including professional organizations involved in guideline preparation for their practitioners ; cases in point are recommendations made by the american college of obstetrics and gynecology regarding cervical cancer screening10 and the american cancer society s recommendations for early cancer detection.11 to illustrate outcomes in a systematic review of a screening test , we present the example of a systematic review about screening for bacterial vaginosis in pregnant women.12 this systematic review was first done for the uspstf in 2001 and was later updated . the authors addressed whether screening for bacterial vaginosis during pregnancy in asymptomatic women reduces adverse pregnancy outcomes . they included a review of the clinical management effects that would result from antibiotic treatment based on screening results . these included adverse effects of therapies and the beneficial effects of reduction in adverse pregnancy outcomes , such as preterm delivery . the authors might also have explicitly included an outcome that examines whether the screening leads to receipt of antibiotic treatment whether screening leads to a change in clinical management . this would be a relevant intermediate outcome on the path between screening and the outcomes attributable to therapy . appropriately , the authors of this review did not include outcomes that are a direct result of the testing process because direct test effects are unlikely in this example ; a vaginal swab will not cause any injury . similarly , the test does not confer any direct benefit either except perhaps contact with clinicians . the authors might have also looked at the emotional , social , cognitive , or behavioral effects from the screening process or from the screening test results . it may have been appropriate to consider outcomes that are associated with screening but are not the result of antibiotic therapy . consideration may have been given to the effects of testing positive for bacterial vaginosis , such as emotional responses to a diagnosis of infection leading to either healthier or riskier prenatal activities , or maternal worry as an outcome . as with any measure , the systematic review team might require that the instrument used to measure emotional response be a validated and appropriate instrument . although specifying ethical issues in screening for bacterial vaginosis ( which is not a sexually transmitted infection ) may seem unnecessary , bacterial vaginosis testing may be done as part of an infectious disease screening for reportable diseases such as syphilis or hiv . therefore , a review of the effects of testing should consider whether the test being reviewed might be administered with concurrent screening tests that could themselves raise ethical issues . the authors of this review did not consider the costs of the test to the patient as an outcome . widespread initiation of screening programs , such as on a population level , may have profound cost implications . the authors of this review considered the effects of screening on the mother and on the fetus or infant . however , they might have also considered other relevant parties ; these might include the mother s partner and society , as antibiotic resistance is a conceivable outcome from widespread testing and treatment of bacterial vaginosis . whereas a diagnostic test is applied to confirm or refute disease in a symptomatic person , a screening test is used in an asymptomatic or pre - symptomatic person . the uspstf mostly makes recommendations about screening tests that may be used in the general population ; other organizations are more concerned with ensuring safe use of diagnostic tests in patient populations . payers are also interested in optimizing use of diagnostic tests , as many are costly . we discuss a review that addressed the diagnostic value of 64-slice computed tomography ( ct ) in comparison to conventional coronary angiography.13 stating that their review concerned the accuracy of ct , the authors aimed to assess whether 64-slice ct angiography might replace some coronary angiography for diagnosis and assessment of coronary artery disease . a broader review may consider the effectiveness of ct angiography , and the investigators would consider the full range of outcomes as below . the authors of the review focused exclusively on detection of occluded coronary arteries and not on any downstream outcomes from identification of occluded coronary arteries . individuals diagnosed with coronary artery disease are subjected to many clinical management changes ; these include medications , recommendations for interventions such as angioplasty or bypass surgery , and recommendations for lifestyle changes ; each of which has associated benefits and harms . if one test under consideration identifies more coronary artery disease than another , this will be reflected in clinical management changes and their consequences . other conceivable clinical management effects relate to the impact of testing on other health maintenance activities . for example , a patient might defer other necessary testing ( e.g. , bone densitometry ) to proceed with the ct . we would expect , however , that this would also be the case in the comparison arm . family members may be affected as well by testing ; for instance , they may be called upon to assist the diagnosed patient with future appointments , which may necessitate time away from work and cause emotional stress . the test under consideration it confers no direct benefit itself ( unlike the comparison procedure in which an intervention can be performed at the time of conventional diagnostic angiography ) . the testing process poses potential harms , including allergic reaction to the intravenous contrast material , renal failure from the contrast material , and radiation exposure . the testing process itself is unlikely to have significant emotional consequences , as it is not an invasive test and is generally comfortable for the tested individual . an individual diagnosed with coronary disease might alter his or her lifestyle to reduce disease progression . on the other hand , an individual might become depressed by the results and engage in less self - care or riskier behavior . however , in this example the emotional or behavioral effects are expected to be similar for both ct and conventional angiography and therefore may not be relevant for this particular review . in contrast , they would be relevant outcomes if ct angiography were being compared with no testing . testing could have legal consequences if the tested individual is in a profession that requires disclosure of health threats for the safety of the public ; this might arise if , e.g. , the tested person were an airline pilot . however again , this outcome is not expected to differ between ct and conventional angiography . the relative costs of the two tests to the insurer and the patient , and the costs of diverting equipment away from other uses , could also be of interest to some stakeholders . prognostic tests are tests used in individuals with known disease to predict outcomes . the procedure itself may be identical to a procedure that is used as a screening test or a medical test , but the results are applied with a different purpose . given this , additional considerations for outcomes for example , consider the use of spirometry for predicting prognosis in individuals with chronic obstructive pulmonary disease ( copd ) . the test is commonly used for making the diagnosis of copd and monitoring response to treatment , but the question has been raised as to whether it might also predict survival . in 2005 , the minnesota epc did a systematic review of this topic on behalf of the american thoracic society , american college of physicians , american academy of family physicians , and american academy of pediatrics.14 the discussion below focuses on one of their key questions , which was whether prediction of prognosis with spirometry , with or without clinical indicators , is more accurate than prediction based on clinical indicators alone . although the prognoses for some diseases are minimally modifiable with current treatments , most prognostic information can be used to alter the course of treatment . in this example , spirometry may suggest a high likelihood of progressing to respiratory failure and prompt interventions to avert this ( e.g. , pulmonary rehabilitation efforts , changes in medication , avoidance of some exposures ) . if the likelihood of dying of respiratory failure is high , patients and their physicians may choose to refrain from colonoscopy and other screening procedures from which the patient is unlikely to benefit . similarly , treatments of other conditions may be of less interest if life expectancy is short . spirometry has few direct test effects , although patients can have adverse reactions to testing particularly if challenged with methacholine as part of the test . in general , it is unlikely that tests used for prognosis are more or less likely to have direct test effects than tests used for other purposes . we doubt that many emotional or cognitive effects would arise in response to the testing process . emotional effects to the results of testing are possible ; emotional effects could even be more pronounced for prognostic tests than for screening or medical tests if the test yields more specific information about mortality risk than is usual from a diagnostic test . this could have a range of effects on behavior including efforts to alter prognosis , like smoking cessation . results of tests that provide prognostic information could have legal outcomes , too , especially if the tested individual acts in ways that belie the information he has received ( e.g. , entering into a contract or relationship that he is unlikely to fulfill ) . in this present example , it is unlikely that the prognostic information from spirometry would actually raise these issues , but in other cases , such as a test that demonstrates widely metastatic cancer , this could be an issue . these legal and ethical effects of testing may reach beyond the tested individual and affect society if many individuals have substantial concealed information that influences their actions . the relative costs of the test to the insurer and the patient , relative to the costs of collecting information from a history and physical examination , may all be of interest to stakeholders . chapter 10 of this guide describes in detail unique issues regarding evaluation of genetic tests . with respect to relevant outcomes , we note a few considerations here . genetic information about the tested individual has direct bearing on family members who share genes . this may affect emotional and behavioral outcomes , and ethical outcomes , if family members feel pressured to proceed with testing to provide better information for the rest of the family . recent legislation in the united states prohibits the use of genetic test results to exclude an individual from health insurance coverage , making this less a relevant outcome than in the past . this policy varies worldwide , however , and may be a relevant consideration in some countries . in specifying and setting priorities for outcomes to address in their systematic reviews , investigators should : consider both outcomes that are relevant to the process of testing and those that are relevant to the results of the test.consider inclusion of outcomes in all five domains : clinical management effects , direct test effects ; emotional , social , cognitive and behavioral effects ; legal and ethical effects , and costs.consider to which group the outcomes of testing are most relevant.given resource limitations , prioritize which outcomes to include . this decision depends on the needs of the stakeholder(s ) , who should be assisted in prioritizing the outcomes for inclusion . consider both outcomes that are relevant to the process of testing and those that are relevant to the results of the test . consider inclusion of outcomes in all five domains : clinical management effects , direct test effects ; emotional , social , cognitive and behavioral effects ; legal and ethical effects , and costs . this decision depends on the needs of the stakeholder(s ) , who should be assisted in prioritizing the outcomes for inclusion .	in this chapter of the evidence - based practice centers methods guide for medical tests , we describe how the decision to use a medical test generates a broad range of outcomes and that each of these outcomes should be considered for inclusion in a systematic review . awareness of these varied outcomes affects how a decision maker balances the benefits and risks of the test ; therefore , a systematic review should present the evidence on these diverse outcomes . the key outcome categories include clinical management outcomes and direct health effects ; emotional , social , cognitive , and behavioral responses to testing ; legal and ethical outcomes , and costs . we describe the challenges of incorporating these outcomes in a systematic review , suggest a framework for generating potential outcomes for inclusion , and describe the role of stakeholders in choosing the outcomes for study . finally , we give examples of systematic reviews that either included a range of outcomes or that might have done so . the following are the key messages in this chapter : consider both the outcomes that are relevant to the process of testing and those that are relevant to the results of the test.consider inclusion of outcomes in all five domains : clinical management effects , direct test effects ; emotional , social , cognitive and behavioral effects ; legal and ethical effects , and costs.consider to which group the outcomes of testing are most relevant.given resource limitations , prioritize which outcomes to include . this decision depends on the needs of the stakeholder(s ) , who should be assisted in prioritizing the outcomes for inclusion .
You are an expert at summarizing long articles. Proceed to summarize the following text: the cloning of atp7b , responsible for wilson s disease ( wd ) , provided evidence that wd is a primary hepatic disease caused by toxic copper retained in the liver.13 in addition , various forms of extra - hepatic organ damage occur in patients . they include an acute hemolytic episode of transient jaundice and anemia , neuropsychiatric disorders , and kayser - fleischer ( kf ) rings.47 wd first appears with diverse clinical features , resulting in a delayed diagnosis and poor prognosis . to promote an early diagnosis and consequently improved prognosis , an international study group for wd ( isgw ) proposed the first clinical features consisting of acute hepatic ( h1 ) , chronic hepatic ( h2 ) and neurological ( n1 , etc . ) phenotypes , and a scoring system for the definite diagnosis of wd.8 the phenotyping of isgw has been proposed to simplify the diverse clinical features for diagnosis at the initial presentation , but it does not describe the chronic organ damage of wd which will require life - long treatment . chronic liver disease , designated as phenotype h2 in the isgw proposal , is a fundamental feature of wd.9 neurological disease is a major chronic complication of patients with phenotype n1,10 and hemolytic episodes are major acute complications of patients with phenotype h1.11 affected siblings who have the same genetic background and exposure to similar environmental factors , including foods and drinking water , during their childhood growth in a familial setting may be a good model for the study of the genotype - phenotype correlation of wd , which has been the main clinical issue since the cloning of atp7b.1216 in this retrospective study , we evaluated the clinical features of 23 affected siblings in 11 families using the phenotypes of the isgw proposal at the first appearance8 and the final diagnosis based on chronic organ damage for the life - long treatment of patients.9,11 this study was approved by the review boards evaluating research involving human subjects at the aichi gakuin university school of pharmacy ( nagoya , japan ) and participating hospitals . informed consent for atp7b analysis was then obtained from each patient according to the study protocols approved . two or more siblings with wd found in a family were selected from our database covering the last 20 years . all plural siblings referred to our institutes underwent atp7b analysis for the final diagnosis of wd.17 homozygotes or compound heterozygotes of atp7b responsible for copper toxicosis were diagnostically definitive for wd . in the patients for who genetic diagnosis was incomplete , the scoring system proposed by isgw8 was applied for the final diagnosis of wd . according to the isgw proposal,8 the clinical features at the initial manifestation were classified into h1 , h2 and n1 phenotypes . briefly , the phenotype h1 causes acute jaundice due to hepatitis - like illness or coombs - negative hemolysis in a previously apparently healthy subject , the phenotype h2 is any type of chronic hepatic disease , and the phenotype n1 refers to chronic hepatic disease associated with neuropsychiatric symptoms being present at diagnosis . information on the chronic liver diseases of our patients was complete at the time of atp7b examination . the chronic organ damage of patients for their life - long management was classified into two types : uncomplicated chronic liver disease of wilson ( wd ) and chronic liver disease complicated with neurological disease of wilson ( wd with n).9,11 phenotype combinations at the first appearance and final combinations of chronic organ damage renewed for life - long management were investigated in the 23 affected siblings of the 11 unrelated families . the clinical features , atp7b mutations , first phenotypes and final diagnoses of the 23 siblings affected with wd in the 11 families are summarized in table 1 . twenty - one affected siblings of 10 families were either homozygous or compound heterozygous for the atp7b mutation responsible for wd . the diagnosis of wd in the 2 heterozygous siblings was confirmed by the clinical characteristics of hypoceruloplasminemia , kf rings and copper contents in the liver and urine.8 a proband in the affected siblings ; both siblings visited a hospital on the same day . abbreviations : m , male ; f , female ; h1 , acute hepatic phenotype ; h2 , chronic hepatic phenotype ; n1 , neurological phenotype with neuropsychiatric symptoms and chronic hepatic disease ; wd , uncomplicated chronic liver disease of wilson ; n , neurological disease of wilson ; wd with n , chronic liver disease complicated with neurological disease of wilson . note : two siblings were affected in 10 families , and 3 siblings in 1 family . a younger sibling was the proband in 6 families , while either older or the oldest sibling being the proband in 4 families . nine affected siblings of 4 families were homozygous , 12 affected siblings of 6 families were compound heterozygous , and 2 affected siblings of 1 family were heterozygous for the atp7b mutation responsible for wd . all 4 patients with the phenotype h1 survived their acute episodes with underlying chronic diseases . according to the phenotypes of isgw,8 the identical combination of h2/h2 was found in 4 families and n1/n1 in 1 family , while the different combination of h1/h2 was found in 2 families , h1/n1 in 2 families , h2/n1 in 1 family , and h2/h2/n1 in 1 family . all the patients survived their acute episodes with conservative treatment and short - term anti - copper regimens . the phenotype h1 first appeared in a younger sibling in 3 of the families , and in 1 older sibling in 1 family . re - evaluation of the h1 phenotypes at the recovery stage showed that wd remained in 3 of the survivors and wd with n in 1 survivor . the phenotype h2 or wd was incidentally found in 4 unrelated patients during the investigation of their biochemical liver damage of unknown etiology . there were 6 siblings affected by phenotype n1 , and they comprised h1/n1 in 2 families , h2/h2/n1 in 1 family , h2/n1 in 1 family , and n1/n1 in 1 family . the final organ damage combinations of siblings renewed for their life - long management were wd / wd in 6 families , wd / wd with n in 2 families , wd / wd / wd with n in 1 family , and wd with n / wd with n in 2 families . in the 3 families with split organ damage combination , neuropsychiatric symptoms appeared age - dependently in the older sibling in 2 of the families and in the oldest sibling in 1 family . the identical organ damage of wd with n was found in the relatively aged siblings after atp7b study of family members . the probands were both younger siblings because the genetic study was the first for the older siblings with neuropsychiatric symptoms . the study results obtained from the plural siblings with the same genetic backgrounds and exposure to similar environmental factors before their late teens provided important information regarding the genotype - phenotype correlation of wd . different from other genetic diseases , the phenotype combinations of the affected siblings with wd were significantly modified by the phenotype h1 complicated with self - limiting hemolysis . the phenotypes at the first manifestation and chronic organ damage requiring lifelong anti - copper management were different in 6/11 and 3/11 family members , respectively . physiologically , the liver takes up dietary copper from the portal blood , synthesizes cuproproteins in hepatocytes , and secretes excess copper into the bile via the essential role of the hepatic copper transporter atp7b.4 dysfunction of atp7b causes a sequence of the copper - induced chronic liver diseases of steatohepatitis , chronic hepatitis and cirrhosis in patients.5,6 therefore , the liver disease stages may be age - dependent in most siblings with the same genetic background and similar environmental factors . copper is a hemolytic agent.18 the hemolytic episode of phenotype h1 may occur in wd patients with excess copper stored in the liver and other organs when these organs are incidentally damaged , releasing the toxic copper into the systemic circulation.46 triggers inducing copper overflow may include infectious agents , toxins and drugs . it may be incidental to whether or not either a younger or older sibling is affected by such an external agent . therefore , the phenotype h1 in a sibling with wd could be determined by an environmental factor rather than the age and genetic background.9,11 when the uncomplicated liver disease of wilson is designated wd , the neurological disease designated n is a major extra - hepatic complication of wd.9,10 non - ceruloplasmin - bound copper accumulates increasingly in the brain of wd patients along with the progression of copper - induced liver disease.46 this process may also be age - dependent in affected siblings . in fact , in our family studies , an older sibling with wd was the first to be complicated by n , followed by the identification of wd in a younger sibling . when wd with n in an older sibling had been missed due to various reasons , a younger sibling presented with wd with n. however , the reverse split combination of a younger sibling with wd with n and an older sibling with wd was not found in our case study . such reverse split , suggesting involvements of the genes other than atp7b , epigenetic factors and environmental agents , was reported in neuropsychiatric disorders of relatively aged patients . a male patient with digenic mutations in prnp and atp7b presented with a severe neuropsychiatric disorder , while his older sister with atp7b mutations alone had asymptomatic liver disease of wd.16 two females from two monozygotic twins were first complicated by neurological disease at the ages of 26 and 36 years respectively ; yet , their siblings remained asymptomatic for more than 2 and 3 years respectively.14 two male siblings , at the ages of 16 and 28 in a single family , and a male patient , at the age of 32 in another family , first presented with neurological symptoms , followed by the identification of older female siblings with asymptomatic wd.12 backgrounds other than atp7b may cause a reverse split of the neurological complications in relatively aged pairs of affected siblings , probably because of the prolonged period of exposure to these factors . the phenotype combinations of the siblings with wd in our case series were significantly modified by acute hemolytic episodes , and split chronic organ damage was determined by the age - dependent neurological complications .	abstractbackground and aims : cloning of atp7b provided evidence that wilson s disease is a hepatic copper toxicosis with a variety of extrahepatic complications . affected siblings with the same genetic background and exposure to similar environmental factors may be a good model for the study of genotype - phenotype correlation.methods : twenty - three affected siblings in 11 families were selected from a database . the first phenotypes were determined according to the international proposal . the final types of chronic organ damage were re - evaluated for life - long management.results : phenotypes were identical in 5 of the families and different in 6 of the families . the acute hepatic phenotype h1 was found in 3 younger siblings and 1 older sibling . all survived an acute episode of hemolysis with underlying chronic liver disease . one also presented complication with neurological disease . the neurological phenotype n1 with neuropsychiatric symptoms and hepatic disease was found in 2 aged siblings of 1 family , in an older sibling in 3 families and in the oldest sibling in 1 family . phenotypes in siblings were mainly split by either h1 occurring in random order or age - dependent n1 . types of chronic organ damage were identical in 8 of the families and different in 3 of the families . the same combination of chronic liver disease was found in 6 families and chronic liver disease complicated with neurological disease in 2 families . split organ damage in siblings was found when an older sibling was complicated by neurological disease . there was no reverse combination of a younger sibling being complicated by neurological disease in any of the families.conclusion : phenotype combinations of siblings were mainly modified by externally - induced hemolytic episodes , while chronic organ damage in siblings was split by age - dependent neurological complications .
You are an expert at summarizing long articles. Proceed to summarize the following text: the third stage of labor is still associated with considerable maternal morbidity and mortality . the major complication is postpartum hemorrhage ( pph ) , which affects about 5% of deliveries [ 1 , 2 ] . therefore it is the leading cause of maternal morbidity and mortality worldwide . in western countries , such as the united kingdom , it is the fifth most common reason for maternal death after thromboembolism , preeclampsia / eclampsia , genital tract sepsis , and amniotic fluid embolism . some ten years ago , an editorial titled the retained placenta new insights into an old problem was raising hopes that this problem is to be solved soon . whereas in the event of pph due to atony of the uterus there exist numerous guidelines , recommendations , and flowcharts for its management ; in the treatment of retained placenta the general consensus is more difficult to establish . retained placenta is an important cause of pph and has an incidence of 1 : 100 to 1 : 300 births [ 6 , 7 ] . with this paper our aim was to attract the obstetricians ' attention to the potential risk of retained placenta in the low risk setting where it occurs without prior warning and to present a possible flowchart for the timing of treatment to reduce blood loss and therefore maternal morbidity . in general it can be said that already the duration of the third stage of labour is contributing to the risk of pph as the risk of major bleeding is believed to increase with time elapsed after birth . hence , active management of third stage of labour using prophylactic oxytocics is accepted as standard of care . active management of the third stage of labour involves administration of intravenous oxytocin , early cord clamping , transabdominal manual massage of the uterus , and controlled traction of the umbilical cord . should this appear insufficient , the next step is usually manual removal of the placenta ( mrop ) . however , the timing of this manoeuver is difficult as the risk of pph from leaving the placenta in situ has to be weighed against the knowledge that manual removal can itself cause hemorrhage . it should also be borne in mind that the placenta may be delivered spontaneously up to 30 minutes or more after delivery of the child , without major additional blood loss . the management questions that thus need answering are when and how to detect increased blood loss ? observation of routine practice shows that mrop is regularly deferred beyond the limits recommended . in the absence of immediate evidence of increased vaginal bleeding , management is often conservative and expectant , open to several different options , and paying little attention to the time elapsed since birth . in a study of over 12,000 births , combs and laros similarly , magann et al . found that the risk of hemorrhage increased with time . in their study , the risk of pph was already significantly increased at 10 minutes and , using a receiving operator characteristic ( roc ) curve , they demonstrated that the optimal cut - off time for the prediction of pph was 18 minutes , with a sensitivity of 31% and a specificity of 90% . however , delaying the manual removal will lead to the spontaneous delivery of many placentas . despite scant evidence it is commonly advised that if the placenta has not been expelled 30 minutes after delivery despite active management , mrop should be carried out under anaesthesia . clearly , in the published recommendations the choice of timing for manual removal depends on the facilities available and the local risks associated with both pph and mrop . thus the 2007 intrapartum guidelines produced for the uk government agency the national institute for health and clinical excellence ( nice ) suggest 30 minutes , whereas the who manual for childbirth suggests 60 minutes . accordingly , a survey in europe showed that time until manual removal of placenta in the absence of bleeding varies widely between different countries , from under 30 minutes ( spain and hungary ) to 60 minutes and more ( the netherlands ) . there are different reasons for retained placenta and there is a wide variety in the nomenclature for disturbances in placental disruption . we believe the following classification is sound : placenta adherens is caused by failed contraction of the retroplacental myometrium , incarcerated placenta is caused by a closed or closing cervix , and placenta accreta is caused by abnormal placental implantation . a part of the placenta or the entire placenta is abnormally adherent to the uterine wall without underlying decidua basalis . in placenta increta the placental villi invade into the myometrium , while percreta placenta is classified as placental villi penetrating through the uterine serosa or the adjacent organs , usually the bladder [ 14 , 15 ] . as there is a fair probability for detecting cases with percreta placenta before the onset of labour due to ultrasound and/or magnetic resonance imaging and therefore operative delivery can be planned with all necessary precautions , it is almost impossible for the clinician to detect or even distinguish between placenta accreta and increta despite numerous attempts to do so with several imaging techniques . weeks observed a considerable variation in the retained placenta rate between countries . in less developed countries it is less common ( about 0.1% of all deliveries ) but has a high fatality rate . in more developed countries it affects about 3% of all vaginal deliveries but is very rarely associated with maternal death . it is suggested that interventions common in the most developed countries such as abortions , uterine intervention , labour induction , and use of oxytocin could be contributing to the increase in retained placenta rate with increasing development . commonly named risk factors for disturbances in placental disruption , such as placenta accreta , are history of retained placenta , previous caesarean section , maternal age over 35 years , preterm labour , induced labour , multiparity , previous uterine injury or surgery , uterine malformations , infection , and preeclampsia [ 1 , 3 , 68 , 1418 ] . it is believed that placenta accreta is becoming more common due to the rising caesarean section rate and advancing maternal age , both independent risk factors for placenta accreta [ 2 , 17 ] . history of caesarean section and placenta previa are often of special interest as risk factors for placenta accreta . in a prospective observational cohort study of over 30,000 women who had caesarean delivery without labour , placenta accreta was present in 0.24% of women undergoing their first up to 6.74% of women undergoing their sixth or more caesarean delivery . in women with placenta previa the risk for placenta accreta was 3% , 11% , 40% , 61% , and 67% for first , second , third , fourth , fifth , and sixth or more caesarean deliveries . with every additional caesarean delivery the risk for emergency hysterectomy was rising as well . hysterectomy was required in 0.65% for their first caesarean delivery and increased up to 8.99% for their sixth or more caesarean delivery . in another study the incidence of placenta accreta in case of placenta previa was 5% . with a previous caesarean section , some studies showed promising results by injecting oxytocin into the umbilical cord , as it increased the rate of spontaneous expulsions of the placenta and fewer manual removals of the placenta , but two cochrane reviews , either investigating umbilical cord injection of saline or oxytocin in the routine management of the third stage of labour or for the reduction of mrop , were not able to detect a significant reduction in the need for mrop . nevertheless , umbilical vein injection of oxytocin solution is an inexpensive and simple intervention that could be performed while placental delivery is awaited . however , high - quality randomized trials show that the use of oxytocin has little or no effect . the same review showed a statistically lower incidence in manual removal of placenta if prostaglandin solution was used . eller et al . published a study including 57 cases with placenta accreta , where all women underwent hysterectomy . in 15 cases an attempt was made to remove the placenta manually , but these entire women required immediate hysterectomy for uncontrollable bleeding . the authors of this study concluded that , in case of suspected placenta accreta , scheduled caesarean hysterectomy without attempting placental removal is associated with a significantly reduced rate of early morbidity compared with cases in which placental removal is attempted . diagnosis of placenta accreta is not based on universally valid standard criteria but rather a diagnosis based on the obstetricians ' impression and subjective judgement . some authors use only clinical criteria for the diagnosis of placenta accreta , while others use histopathological criteria , which is not always possible for obvious reasons . some authors distinguish between total and partial placenta accreta , a diagnosis even more difficult to make . as well for the term placenta adherens there is no consensus regarding exact criteria for the definition . this may also be contributing to the highly variable incidence of placenta accreta , with rates reported in literature between 1 : 93,000 and 1 : 110 . aside from patients with placenta previa and patients with a high risk of morbidly adherent placenta due to obstetrical history , the diagnosis of placenta accreta is usually made at the time of delivery . a prenatal screening for placenta accreta , especially for woman with risk factors , would be eligible . a prenatal diagnosis would allow a more planned approach and minimize maternal blood loss . in literature greyscale ultrasonography , colour doppler imaging , and magnetic resonance imaging ( mri ) have been described as alleged successful approaches to diagnose placenta accreta antenatally [ 6 , 15 , 17 , 18 ] . esakoff and colleagues stated that ultrasound examination is a good diagnostic test for accreta in women with placenta previa and found this in consistency with most other studies in the literature . a recent meta - analysis involving 3707 pregnancies showed a sensitivity of 90.72% and a specificity of 96.94% of ultrasound for the antenatal detection of invasive placentation . there is a general consensus that sensitivity and specificity of ultrasound are superior to those of mri ( sensitivity 8085% , specificity 65100% ) , but often both imaging techniques are used in conjunction in women at risk . however , prenatal diagnosis of placenta accreta in absence of further abnormalities of placentation remains a challenge . there are also few biochemical markers named which are thought to have a diagnostic potential , such as elevated levels of maternal serum creatinine kinase , alpha fetoprotein , and -human chorionic gonadotropin . others promisingly described cell - free fetal dna , placental mrna , and dna microarray as potential tools for the diagnosis of abnormalities of placental invasion [ 15 , 26 , 27 ] . but so far there exists no diagnostic tool ready to use in daily routine for prenatal diagnosis of placenta accreta . the sensitivity of theoretically possible test methods also depends on the degree and extent of the abnormal placental invasion . in our experience prenatal diagnosis is almost impossible in the low risk population , where often the parturient is seen in the maternity hospital only for childbirth . we only can assume that these patients most probably have not undergone prenatal ultrasound examination with the very question of morbidly adherent placenta . nevertheless , the use of colour doppler sonography in the third stage of labour has been promisingly introduced by krapp et al . they examined the third stage of labour using greyscale and colour doppler ultrasound . in cases with normal placental separation they found cessation of blood flow between placenta and myometrium immediately after birth . suggestive of placenta accreta was persistent blood flow from the myometrium deep into the placenta demonstrated by colour doppler ultrasound . according to the authors this method allows a quicker diagnosis of placenta accreta and maternal blood loss can be minimized by early manual removal . as an ultrasound machine should be easily available in a well - equipped delivery unit it is advisable to use ultrasound in the third stage of labour complicated by retention of the placenta . with a gain of experience in judging the separation of the placenta from the uterine muscle ultrasound imaging may develop into a useful tool in the management of pathologic third stage of labour . audureau et al . were able to show that the implementation of a multifaceted intervention scheme for the prevention and management of postpartum hemorrhage can be successful . in such way the median delay for second - line pharmacological treatment was significantly shortened from 80 min before introduction to 32.5 min afterwards . comparable to a strict work flow as already developed and implemented in most large delivery units for emergency caesarean section ( target of decision delivery time < 20 min ) a similar standardized protocol for manual removal of placenta might be useful . in figure 1 we present a showcase flowchart for cases with retained placenta with special emphasis on the time frame . we believe that already strict observation of time , use of ultrasound for evaluation of the grade of placental detachment , and early involvement of support staff ( i.e. , second midwife , anaesthesiologist ) might contribute to a reduction of maternal morbidity . needless to say , the suggested time frame is only applicable in the absence of increased vaginal bleeding , and its efficacy has to be proven in a controlled trial . in case of an increased blood loss during third stage of labour ideally standardized operating procedures are already implemented . in conclusion , retained placenta remains a problem of the third stage of labour , which in the low risk setting usually is occurring without prior warning . in daily routine adherence to a strict protocol of active management of third stage of labour may be helpful to minimize time interval between birth and delivery of placenta and therefore minimize postpartum complications .	the third stage of labor is associated with considerable maternal morbidity and mortality . the major complication is postpartum hemorrhage ( pph ) , which is the leading cause of maternal morbidity and mortality worldwide . whereas in the event of pph due to atony of the uterus there exist numerous treatment guidelines ; for the management of retained placenta the general consensus is more difficult to establish . active management of the third stage of labour is generally accepted as standard of care as already its duration is contributing to the risk of pph . despite scant evidence it is commonly advised that if the placenta has not been expelled 30 minutes after delivery , manual removal of the placenta should be carried out under anaesthesia . pathologic adhesion of the placenta in the low risk situation usually is diagnosed at the time of delivery ; therefore a pre- or intrapartum screening opportunity for placenta accreta would be desirable . but diagnosis of abnormalities of placentation other than placenta previa remains a challenge . nevertheless the use of ultrasound and doppler sonography might be helpful in the third stage of labor . an improvement might be the implementation of standardized operating procedures for retained placenta which could contribute to a reduction of maternal morbidity .
You are an expert at summarizing long articles. Proceed to summarize the following text: the property that most clearly separates tnts from other cellular protrusions in vitro is their straight , bridge - like structure , interconnecting cell pairs . in vitro imaging has shown that the length of tnts displays large variation , differing between cell lines ( table 1 ) . tnts connecting t cells , for example , were reported to have an average length of 22 m , whereas in pc12 cells , the length was found to be much less . the tnt lengths can vary as the connected cells migrate and the distances between them change , indicating that tnt length can be dynamically regulated . in addition , some cells show a negative correlation between the tnt lifetime and the cell migration speed . statistical analysis of tnt length will provide information about the effective distance for tnt formation , and also the threshold distance for tnt - dependent cell - to - cell communication . wga , wheat germ agglutinin ; did , vybrant did celllabeling solution ; sem , scanning electron microscopy ; tem , transmission electron microscopy ; dic , differential interference contrast ; afm , atomic force microscopy . * unpublished data ; * * all nanotubes contained actin , and a subgroup also contained microtubules ; * * * all nanotubes contained microtubules , and a subgroup also contained actin ( 65% ) . measuring the diameter of tnts using light microscopy can not be done with adequate accuracy due to the resolution limit . transmission electron microscopy analysis has revealed that tnts have a diameter in the range of 50200 nm in pc12 cells and 180380 nm in t cells ( table 1 ) . however , to preserve and search for intact tnts in series of sample slices is laborious . confocal microscopy has shown that some tnts reach thicknesses of over 700 nm , which could be due to incorporation of additional components inside the tnts , such as microtubules . it should also be noted that multiple thin tnts could stick together to form what looks like a single , thick tnt ( unpublished data ) . since cells after division sometimes form transient thin intercellular connections containing a midbody ring , a double labeling can help to distinguish tnts from incompletely divided cells . tnts are not empty membrane tubes , but filled with cytoskeletal filaments ( table 1 ) . f - actin is found in most tnts , spanning uniformly along their entire length , and is thus an important labeling target in tnt - imaging . f - actin also plays a crucial role in the formation of tnts , as shown in experiments where incubation with f - actin depolymerization drugs , such as cytochalasin b , inhibit tnt formation . in addition , evidence show that various cellular components are transported inside tnts in the speed range of f - actin - associated myosin - motors . besides f - actin , microtubules are also detected in tnts in a few cell lines , such as immune cells , between primary neurons and astrocytes , and in huvec cells during cancer - induced angiogenesis . why and how microtubules are present in some tnts remains to be investigated . as with f - actin and myosin , microtubules could serve as tracks for transport of cargo via a kinesin / dynein - mechanism . furthermore , microtubule - filaments have shown a bending stiffness many orders of magnitude higher than that of actin - filaments . thus , incorporation of microtubules could provide a high degree of rigidity and longer lifetime to the tnt . transmembrane proteins and membrane - binding proteins , such as n - cadherin and myosin x , are considered necessary in the recognition of and attachment to target cells during tnt formation ( unpublished data ) . for example , interposed gap junctions on the tnt / cell - contact site allow transmission of depolarization signals . moreover , the accumulation of mhc class i chain - related protein a ( mica ) at the tip of nanotubes of natural killer cells can induce immune responses in target cells . in t cells , the transfer of endogenous fasl from effector cells can result in apoptosis in the receiving cell . finally , certain membrane components could accumulate passively along tnts , thus becoming a potential marker for the imaging of tnt - like structures . in support of this theory , experiments with liposomes have shown that both specific proteins and lipids can be sorted spontaneously into artificial nanotubes . in addition to the tnts described above , some extracellular protrusions can be considered tnt - like , as they share some of the characteristics of tnts ( table 2 ) . besides filopodia cytonemes are long and thin protrusions containing f - actin found in the drosophila imaginal disc . they facilitate peripheral uptake and subsequent transport of extracellular signaling molecules toward the cell body . it is not clear if these protrusions attach to other cells like tnts do , or if they just act as periscope - like sensors . another tnt - like structure was observed in cultured b cells upon antibody - opsinization . the streamers also contain f - actin , and do not form when incubated with the f - actin depolymerization drug cytochalasin d. however , the streamers , like cytonemes , do not necessarily attach to other cells . when they do make contact to other cells , evidence suggests that this confers a protective effect to complement - mediated cell lysis . in 2011 , a new kind of thin extracellular protrusions , called a nanopodium , was reported . nanopodia emanate from endothelial cells expressing the tetraspanin - like protein tm4sf1 , which is also found in puncta along the protrusions . being relatively deficient in f - actin , and also not necessarily attached to other cells , their function in intercellular communication is uncertain . although the morphology of the structures discussed above resembles tnts closely , they usually do not interconnect cells . however , it is reasonable to believe that the cell employs much of the same machinery to form these structures , and some could possibly be tnt - precursors under certain conditions since tnts can derive from filopodia . the search for tnts in tissue is important not only to establish their presence in multicellular organisms , but also as a key step to understand their physiological functions . the development of fluorescent protein tags and advanced confocal microscopy have facilitated tnt identification in vivo by enabling the labeling of specific proteins only present in a certain subpopulation of cells . in chick embryos , tnt - like structures were successfully identified between neural crest cells after this specific cell population had been labeled with fluorescent fusion proteins . there are similar findings from gastrulation - stage embryos , and in immune cells in the adult mouse cornea . tnts between neural crest cells have been shown to mediate the transfer of cellular material , indicating that tnts play a functional role also in vivo . to date , most of the reports on tnts in vivo are studies on embryos , which might hint at a role of tnts during the development of multicellular organisms . although the tnts in tissue share many morphological features with tnts in vitro , there are still dissimilarities that can be ascribed to the complex microenvironment in vivo . contorted tnts have been found both in healthy tissue , such as in the mouse cornea , as well as in tumor tissue . this effect could possibly be due to obstacles , such as other cells and dense extracellular matrix , preventing the protrusion from connecting at the shortest distance between the two cells . indeed , when there are no obstacles between the cells , such as between the rims of the neural folds during neural tube closure , straight tnt - like structures can be found in vivo . in addition , extracellular matrix could in principle provide structural stability and protection from external forces when it is surrounding the tnts . in a recent paper elaborating on tnts in the cornea , the lifetime was measured to be more than 90 min . a study on primary t cells inside an artificial 3d matrix revealed long - lived tnts with curved morphologies , in accordance with in vivo studies . due to the difficulty of imaging tnts in vivo , details about the composition and structure of tnts in complex tissue environments remain to be investigated . certainly , such research will expand our knowledge about tnts , and may also give information about their function and the mechanism of formation in tissue . the heterogeneous morphology and composition of tnts suggests that tnts may form in different ways . according to live imaging of cultured cells , the formation of tnts occurs by filopodial interplay ( making contact ) or cell dislodgement ( keeping contact ) . recent research on the molecular level has revealed that the unconventional motor protein myosin x , usually associated with filopodia , promotes tnt formation by interacting with several transmembrane proteins , supporting the notion that tnts can derive from filopodia ( unpublished data ) . moreover , the m - sec protein has been demonstrated to be an important regulator of tnt formation . it was also involved in p53 and mhc class iii protein lst1 induced tnt formation . however , recent observation that p53 is not a master protein for tnt formation in every cell type , and the fact that tnts have been observed between cells that do not express m - sec , suggest that different mechanisms of tnt formation may exist in different cell types . therefore , there may be a certain level of one - to - one relationship between the diversity of tnts and the molecular determinants of their formation . several studies support the notion of a correlation between the structure and the function of tnts . an interesting example is the tnt - dependent propagation of calcium - fluxes between cells , for which at least three ways have been described in different cell lines . first , tnt - mediated electrical coupling can transfer depolarization from one cell to another via interposed gap junctions , and thus elicit a depolarization - dependent calcium uptake in the connected cell through voltage - gate channels . second , ins(1,4,5)p3 receptors bound to the endoplasmic reticulum inside tnts actively propagate intercellular calcium signals along tnts via calcium - induced calcium release . lastly , calcium itself can diffuse directly through tnts , thereby rising calcium level in the receiving cell . obviously , in mediating this calcium flux , the tnts need to fulfill certain structural requirements . in the first model , the presence of gap junctions on the contact site between the tnt and the target cell is necessary to transfer electrical depolarization . in the two latter models , a tnt with a larger luminal diameter is required to allow the entry of endoplasmic reticulum or efficient diffusion of calcium from one cell to another . in addition , motor proteins might be necessary to mediate the entry of endoplasmic reticulum into tnts . thus , the structural and compositional properties of the tnts determine the possibilities and limitations of their functions that can be performed . future research on tnts will inevitably increase our knowledge about the morphological and structural diversity of tnts during embryo development , tissue homeostasis , and pathological processes . however , to resolve the molecular mechanisms underlying the diversity , as well as to depict the genetic , cellular and species - specific variation , represents a great challenge . in addition , to determine the relationship between the diversity and the different functions of the tnts , particular at the tissue level , is an important task in this research field .	tunneling nanotubes ( tnts ) are recently discovered thin membranous tubes that interconnect cells . during the last decade , research has shown tnts to be diverse in morphology and composition , varying between and within cell systems . in addition , the discovery of tnt - like extracellular protrusions , as well as observations of tnts in vivo , has further enriched our knowledge on the diversity of tnt - like structures . considering the complex molecular mechanisms underlying the formation of tnts , as well as their different functions in intercellular communication , it is important to decipher how heterogeneity of tnts is established , and to address what roles the compositional elements have in the execution of various functions . here , we review the current knowledge on the morphological and structural diversity of tnts , and address the relation between the formation , the structure , and the function of tnts .
You are an expert at summarizing long articles. Proceed to summarize the following text: the presence of cardiovascular dysfunction is an early and fatal complication for patients with sepsis or septic shock , with the mortality rate of 70% to 90% compared with 20% in septic patients without cardiovascular impairment [ 1 , 2 ] . and approximately 64% of patients with severe sepsis or septic shock presented with myocardial depression including left ventricular diastolic and systolic dysfunction and right ventricular dysfunction . cardiovascular dysfunction during sepsis is probably multifactorial , and inflammatory cytokines have been shown to be clearly associated with it , but the precise molecular mechanisms remain unclear . furthermore , the previous study showed that proinflammatory mediators , such as tumor necrosis factor- ( tnf- ) and interleukin-1 ( il-1 ) , played a critical part in myocardial dysfunction during sepsis . triggering receptor 1 expressed on myeloid cells ( trem-1 ) , a newly discovered receptor of the immunoglobulin superfamily , activates neutrophils and monocytes / macrophages through an associated signal transduction molecule dap12 . engagement of trem-1 has been reported to trigger the synthesis of proinflammatory cytokines such as tnf- and il-1 in the presence of lps , leading to systemic inflammatory response syndrome [ 79 ] . as described above , the release of proinflammatory cytokines ( tnf- and il-1 ) leads to cardiac depression or dysfunction following sepsis . hence , we hypothesize that trem-1 could play an important role in lipopolysaccharide ( lps)-induced cardiac dysfunction . furthermore , our previous observation found that serum strem-1 level was significantly associated with cardiac index ( ci ) , global ejection fraction ( gef ) , and left ventricular systolic index ( dp / dt max ) in patients with severe sepsis . thus , the purpose of this study was to investigate the association of myeloid cells of triggering teceptor-1 with left ventricular systolic dysfunction in balb / c mice with sepsis . lps ( escherichia coli 055:b5 ) was purchased from sigma chemical co. ( st . louis , mo , usa ) ; mouse trem-1 monoclonal antibody ( mab ) was purchased from r&d systems inc . lps was dissolved in sterile saline to a concentration of 1.0 mg / ml , and trem-1 mab was reconstituted at 0.5 mg / ml in sterile pbs . all experiments were conducted in accordance with the guidelines for the care and use of laboratory animals by the experimental animal care and ethical committee of nanfang medical university . ninety male balb / c mice ( age : 7 to 9 weeks and weight : 20 to 25 g ) were purchased from nanfang medical university experimental animal center ; they were randomly divided into 3 groups , each of 30 animals . lps , lps / trem-1 , and control groups were injected intraperitoneally with 25 mg / kg lps , 5 g trem-1 mab 1 h after lps challenge , and sterilized normal saline , respectively . each group was further divided into 3 subgroups according to different time - points , that is , 6 h , 12 h , and 24 h. the left ventricular systolic function was evaluated in 5 mice of each subgroup , and the myocardium and blood sample were collected in the remainder at 3 time - points . all animals were housed in plastic cages ( 5 mice per cage ) under standard laboratory conditions and mice were maintained on a 12 h light / dark cycle with free access to regular rodent chow and tap water . the experimental animal models were redone to get the blood and tissue specimens or echocardiographic assessment if the mice died before / at 3 time - points . according to the report of ha et al . , 2d guided m - mode echocardiography with 15l8 probe ( contrast pulse sequencing , sequoia 512 , siemens , germany ) was used to assess mouse left ventricular systolic function at 3 time - points . m - mode tracings were used to measure the left ventricular end - systolic diameter ( lvesd ) and left ventricular end - diastolic diameter ( lvedd ) from 5 consecutive cardiac cycles . left ventricular fractional shortening ( fs ) index and ejection fraction ( ef ) index were calculated as described previously . all measurements were made by the same observer who was blinded with the experimental grouping . serum and myocardial concentrations of tnf- , il-1 , and strem-1 in all mice at 3 time - points were determined using elisa kits ( jianglai , shanghai , china ) according to the manufacturer 's protocols . total rna was extracted from frozen cardiac tissues obtained from all mice at 3 time - points , using rnazol solution ( biogenesis ) according to the manufacturer 's instructions . myocardial tnf- , il-1 , and strem-1 expression were measured by quantitative real - time rt - pcr with icycler iq real - time pcr system ( bio - rad ) . primer pairs used were as follows : tnf- : forward 5-ccccaaagggatgagaagttc-3 , reverse 5-ggcttgtcactcgaattttgaga-3 ; il-1 : forward 5-ccaaaagatgaagggctgctt-3 , reverse 5-gaaaagaaggtgctcatgtcctc-3 ; strem-1 : forward 5-gacgggaaggaacccttga-3 , reverse 5-catggcctcactagggtcatgt-3 ; gapdh : forward 5-cgtgttcctacccccaatgt-3 , reverse 5-tgtcatcatacttggcaggtttct-3. tnf- : forward 5-ccccaaagggatgagaagttc-3 , reverse 5-ggcttgtcactcgaattttgaga-3 ; il-1 : forward 5-ccaaaagatgaagggctgctt-3 , reverse 5-gaaaagaaggtgctcatgtcctc-3 ; strem-1 : forward 5-gacgggaaggaacccttga-3 , reverse 5-catggcctcactagggtcatgt-3 ; gapdh : forward 5-cgtgttcctacccccaatgt-3 , reverse 5-tgtcatcatacttggcaggtttct-3. each sample was tested in triplicate for each gene ; relative levels of gene expression were normalized to gapdh expression levels . paraffin embedded heart tissues were sectioned and stained with hematoxylin and eosin as previously described . histopathological examination was observed by an expert who was unaware of the experimental allocation of the animals . statistical analysis was performed using graphpad prism 5.0 ( graphpad software , san diego , california , usa ) . to study the potential function of trem-1 in the pathogenesis of myocardial dysfunction in septic mice , we observed that lps / mabtrem-1-treated and lps - treated mice showed signs of sepsis such as absence of grooming activities with resulting ruffled fur , olfactory discharge , and diarrhea , no oral uptake of food or water , and lethargy compared with saline - treated controls after lps administration at different times . besides , few of mice in lps and lps / mabtrem-1 group died within the first 24 h. to investigate the effect of trem-1 in the pathogenesis of left ventricular systolic dysfunction in septic mice , we employed echocardiography as a noninvasive approach to assess left ventricular systolic function at 6 h , 12 h , and 24 h in 3 groups . fs and ef were severely decreased in both lps - treated and lps / mabtrem-1-treated mice , when compared to the control group . lps - induced left ventricular systolic dysfunction was evident at 6 h , pronounced at 12 h , and gradually returned to near baseline values at 24 h after lps challenge . as expected , 12 h after lps / mabtrem-1 challenge , the mice had a lower fs and ef than did lps - treated mice ( fs : 50.7% versus 36.1% at 12 h and ef : 87.5% versus 72.3% , both p < 0.05 , figure 1 ( 12c ) and ( 12d ) ) . however , there was no significant difference of both fs and ef between lps - treated and lps / mabtrem-1-treated mice at 6 h and 24 h ( figure 1 ( 6c ) , ( 24c ) , ( 6d ) , and ( 24d ) , p > 0.05 ) . interestingly , no significant differences were observed on lvesd and lvedd between lps and lps / mabtrem-1 groups at 3 time - points ( figure 1 ( 6a)(24a ) and ( 6b)(24b ) , p > 0.05 ) . to investigate the effect of trem-1 pretreatment on production of cytokines in septic mice induced by lps , the levels of tnf- , il-1 , and strem-1 in serum and myocardium were determined at protein levels by elisa at 3 time - points among 3 groups . as shown in figure 2 , the basic serum and myocardial tnf- , il-1 , and strem-1 levels in control mice were lower detectable ; normal saline did not influence these values . the levels of tnf- , il-1 , and strem-1 were significantly increased in myocardium and serum of both lps / mabtrem-1-treated and lps - treated mice compared with saline - treated mice at 3 time - points , respectively ( figures 2(a)2(f ) , p < 0.05 ) . myocardium tnf- expression was markedly increased in lps / mabtrem-1-treated mice in comparison with lps - treated at 6 h and 24 h ( figure 2(a ) , p < 0.05 ) . however , no significant differences were found in serum tnf- expression between lps and lps / mabtrem-1 mice at 3 time - points . serum il-1 expression in lps / mabtrem-1-treated mice exhibited marked increases compared with the lps - treated mice at 6 h ( figure 2(b ) , p < 0.05 ) and indicated that administration of mabtrem-1 to endotoxemic mice markedly increased in il-1 expression of serum . but there were no significant differences in myocardium il-1 expression between lps - treated and lps / mabtrem-1-treated mice at 3 time - points . serum strem-1 expression in lps / mabtrem-1-treated mice was significantly increased compared with lps - treated mice at 12 h and 24 h ( figure 2(c ) , p < 0.05 ) , and the same trend was observed in myocardium at 6 h between lps / mabtrem-1-treated and lps - treated mice ( figure 2(e ) , p < 0.05 ) . as shown in figure 3 , myocardial tnf- , il-1 , and strem-1 expression among 3 groups at 3 time - points were examined at transcript levels by real - time pcr . the levels of myocardial tnf- , il-1 , and strem-1 mrna were increased between lps / mabtrem-1-treated and lps - treated mice after lps exposure compared to those of saline - treated controls . cardiac tnf- mrna expressions in lps - treated and lps / mabtrem-1-treated mice were markedly higher than in saline - treated mice at 12 h and 24 h. and the same trend on il-1 mrna expression was found at 3 time - points between the two groups . similarly , strem-1 mrna expression was significantly greater in lps / mabtrem-1-treated mice and lps - treated mice than in saline - treated mice at 24 h , respectively ( figures 3(a ) , 3(b ) , and 3(c ) , p < 0.05 ) . myocardial tnf- mrna expression in lps / mabtrem-1-treated mice was markedly higher than in lps - treated mice at 24 h ( figure 3(a ) , p < 0.05 ) . however , there were no significant differences at the other 2 time - points ( figure 3(a ) , p > 0.05 ) . although the levels of myocardial il-1 and strem-1 mrna in lps / mabtrem-1-injected were higher than in lps - treated mice at 6 h , 12 h , and 24 h , there were no statistical differences ( figures 3(b ) and 3(c ) , p > 0.05 ) . to evaluate the role of trem-1 in the pathogenesis of myocardial impairment during sepsis , morphological examination displayed no significant difference in heart tissues of control group mice under light microscopy levels at 3 time - points . hearts from lps - treated mice displayed a mild feature of myocardial damage to some degree , including irregular arrangement and interstitial edema , infiltration of inflammatory cells , and slight hemorrhage at 3 time - points . moreover , hearts from mice pretreatment with lps / mabtrem-1 also displayed myocardial damage to a certain extent at 6 h , 12 h , and 24 h. as expected , compared with these changes found in time - matched lps mice , these lesions were considerably more severe in lps / mabtrem-1-treated mice ( figure 4 ) . sepsis is the most important cause of morbidity and mortality in intensive care units ( icus ) , and myocardial dysfunction frequently accompanies severe sepsis and septic shock [ 1 , 2 ] . the previous study has showed that myocardial depression in septic shock has been well characterized in both spontaneous human septic shock and in experimental animal models of septic shock [ 1618 ] . similarly , this study indicated that left ventricular systolic dysfunction was observed in septic mice . we also observed that lps treatment caused significant decrease of both ef and fs when compared with saline - treated mice at 6 h and 12 h. although the pathophysiology responsible for lps - induced myocardial dysfunction remains unclear , several studies have shown that the large release of inflammatory mediators was an important factor . we observed that serum tnf- , il-1 , and strem-1 production was markedly increased in mice pretreatment with lps which accompanied a significant decrease of both left ventricular ef and fs compared with saline - treated mice at 6 h and 12 h. furthermore , the previous experiments have showed hemodynamic changes and left ventricular systolic and diastolic dysfunction in mice after tnf- or il-1 administration . besides , inhibition of tnf- or il-1 can mitigate multiple organ dysfunctions and improve survival in septic mice [ 2022 ] . thus , it is suggested that tnf- , il-1 , and strem-1 in serum played an important part in lps - induced left ventricular systolic dysfunction . the previous experiments showed that tnf- and il-1 were important inflammatory cytokines during sepsis , which were mainly produced by activated macrophages and cardiomyocytes . as shown in figures 1 and 3 , the data indicating that lps treatment caused marked increase of both myocardial tnf- , il-1 , and strem-1 protein and gene expression were paralleled by significant decrease of both ef and fs when compared with saline - treated mice at same time - points . as described above , lps treatment did cause significantly decrease of both ef and fs in comparison with the saline - treated mice , the inflammatory cytokines in serum and myocardium expression were markedly increased at the same time . hence , we concluded that inflammatory cytokines played a critical part in left ventricular systolic dysfunction during sepsis . due to various metabolic cycles , the difference of inflammatory cytokines between protein and gene expression appeared at different time - points . trem-1 is a newly discovered cell receptor , expressed on the surface of neutrophil and monocyte / macrophage , and can activate inflammation through trem-1/dap12 inflammatory signaling pathway [ 68 ] . as observed in this study , serum il-1 ( 6 h ) and strem-1 ( 12 h and 24 h ) expression were significantly higher in lps / mabtrem-1-treated mice than in time - matched lps - treated mice ; meanwhile , we also observed the same trend on tnf- ( 6 h and 24 h ) and strem-1 ( 6 h ) in myocardium between the 2 groups . trem-1mab that amplified the inflammation during sepsis was confirmed again in our study . on the other hand , the previous study indicated that blocking of trem-1 can protect mice against lps- or clp - induced sepsis by mtrem-1/iggl or lpl7 . moreover , the mechanisms of lps and trem-1 signaling pathway reported that lps binds to an acute - phase protein , lps - binding protein ( lbp ) , and activates cd14 on cell surfaces [ 24 , 25 ] . subsequently , a complex is formed among lps / lbp , cd14 , and a secreted protein ( md2 ) , with toll - like receptor ( tlr)-4 , one of the recently described family of toll - like receptors [ 26 , 27 ] . next , expression of trem-1 may be under the control of nuclear factor-b , with engagement of trem-1 during sepsis possibly leading to activation of several transcription complexes that synergize with nf-b in order to elicit transcription of proinflammatory genes . at last , large amounts of inflammatory cytokines release from activated inflammatory cells result in inflammation . obviously , the similar reaction occurred in myocardium and serum during sepsis as this study . as our data indicated , mabtrem-1 treatment indeed caused marked decrease of both ef and fs when compared with the lps - treated mice ; the same trend on serum / myocardium proinflammatory cytokines expression was observed at the same time . as mentioned above , mab trem-1 stimulates the expression of large amounts of trem-1 during endotoxemia . subsequently , trem-1 can activate a cascade of proinflammatory cytokines in serum or myocardium ( such as tnf- , il-1 , or strem-1 ) , which appears to be a mediator of cytokine - induced left ventricular systolic dysfunction ( fs and ef ) . besides , we have previously reported the close relationship between cardiac dysfunction and serum strem-1 . furthermore , trem-1 that aggravated lps - induced myocardial damage was observed by histopathological examination . thus , trem-1 as an amplifier of the inflammatory response played a pivotal role in the pathogenesis of lps - induced left ventricular systolic dysfunction . taken together , our present study demonstrated for the first time the association of trem-1 with myocardial function in septic mice ; as described above , trem-1 had indeed proinflammatory properties and played an important role in the pathogenesis of lps - induced left ventricular systolic dysfunction . limitation of the present study : our work is focused on the characteristics of trem-1 in lps - induced left ventricular systolic dysfunction in vivo . to understand the mechanism of trem-1 in lps - induced cardiac dysfunction , further studies with isolated myocardium from trem-1 and wild mice under lps stimulation are needed , which may offer direct evidence for the effects of trem-1 under lps . in summary , our results suggest that trem-1 is significantly associated with lps - induced left ventricular systolic dysfunction in balb / c mice . the overexpression of trem-1 plays an important role in the pathogenesis of lps - induced left ventricular systolic dysfunction in mice . thus , trem-1 might be a therapeutic target for the treatment of cardiovascular dysfunction in sepsis .	objective . to investigate the correlation between trem-1 and lps - induced left ventricular systolic dysfunction in balb / c mice . methods . male balb / c mice were randomly divided into 3 groups : lps , lps / trem-1 , and control groups which were injected intraperitoneally with 25 mg / kg lps , 5 g trem-1mab 1 h after lps challenge , and sterilized normal saline , respectively . left ventricular systolic function was monitored by echocardiography at 6 h , 12 h , and 24 h. meanwhile , tnf- , il-1 , and strem-1 in serum and myocardium were determined by elisa or real - time pcr ; at last left ventricles were taken for light microscopy examination . results . fs and ef in lps / mabtrem-1 group , significantly declined compared with lps and control group at 12 h , were accompanied with a markedly increase in serum il-1 ( at 6 h ) and strem-1 ( at 12 h and 24 h ) expression . myocardium tnf- ( at 6 h and 24 h ) and strem-1 ( at 6 h ) were significantly higher in lps / mabtrem-1-treated mice than in time - matched lps - treated mice ; meanwhile myocardium tnf- mrna were markedly increased in comparison with lps - treated or saline - treated mice at 24 h. besides , mabtrem-1 aggravated lps - induced myocardial damage was observed . conclusions . our results suggest that trem-1 is significantly associated with lps - induced left ventricular systolic dysfunction in balb / c mice .
You are an expert at summarizing long articles. Proceed to summarize the following text: pregnancy is characterized by dramatic and reversible changes in cardiovascular hemodynamic to meet the increased metabolic demands of the mother and the fetus . early changes are due , in part , to the metabolic demands brought on by the fetus , placenta and , to the increasing levels of pregnancy hormones , particularly those of progesterone and estrogen . later changes , starting in mid - pregnancy , are anatomical in nature and are caused by mechanical pressure from the expanding uterus ( 15 ) . heart rate increases by 10% t o 20% , and stroke volume is significantly increased as early as the eighth week of pregnancy . a 30% to 60% increase in cardiac output begins to appear early in the first trimester ( 6 ) . blood volume increases by 30% to 40% , so that it is 1200 to 1600 ml greater than it is in the non - pregnant state ( 7 ) . this increase begins as early as 6 weeks of gestation and plateaus by the third trimester . this dramatic change seems to be driven by increase in preload and stroke volume early in pregnancy and is maintained by an increase in heart rate late in pregnancy . particularly in the last trimester when the patient is in the supine position , the gravid uterus compresses the inferior vena cava , decreasing systemic venous return and preload and causing an acute reduction in cardiac output of up to 25% . use of the left lateral decubitus position can prevent this and allows reliable and reproducible hemodynamic measurements ( 1 , 5 ) . the compliance of the arterial and venous systems increases in parallel with the expanding blood volume , suggesting that , acting as capacitor , this compliance is one of the body 's adaptive mechanisms to accommodate the increase in volume ( 7 , 8) . increased heart rate is one of the physiological adaptations , which can be exaggerated , in some pregnant women . these palpitations can be severe enough to affect the daily activity of pregnant women . despite the availability of treatment for symptomatic palpitation aim of this study is to determine the number of women with benign symptomatic palpitations who receive treatment . we did a retrospective chart review of all pregnant patients who were referred to the obstetric - medicine clinic for evaluation of palpitation from january 2009 to december 2009 in one major maternity hospital in kuwait . the patients were either referred from the obstetric clinics or from their family physicians at the polyclinics . we defined benign palpitation as sinus tachycardia without any evidence of structural heart disease or systemic causes . data collected included : age of patient , gestational age ( ga ) at referral and also at delivery , symptoms at presentation , past medical history , heart rate ( hr ) recorded in initial visit , electrocardiogram ( ecg ) , thyroid stimulating hormone ( tsh ) , treatment given and apgar score . digoxin was used in those patients who had systolic blood pressure < 100 mmhg at presentation . response to treatment defined as subjective relief of palpitation with documented hr in antenatal visits was obtained from the charts of those patients who were started on treatment . there were a total of 822 women referred to our obstetric - medicine clinic for various reasons from january 2009 to december 2009 . two patients were referred at first trimester ( ga 10 and 11 ) , 9 were referred at late 3 trimester ( ga 35 - 38 ) and the other 18 patients were in their 2 and early 3 trimester at referral . the major complaints of all 27 women were palpitation , associated with chest tightness , nausea and presyncope . twenty two women were previously healthy , 2 had preexisting hypertension , 1 with bronchial asthma , 1 had hypothyroidism and 1 had preexisting mitral valve prolapse . except for 3 patients , all others had ecg done and available in the charts , which was normal apart from sinus tachycardia with hr ranging from 76 beats / min to 132 beats / min . echocardiogram , which was done for all women , showed normal heart structures except for 2 patients who had mitral valve prolapse ( mvp ) . 1 ) . three women ( 11.1% ) were started on propranolol , two women ( 7.4% ) received digoxin , one woman ( 3.7% ) received verapamil and one ( 3.7% ) received both propranolol and digoxin . one woman with mvp was not given any treatment while the other one was given digoxin because of borderline low blood pressure . all seven women reported symptomatic relief with treatment given during antenatal follow up ( table 1 ) . nine ( 33.3% ) of the 27 women delivered in private hospitals and therefore their postpartum data were not available in the charts . eighteen ( 66.6% ) women had normal deliveries with healthy babies and apgar score at 1 minute was 6 - 9 and apgar score at 5 minute was 8 - 9 . the mother of the one baby with the lowest apgar score of 6 at 1 minute and 8 at 5 minute was not on any treatment . data evaluating the use of medications to control symptomatic benign palpitation in pregnant women are limited . however , some of them would have severe tachycardia requiring medical attention and intervention . in our study , we found that only about 25% of pregnant women with symptomatic tachycardia or palpitation receive treatment , despite the available evidence of the safety of drugs being used ( 9 , 10 ) . drugs commonly used are rate control medications , which include beta - blockers , calcium channel blockers and other antiarrhythmic agents like digoxin . propranolol , one of the commonest beta - blockers , which is used during pregnancy for maternal and fetal indications , has been shown not to be teratogenic . verapamil , which is used as an antiarrhythmic agent , has also been shown to have a very low risk or nonexistent risk when used in any portion of pregnancy . digoxin has no teratogenic effect and pregnancy recommendations show compatibility ( 11 ) . in our study , all seven women who were given treatment reported resolution of their symptoms , ability to perform their activities of daily living and all delivered healthy babies . the one baby with the lowest apgar score was not exposed to any medication . however , only a small number of patients receive treatment despite the safety of drugs that are used to control symptomatic tachycardia . the number of patients in our study is small and therefore , further larger studies are needed to evaluate the management of symptomatic benign palpitation in pregnancy .	backgroundincreased heart rate is a normal physiological adaptation occurring during pregnancy . some women have severe tachycardia requiring medical attention . aim of this study is to determine the number of women with benign symptomatic palpitations who receive treatment.methodswe performed a retrospective chart review of all women who were referred to our obstetric - medicine clinic for evaluation of palpitation from january 2009 to december 2009 in one major maternity hospital in kuwait.resultsa total number of 27 women were identified . of these , only 7 ( 25.9% ) were given treatment for palpitation . two were started on digoxin , 3 given propranolol , 1 woman on both propranolol and digoxin and 1 was started on verapamil . eighteen women had normal deliveries with healthy babies.conclusionpalpitation is a common symptom during pregnancy . however , only a small number of patients receive treatment despite safety of drugs that are used to control tachycardia .
You are an expert at summarizing long articles. Proceed to summarize the following text: for example , between 1970 and 2009 , the proportion of csections recorded in the united states of america ( usa ) increased from 5.0% to 32.9% 1 . one notable complication of csection is uterine scar dehiscence ( usd ) , in which scar tissue remaining from a previous csection is disrupted and separates . although usd has not been precisely defined , the reported incidence of this condition ranges between 0.2% and 4.3% of all pregnancies associated with a previous csection 1 . a previous csection with usd is a wellknown risk factor for uterine rupture during vaginal delivery . moreover , the relative importance of screening for this complication of csection and the appropriate methodologies for such screening as well as the best surgical approach for repairing usd or the tearing of the anterior uterine wall during delivery have yet to be established . obstetricians often encounter usd during delivery by csection . however , usd extending over onethird of the anterior uterine wall is rare . furthermore , we have yet to establish the best surgical approach for repairing this rare complication of csection or repairing a torn anterior uterine wall during delivery . herein , we report two cases of usd with large defects of the anterior uterine wall and discuss the challenges associated with this complication . although the anterior uterine wall was torn and extensively damaged in these patients , the repairs were successful in both . a 38yearold woman , gravida 2 , para 1 , underwent csection via transverse incision in the lower uterine segment ( lus ) at gestational week 32 in her previous pregnancy because of concerns over fetal status . intraabdominal findings during this procedure included endometriosis , and abnormally strong adhesions were observed between the posterior uterine wall and the rectum . the patient had experienced a previous premature delivery but did not suffer a recurrence during the current pregnancy despite cervical shortening . ultrasonography reports showed that fetal growth was appropriate for gestational age , and transabdominal and transvaginal ultrasonography reports showed no indications of usd . at 38 weeks and 1 day of gestation , a repeat csection was performed . the myometrium of the anterior uterine wall had thinned over an extensive area , thus rendering the fetal membrane and fetus visible ( fig . surgical records from the patient 's previous csection reported endometriosis and the existence of abnormal adhesions between the posterior uterine wall and the rectum . we anticipated that abnormal adhesions would introduce challenges for the current csection and had concerns that a transverse incision on lus might damage the tissue lateral to the uterus , making it difficult to repair the uterine incision . consequently , we prioritized safety and performed a vertical incision in the uterine body to deliver a healthy male infant weighing 3150 g with apgar scores of 4 and 9 at 1 and 5 min after birth , respectively . ( b ) a vertical uterine incision was made , which extended to a tear in the lower segment of the uterine wall . ( c ) square sutures were first inserted for relaxation suturing of the thinned and weakened myometrium . ( d ) minute zsuturing between the square sutures was performed to repair and reinforce the wound . however , the lus was torn and appeared to be very difficult to repair ( fig . we performed a single interrupted suturing technique with size 0 polydioxanone ( pds ) thread to prevent bleeding from the transverse uterine layers . to repair the thinned and vertically torn lus , we performed extensive relaxation suturing on the first layer with square sutures ( fig . these sutures allowed repair of the incision without any leakage of the lochia ( fig . because the lower margin of the torn vertical uterine area was extremely close to the bladder , we filled the bladder with diluted indigo carmine dye to confirm that it had not been damaged . a 30yearold woman , gravida 3 , para 2 , had experienced two previous csections . during the second delivery , surgical findings showed that the fascia had thinned directly above the wound from the first csection , and the fetus was visible . therefore , the fetus was delivered via a transverse lus incision approximately 1 cm cranially from the previous incision . ultrasonography reports showed that fetal growth was appropriate for gestational age and transabdominal and transvaginal ultrasonography reports showed no indications of usd . a repeat csection was performed at 37 weeks and 3 days of gestation at the patient 's request . intraoperative findings revealed that extensive areas of the uterine anterior myometrium were missing at the previous uterine incision site , and the fetal membrane was visible . a transverse lus incision had the potential to damage the uterine cervix and bladder if extended downward . we therefore made a transverse lus incision in an upward direction from the thinning area of the uterine body to deliver a healthy female infant weighing 2872 g with apgar scores of 8 and 9 at 1 and 5 min after birth , respectively . however , following delivery , the uterine incision longitudinally extended in a tshape to a point directly above the internal ostium of the uterus ( fig . 2a and b ) . therefore , to perform the initial extensive relaxation suturing , we used size 0 pds thread to insert square sutures at four locations . we selected this technique because the myometrium was very thin and difficult to repair using traditional methods ( fig . 2c ) . then , we used size 0 pds thread to insert interrupted sutures to reinforce the wound site . the two layers of the transverse incision of the uterine segment were then closed with interrupted sutures ( fig . finally , the bladder was filled with diluted indigo carmine dye to confirm that it had not been damaged . ( a , b ) a transverse incision was made in an upward direction from the thinned area of the uterine body to deliver the fetus . however , after delivery , the uterine wound extended to a point directly above the internal ostium of the uterus . ( c , d ) as in case 1 , square sutures were inserted with minute zsuturing in between . square sutures were used for relaxation suturing of the thinned and weakened myometrium because normal suturing alone would have been insufficient for adequate repair . however , square suturing could not sufficiently prevent leakage of the lochia into the peritoneum . minute zsuturing was performed between the square sutures to repair and reinforce the wound . a 38yearold woman , gravida 2 , para 1 , underwent csection via transverse incision in the lower uterine segment ( lus ) at gestational week 32 in her previous pregnancy because of concerns over fetal status . intraabdominal findings during this procedure included endometriosis , and abnormally strong adhesions were observed between the posterior uterine wall and the rectum . the patient had experienced a previous premature delivery but did not suffer a recurrence during the current pregnancy despite cervical shortening . ultrasonography reports showed that fetal growth was appropriate for gestational age , and transabdominal and transvaginal ultrasonography reports showed no indications of usd . at 38 weeks and 1 day of gestation , a repeat csection was performed . the myometrium of the anterior uterine wall had thinned over an extensive area , thus rendering the fetal membrane and fetus visible ( fig . surgical records from the patient 's previous csection reported endometriosis and the existence of abnormal adhesions between the posterior uterine wall and the rectum . we anticipated that abnormal adhesions would introduce challenges for the current csection and had concerns that a transverse incision on lus might damage the tissue lateral to the uterus , making it difficult to repair the uterine incision . consequently , we prioritized safety and performed a vertical incision in the uterine body to deliver a healthy male infant weighing 3150 g with apgar scores of 4 and 9 at 1 and 5 min after birth , respectively . ( b ) a vertical uterine incision was made , which extended to a tear in the lower segment of the uterine wall . ( c ) square sutures were first inserted for relaxation suturing of the thinned and weakened myometrium . ( d ) minute zsuturing between the square sutures was performed to repair and reinforce the wound . however , the lus was torn and appeared to be very difficult to repair ( fig . we performed a single interrupted suturing technique with size 0 polydioxanone ( pds ) thread to prevent bleeding from the transverse uterine layers . to repair the thinned and vertically torn lus , we performed extensive relaxation suturing on the first layer with square sutures ( fig . these sutures allowed repair of the incision without any leakage of the lochia ( fig . because the lower margin of the torn vertical uterine area was extremely close to the bladder , we filled the bladder with diluted indigo carmine dye to confirm that it had not been damaged . a 30yearold woman , gravida 3 , para 2 , had experienced two previous csections . during the second delivery , surgical findings showed that the fascia had thinned directly above the wound from the first csection , and the fetus was visible . therefore , the fetus was delivered via a transverse lus incision approximately 1 cm cranially from the previous incision . ultrasonography reports showed that fetal growth was appropriate for gestational age and transabdominal and transvaginal ultrasonography reports showed no indications of usd . a repeat csection was performed at 37 weeks and 3 days of gestation at the patient 's request . intraoperative findings revealed that extensive areas of the uterine anterior myometrium were missing at the previous uterine incision site , and the fetal membrane was visible . a transverse lus incision had the potential to damage the uterine cervix and bladder if extended downward . we therefore made a transverse lus incision in an upward direction from the thinning area of the uterine body to deliver a healthy female infant weighing 2872 g with apgar scores of 8 and 9 at 1 and 5 min after birth , respectively . however , following delivery , the uterine incision longitudinally extended in a tshape to a point directly above the internal ostium of the uterus ( fig . therefore , to perform the initial extensive relaxation suturing , we used size 0 pds thread to insert square sutures at four locations . we selected this technique because the myometrium was very thin and difficult to repair using traditional methods ( fig . 2c ) . then , we used size 0 pds thread to insert interrupted sutures to reinforce the wound site . the two layers of the transverse incision of the uterine segment were then closed with interrupted sutures ( fig . finally , the bladder was filled with diluted indigo carmine dye to confirm that it had not been damaged . ( a , b ) a transverse incision was made in an upward direction from the thinned area of the uterine body to deliver the fetus . however , after delivery , the uterine wound extended to a point directly above the internal ostium of the uterus . ( c , d ) as in case 1 , square sutures were inserted with minute zsuturing in between . square sutures were used for relaxation suturing of the thinned and weakened myometrium because normal suturing alone would have been insufficient for adequate repair . however , square suturing could not sufficiently prevent leakage of the lochia into the peritoneum . minute zsuturing was performed between the square sutures to repair and reinforce the wound . longterm complications include placenta accreta , peritoneal adhesions , infertility , and myometrial thinning with uterine rupture 2 , 3 , 4 . literature shows that there are numerous definitions of myometrial thinning ; however , there is no consistent definition of this condition with respect to uterine incision 5 , 6 , 7 . myometrial thinning is observed in 0.2%4.3% of postcesarean pregnancies 1 and is thought to occur as a result of usd . however , no clear consensus has been reached regarding the precise underlying mechanism of this condition after csection . the major complication of usd is uterine rupture , which is reported in approximately 1 in 16,00019,000 women without a history of previous uterine surgery 8 . the incidence of uterine rupture is markedly increased among women with previous csection and is observed in approximately 0.3% of trials of labor ( tol ) in such women 9 . if these patients request tol , it may be crucial to screen for usd . recent studies have demonstrated the efficacy of predicting uterine tears using sonographic lus measurements before tol in women with previous csection 10 , 11 . a recent metaanalysis provides support for the use of antenatal lus measurements to predict uterine ruptures during tol 10 . although screening for usd during tol has been reported previously , the efficacy of this technique remains unknown for patients with planned repeat csection . in addition , there is no consensus about diagnosing usd by measuring the lus , and little is known about the ideal treatment for usd or the optimal timing of delivery in a patient with this complication . we suspect that excessive diagnosis of usd might unnecessarily increase the incidence of preterm birth . therefore , we conclude that screening for usd is not required for patients requesting repeat csection . although usd may be diagnosed intraoperatively for the first time , it is not harmful for the patient . however , in case 1 , a retrospective analysis of the vaginal sonography reports identified a large defect on the anterior uterine wall ( fig . when measuring cervical length , clinicians should examine the uterine anterior wall in patients who have undergone a previous csection . if clinicians identify large defects in the anterior uterine wall , they will necessarily perform the csection more cautiously . we have treated patients with extensive thinning of the uterus . in these patients , it is difficult to make a safe incision . in case 1 , we had concerns that there would be insufficient myometrial tissue for the closure of the uterine incision following delivery if we performed a transverse incision in the uterine body . therefore , the fetus was delivered via a vertical incision . however , this uterine incision was extended , and the lower margin of the wound was close to the bladder . in case 2 , myometrial thinning was evident , and although the fetus was carefully delivered via transverse uterine incision , the incision extended too close to the internal ostium of the uterus . these two cases show that careful delivery of a fetus can not avoid the extension of a uterine incision in severe cases of usd . notably , we found no evidence of severe complications , such as injury to the bladder or uterine cervix due to the extension of the incision or massive hemorrhage as a result of damage to the uterine parametrium . in the present study , we failed to demonstrate the benefits of a surgical approach involving uterine incision because both patients experienced major complications with extension to the bladder and cervix . however , a lower transverse uterine incision in patients with severe usd might have induced severe complications , as mentioned above . therefore , it is important for clinicians to make the uterine incision in an upward direction from extensively thinned areas of the uterine body during delivery . csection always includes a risk of encountering unexpected and extensive myometrial thinning . as observed in the cases herein although the fetus can be delivered carefully , wounds to the lus can extend downward or transversely . consequently , there is high risk of damage to the bladder and other structures in its vicinity . therefore , when extensive thinning of the myometrium is encountered in the lus , two extremely important questions must be considered in decisions regarding its clinical management . the use of a vertical uterine incision may be considered a protective measure to prevent damage to the uterine parametrium . however , the risk of increased bleeding associated with a longitudinal uterine incision and its potential effects on subsequent pregnancies must also be considered 12 . if an area of thinned myometrium tears unavoidably , it can be very difficult to repair . however , we believe that the aforementioned method could be extremely useful to clinicians . further largescale studies with longterm followup are now required to investigate the standardization of these strategies for safely maintaining fertility , performing uterine incisions for delivery , and completing appropriate repairs in cases of extensive myometrial thinning . to date , no previous reports or case studies have addressed the efficacy of various methods of surgical repair for a thinning or torn anterior uterine wall . therefore , we believe that the discussion of the various repair techniques described herein will be of significant clinical value . ms , sm , rn , and me : made substantial contributions to conception and design , collected the clinical data , and drafted as well as revised the manuscript . ti , ak , kk , and yu : helped in drafting the manuscript and responded to the submission work . tk : conceived and generally supervised of this study , and gave final approval of the version to be published .	key clinical messagethe incidence of cesarean section ( csection ) has increased worldwide . because the major risk factor for uterine scar dehiscence ( usd ) is a previous csection , the rate of this complication has also increased . its clinical significance and management strategies are unclear . here , we discuss usd particularly pertaining to its surgical treatment .
You are an expert at summarizing long articles. Proceed to summarize the following text: congenital hallux varus of secondary type is associated with polydactyly , syndactyly or other congenital deformities of the foot . such congenital deformities can be addressed in childhood with soft tissue reconstructive procedures . in adulthood , treatment of these deformities is challenging because of soft tissue contractures and rigid bony deformities . to our knowledge , this is the first case report demonstrating the management of neglected secondary congenital hallux varus in adults . we present here a case of a 23 years old male patient who presented to us with untreated congenital hallux varus of secondary type . first stage consisted of excision of the extra great toe and gradual correction of medial great toe . in second stage , metatarsophalangeal joint fusion was done . severe neglected congenital deformities presenting in adulthood pose unique problems of soft tissue contractures and permanent bony deformities . a staged approach of gradual soft tissue distraction and then metatarsophalangeal joint fusion has resulted in satisfactory aesthetic and functional outcome . different etiological factors have been explained in the literature for the development of hallux varus . the most common variety is due to surgical overcorrection of hallux valgus[1 - 4 ] . other causes include congenital , idiopathic , spontaneous , post - traumatic and inflammatory arthropathies[8 , 9 ] . congenital hallux varus is less commonly noted than the acquired type and is best addressed in infancy . in the developing countries , untreated severe congenital deformities are seen due to socioeconomic issues . while treating neglected hallux varus deformity in adults , soft tissue contractures , bony deformities and arthritis of joints are the issues to be considered . in the case presented here , all these points are addressed in stepwise manner to give successful outcome . a 23-year - old male patient presented with right congenital hallux varus with duplicate great toe and syndactyly untreated since childhood . apart from bad cosmetic appearance , patient was unable to wear regular footwear and had difficulty in pulling the trousers up in his routine life . clinical examination : patient had polydactyly with syndactyly of right great toe [ fig 1 ] . these two great toes were almost at right angle to the first metatarsal [ fig 2a and b ] . each great toe had a separate nail plate and medial great toe was little longer than lateral one [ fig 1 ] . preoperative clinical photograph : preoperative clinical photograph of a 23 year old male showing hallux varus deformity of right foot . plantar flexion and dorsiflexion of 20 degrees each was possible at first metatarsophalangeal ( mp ) joint . other than foot , there was no musculoskeletal or neurological abnormality . radiological evaluation : weight bearing anteroposterior , oblique and lateral radiographs of right foot were taken [ fig 3 ] . pre - operative adiographs : pre - operative anteroposterior , oblique and lateral radiographs showing short first metatarsal with deformed head articulating with two proximal phalanges . first stage : lateral great toe needed to be excised as it was smaller than the medial one and had less vascularity on portable audio doppler study . then gradual correction of medial great toe was planned with gradual soft tissue distraction using external fixator . two distractors were planned , one at mp joint and another at tarsometatarsal ( tm ) joint . second distractor was needed as it was metatarsus adductus and we wanted to include tm joint in future surgical procedure . second stage : as the patient was skeletally mature with deformed first metatarsal head and abnormal mp joint , we decided to opt for mp joint fusion after achieving correction of the deformity . dorsal incision of approximately 3 cm was taken on lateral great toe centering head of first metatarsal . lateral great toe consisting of proximal and distal phalanges was excised [ fig 4 , a ] . kirschner wires ( k - wires ) were inserted in the following manner - first axial 1.5 mm k - wire was inserted from distal to proximal phalanx of medial great toe . two more 1.8 mm k- wires were inserted through medial cuneiform into the middle cuneiform . one distractor was applied between axial wire of great toe bent at 90 degrees and metatarsal k - wires . distraction of 1 mm per day ( one complete turn of 360 degrees of the distractor knob ) was started at tm and mp joints from third postoperative day . the patient was taught the procedure of external fixator distraction and did the distraction in increments of 1 mm per day at both distarctors . he was also i supervised every week in the outpatient i department . during the period of distraction , i patient was kept non - weight bearing and care i was taken to avoid pin tract infection . aintraoperative photographs , first stage : 4a - based on vascularity , lateral great toe was excised . 4b and 4c- k - wires & two distractors were applied to distract metatarsophalangeal and tarsometatarsal joints . post - operative radiographs : post - operative anteroposterior , oblique and lateral radiographs at four weeks follow - up showing distraction at metatarsophalangeal and tarsometatarsal joints . after six weeks , the external fixator was removed and one week of waiting period was observed for pin tract healing . dorsomedial incision of approximately 10 cm was taken starting 2 cm distal to metatarsal head and extending proximally along its medial border crossing first tm joint [ fig 6 , a ] . tricortical bone graft was harvested from iliac crest and grafted at mp joint [ fig 6 , c and d ] . a 2.7 mm dynamic compression plate with 2.7 mm screws was applied on the medial side [ fig 7a to c ] . medial plating was done to provide biomechanically better support during fusion and prevent recurrence of deformity . one axial k - wire was inserted from distal phalanx , proximal phalanx , bone graft and first metatarsal into the medial cuneiform before applying the plate[fig 7 b and c ] . patient was kept non weight bearing for two months and monitored for radiological signs of fusion at mp joint . intraoperative photographs , second stage : 6a- dorsomedial incision for approach to metatarsophalangeal and tarsometatarsal joints . joint preparation intraoperative photographs , second stage : 2.7 mm dynamic compression plate applied medially across metatarsophalangeal and tarsometatarsal joints with an axial k - wire in situ . anteroposterior and lateral radiographs taken at three years follow up show satisfactory alignment and fusion [ fig 8 ] . though first toe was short but still it was in the acceptable plantigrade alignment [ fig 9 ] . after correction , patient was painless on weight bearing and can carry out all daily activities without discomfort . unlike preoperative status , now he can wear regular shoes and pull the trousers up without difficulty [ fig 10a and b ] . the revised foot function index ( ffi - r ) improved to 65 from 120 . post operative radiographs : post - operative radiographs at 2.5 years follow up showing sound fusion at metatarsophalangeal joint . clinical photograph at 2.5 years follow- up : clinical photograph of the same patient at 2.5 years follow - up showing satisfactory correction of the deformity & good soft tissue healing . comparative clinical photograph : 10a , pre - operative and 10b post - operative clinical photographs showing correction of the deformity . there are three types of congenital hallux varus- primary type is not associated with any other deformity . secondary type is associated with anomalies like polydactyly , syndatyly , metatarsus adductus , clubfoot etc [ 12 - 15 ] . this deformity can be addressed in childhood with soft tissue procedures like kelikian and farmers technique . when neglected , they present in adulthood with severe and rigid deformity with soft tissue contractures , bony deformities and arthritis . a single stage correction of such deformity can cause neurovascular compromise . hence stepwise approach and gradual correction of the deformity it was so disfiguring that he came to us for amputation of the great toe . but we offered him the option of correction of deformity and kept amputation as a salvage procedure . we excised the lateral great toe and used external fixator for gradual correction of medial great toe . two distractors were used in the external fixator assembly ; one to correct hallux varus deformity at mp joint and other to correct metatarsus adductus deformity at tm joint . , soft tissues on the medial aspect of foot were stretched gradually ; hence this case did not require any plastic surgery for the coverage . after correction , the great toe was shorter compared to normal side due to congenitally hypoplastic and deformed first metatarsal . we offered the patient option of lengthening of first metatarsal using external fixator , but patient refused it and is happy with the present results . management of this case demonstrates that gradual distraction using external fixator and a stepwise approach has resulted in satisfactory correction and excellent functional outcome in a rigid neglected congenital hallux varus with polydactyly and syndactyly in adulthood . neglected congenital deformities of hallux are not uncommon in the developing countries but they are under - reported . with encouraging results of the presented case , we propose such stepwise approach and gradual correction of the deformity in the management of neglected congenital hallux varus in adults .	introduction : congenital hallux varus of secondary type is associated with polydactyly , syndactyly or other congenital deformities of the foot . such congenital deformities can be addressed in childhood with soft tissue reconstructive procedures . in adulthood , treatment of these deformities is challenging because of soft tissue contractures and rigid bony deformities . to our knowledge , this is the first case report demonstrating the management of neglected secondary congenital hallux varus in adults.case report : we present here a case of a 23 years old male patient who presented to us with untreated congenital hallux varus of secondary type . patient had an extra great toe ( polydactyly ) with syndactyly . we have treated this patient in two stages . first stage consisted of excision of the extra great toe and gradual correction of medial great toe . in second stage , metatarsophalangeal joint fusion was done . we have follow up of this case for more than 2.5 years.conclusion:severe neglected congenital deformities presenting in adulthood pose unique problems of soft tissue contractures and permanent bony deformities . congenital neglected hallux varus with polydactyly and syndactyly is a rare deformity . its correction was challenging as patient presented to us in adulthood . a staged approach of gradual soft tissue distraction and then metatarsophalangeal joint fusion has resulted in satisfactory aesthetic and functional outcome .
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Proceed to summarize the following text: determination of biotin , especially free - form biotin , in foodstuffs is important , because appropriate biotin intake is beneficial in attaining a good quality of life ( qol ) , better health and development of children and adults , improved physical mechanisms that combat aging and disease and efficient mental capacity . recently , we developed a new high - performance affinity chromatographic ( hpac ) determination method for biotin using a trypsin - treated avidin - bound column ( 1 ) . in this new method , biotin is derivatized by 9-anthryldiazomethane ( adam ) to an ester of fluorimetric biotin - adam and detected fluorimetrically at an excitation wavelength of 365 nm and emission wavelength of 412 nm ( 1,2,3 ) . we recently found that avidin is a bifunctional binding protein ; i.e. , avidin ( a well - known biotin - binding protein ) can also strongly recognize lipoic acid ( 4 ) . however , biotin and lipoic acid can be separated and measured safely using this new chromatographic technology . it is a rapid ( analysis requires one day per sample ) , reliable and sensitive fluorometric detection method that makes use of the linear calibration line through the origin . furthermore , other nutrients and antibiotics do not interfere with this chemical method ; i.e. , other biological biotin assay methods are sensitive to nutrients and antibiotics in serum samples . herein , we describe the wide ranges of biotin contents detected among various foodstuffs depending on the species ( strain ) , season , organ ( of plants and of animals ) , geographical area , freshness and preparation and storage methods . the importance of the free biotin content in powdered milk in relation to babies , who have unstable biotinidase and exhibit biotin deficiency symptoms , is also discussed . highly pure form of methanol , acetonitrile , ethanol and ethyl acetate ( > 99.8% ) , d - biotin , activated charcoal ( acid washed ; for column chromatography ; p / n 035 - 18081 ) , 2-propanol ( hplc grade ) , ethylene glycol ( amino acid analysis grade ) , 25% ammonia water ( metal analysis grade ) , sulfuric acid , sodium chloride , lithium chloride ( anhydrous ; amino acid analysis grade ; > 97% ) and sodium dihydrogen phosphate dihydrate were purchased from wako pure chemical industries ( osaka , japan ) . d - desthiobiotin ( 5-methyl-2-oxo-4-imidazolidine hexanoic acid ; d 1411 ) , biocytin ( -n - biotinyl - l - lysine , mr 372.5 ; b 4261 ) and biotin methyl ester ( b 7883 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) , and 9-anthryldiazomethane ( adam ) was purchased from funakoshi pharmaceutical ( tokyo , japan ) . a 0.25% ( w / v ) trypsin - edta solution was purchased from invitrogen corporation ( grand island , ny , usa ) . a light - intercepting microtube with a cap ( 2 ml ; p / n 72.693.018 ) and a microtube with cap ( 2 ml ; p / n 72.694.007 ) were obtained from sarstedt aktiengesellschaft & co. ( nmbrecht , germany ) . microcentrifuge tubes ( 1.5 ml , polypropylene , lock - cap ; p / n 96.8668.9.01 ) were obtained from treff ag ( degersheim , switzerland ) . ekicrodisc 13 cr ( 0.2 m ; ptfe ; p / n e135 ) , ekicrodisc 13 ( 0.2 m ; versapor ; p ( 0.2 m ; versapor ; p / n e254 ) were obtained from nihon pall ltd . paper ph indicator ( ph 6.48.0 , narrow range ) were obtained from whatman ltd . blades and disposable scalpels were obtained from feather safety razor co. ( osaka , japan ) . an affinity column , bioptic av-1 ( 250 4.6 mm i.d . ; with chicken egg - white avidin bound to a 5 m diameter silica gel ) , was purchased from gl sciences inc . bioptic av-1 affinity gels ( 5 m diameter silica gel ) are now available ( 1 g and/or 10 g ) from gl sciences inc . trypsin - treated avidin - bound gel was prepared as described previously ( 1 ) . a trypsin - treated avidin - bound column ( 33 4.6 mm i.d . ) ten types of natto ( a japanese food made from fermented soybeans ) , thirteen sakes ( rice wines ) , ten beers , four coffees , three red wines , four breads , four cheeses , three vinegars , four bananas ( three from the philippines and one from formosa ) , two peanut butters , four edible oils ( salad oils including soybean and rapeseed , rice bran , olive , and sesame oils ) , seven sea weeds , ( aosa [ ulva pertusa ] , me - hijiki [ the sporophylls of hijiki seaweed [ sargassum fujifome ] , hijiki [ sargassum fujifome ] , kinu - mozuku [ nemacystis decipiens ] , ne - kombu [ root of the sea tangle ; laminaria japonica ] , ao - nori [ green laver ; enteromorpha compressa ] , and nori [ laver ; porphyra tenera ] , three bovine milks ( purchased in february [ winter ] and may [ summer ] ) , four flours ( buckwheat , potato and weak and strong wheat flours ) , five root crops ( onion , carrot , scallion , bamboo shoot and garlic ) , sauerkraut ( hengstenberg , esslingen , germany ) , shiitake ( mushroom ) , soy sauce ( kikkoman corporation , noda city , chiba , japan ) , miso ( soybean paste ) , chicken eggs , sujiko ( salmon roe ) , sea urchin roe , black pepper , rice bran , yakult ( purchased in february and may , yakult honsha co. ltd . , tokyo , japan ) , peanuts ( parched ) , soybeans ( parched ) , soy milk , pickles , nukamiso - zuke ( vegetables pickled in fermented rice bran , lactobacillus and yeast ) , tofu ( bean curd ) , honey , komatsuna ( brassica rapa var . pervidis ) , spinach , japanese pepper ( zanthoxylum piperitum ) , pork ( thigh ) , corned beef and chocolate were purchased from grocery stores . dried yeast ( the japan pharmacopoeia ; ebios ; tanabe pharmaceutical co. , osaka , japan ) was purchased from a drugstore . royal jellies were purchased from apiaries ( yamada apiary corp . , kagamino - cho , okayama , japan ; bushu apirary co. , kumagaya , saitama , japan ; and san ken co. , tokyo , japan ) . bee pollen ( imported from spain ) was purchased from kano apiary co. , yame , fukuoka , japan . human breast milk , milk powders and special milk powders for diseases were kindly donated by our institution . lew rats ( 9 wk of age ; male ) were purchased from sankyo labo service corporation ( tokyo , japan ) . lactobacillus casei ( shirota ) and bacillus natto cells were prepared as described in a previous study ( 5 ) . the hplc system used was as described previously ( 1 ) . a six - bored high - pressure valve ( gl sciences inc . ) was used with a 0.1 ml sample - loading loop . biotin - adam was detected with a fluorescence detector ( shimadzu model rf-10axl with a cell temperature controller ) at an excitation wavelength of 365 nm , emission wavelength of 412 nm and flow - through cell temperature of 28c . the parameters used for the fluorescence detector were gain of 1 , sensitivity of 1 and range of 4 . one analysis cycle took 80 min using the program shown in table 1table 1 . typical elution program for the trypsin - treated avidin - affinity column used for the biotin analysis with an analysis time of 80 min. hydrolysis treatment was performed as follows . first , 0.35 ml of sample solution ( dispersed in distilled water ) and 0.05 ml of concentrated sulphuric acid were mixed together ( final concentration of sulphuric acid of 2.25 m ) . the mixture was placed in a light - intercepting microtube with a cap and autoclaved at 120c for 1 h ( 1 ) . normally , 0.050.2 ml of liquid samples were adjusted to 0.35 ml with distilled water , and 550 mg of powdered or wet solid samples were dispersed in 0.35 ml of distilled water ; the resulting samples were then hydrolyzed after adding 0.05 ml of concentrated sulphuric acid . after hydrolysis , the samples were treated and derivatized as described previously ( 1 ) . the samples ( 0.2 ml of milk , 0.1 ml of serum and 10100 mg of powdered dry and/or minced wet foodstuffs were suspended in 95% methanol and ultrasonicated for 5 min . after filtration through ekicrodisc 13cr or 25 fiter , the filtrate was dried under a stream of nitrogen gas . the dried methanol extract was dissolved in 1 ml of distilled water , and 0.015 ml of 2.25 m naoh was added to the dissolved free biotin in water . after 0.06 ml of 1.40 m phosphoric acid was added and mixed with the extract ( ph of approximately 5.4 ) , activated charcoal ( ca . 4 mg ) was added to adsorb the biotin onto the surface of activated charcoal . the charcoal was then washed by centrifugation with pbs ( phosphate - buffered saline ) and distilled water 3 times each . the free biotin adsorbed by the charcoal was extracted with 1 ml of 5% ammonia - ethanol , and the extracted solution was dried under a stream of nitrogen gas . the dried free biotin was dissolved by adding 0.1 ml of methanol and was derivatized by adding 0.08 ml of 0.1% adam solution as described previously ( 1 ) . since the numbers of foodstuff and biological samples were insufficient for estimating the distribution pattern , non - parametric analysis was applied in this text . highly pure form of methanol , acetonitrile , ethanol and ethyl acetate ( > 99.8% ) , d - biotin , activated charcoal ( acid washed ; for column chromatography ; p / n 035 - 18081 ) , 2-propanol ( hplc grade ) , ethylene glycol ( amino acid analysis grade ) , 25% ammonia water ( metal analysis grade ) , sulfuric acid , sodium chloride , lithium chloride ( anhydrous ; amino acid analysis grade ; > 97% ) and sodium dihydrogen phosphate dihydrate were purchased from wako pure chemical industries ( osaka , japan ) . d - desthiobiotin ( 5-methyl-2-oxo-4-imidazolidine hexanoic acid ; d 1411 ) , biocytin ( -n - biotinyl - l - lysine , mr 372.5 ; b 4261 ) and biotin methyl ester ( b 7883 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) , and 9-anthryldiazomethane ( adam ) was purchased from funakoshi pharmaceutical ( tokyo , japan ) . a 0.25% ( w / v ) trypsin - edta solution was purchased from invitrogen corporation ( grand island , ny , usa ) . a light - intercepting microtube with a cap ( 2 ml ; p / n 72.693.018 ) and a microtube with cap ( 2 ml ; p / n 72.694.007 ) were obtained from sarstedt aktiengesellschaft & co. ( nmbrecht , germany ) . microcentrifuge tubes ( 1.5 ml , polypropylene , lock - cap ; p / n 96.8668.9.01 ) were obtained from treff ag ( degersheim , switzerland ) . ekicrodisc 13 cr ( 0.2 m ; ptfe ; p / n e135 ) , ekicrodisc 13 ( 0.2 m ; versapor ; p ( 0.2 m ; versapor ; p / n e254 ) were obtained from nihon pall ltd . paper ph indicator ( ph 6.48.0 , narrow range ) were obtained from whatman ltd . blades and disposable scalpels were obtained from feather safety razor co. ( osaka , japan ) . an affinity column , bioptic av-1 ( 250 4.6 mm i.d . ; with chicken egg - white avidin bound to a 5 m diameter silica gel ) , was purchased from gl sciences inc . bioptic av-1 affinity gels ( 5 m diameter silica gel ) are now available ( 1 g and/or 10 g ) from gl sciences inc . trypsin - treated avidin - bound gel was prepared as described previously ( 1 ) . a trypsin - treated avidin - bound column ( 33 4.6 mm i.d . ) ten types of natto ( a japanese food made from fermented soybeans ) , thirteen sakes ( rice wines ) , ten beers , four coffees , three red wines , four breads , four cheeses , three vinegars , four bananas ( three from the philippines and one from formosa ) , two peanut butters , four edible oils ( salad oils including soybean and rapeseed , rice bran , olive , and sesame oils ) , seven sea weeds , ( aosa [ ulva pertusa ] , me - hijiki [ the sporophylls of hijiki seaweed [ sargassum fujifome ] , hijiki [ sargassum fujifome ] , kinu - mozuku [ nemacystis decipiens ] , ne - kombu [ root of the sea tangle ; laminaria japonica ] , ao - nori [ green laver ; enteromorpha compressa ] , and nori [ laver ; porphyra tenera ] , three bovine milks ( purchased in february [ winter ] and may [ summer ] ) , four flours ( buckwheat , potato and weak and strong wheat flours ) , five root crops ( onion , carrot , scallion , bamboo shoot and garlic ) , sauerkraut ( hengstenberg , esslingen , germany ) , shiitake ( mushroom ) , soy sauce ( kikkoman corporation , noda city , chiba , japan ) , miso ( soybean paste ) , chicken eggs , sujiko ( salmon roe ) , sea urchin roe , black pepper , rice bran , yakult ( purchased in february and may , yakult honsha co. ltd . , tokyo , japan ) , peanuts ( parched ) , soybeans ( parched ) , soy milk , pickles , nukamiso - zuke ( vegetables pickled in fermented rice bran , lactobacillus and yeast ) , tofu ( bean curd ) , honey , komatsuna ( brassica rapa var . pervidis ) , spinach , japanese pepper ( zanthoxylum piperitum ) , pork ( thigh ) , corned beef and chocolate were purchased from grocery stores . dried yeast ( the japan pharmacopoeia ; ebios ; tanabe pharmaceutical co. , osaka , japan ) was purchased from a drugstore . royal jellies were purchased from apiaries ( yamada apiary corp . , kagamino - cho , okayama , japan ; bushu apirary co. , kumagaya , saitama , japan ; and san ken co. , tokyo , japan ) . bee pollen ( imported from spain ) was purchased from kano apiary co. , yame , fukuoka , japan . human breast milk , milk powders and special milk powders for diseases were kindly donated by our institution . lew rats ( 9 wk of age ; male ) were purchased from sankyo labo service corporation ( tokyo , japan ) . lactobacillus casei ( shirota ) and bacillus natto cells were prepared as described in a previous study ( 5 ) . the hplc system used was as described previously ( 1 ) . a six - bored high - pressure valve ( gl sciences inc . ) biotin - adam was detected with a fluorescence detector ( shimadzu model rf-10axl with a cell temperature controller ) at an excitation wavelength of 365 nm , emission wavelength of 412 nm and flow - through cell temperature of 28c . the parameters used for the fluorescence detector were gain of 1 , sensitivity of 1 and range of 4 . one analysis cycle took 80 min using the program shown in table 1table 1 . typical elution program for the trypsin - treated avidin - affinity column used for the biotin analysis with an analysis time of 80 min. first , 0.35 ml of sample solution ( dispersed in distilled water ) and 0.05 ml of concentrated sulphuric acid were mixed together ( final concentration of sulphuric acid of 2.25 m ) . the mixture was placed in a light - intercepting microtube with a cap and autoclaved at 120c for 1 h ( 1 ) . normally , 0.050.2 ml of liquid samples were adjusted to 0.35 ml with distilled water , and 550 mg of powdered or wet solid samples were dispersed in 0.35 ml of distilled water ; the resulting samples were then hydrolyzed after adding 0.05 ml of concentrated sulphuric acid . after hydrolysis , the samples were treated and derivatized as described previously ( 1 ) . free - form biotin was measured as follows . the samples ( 0.2 ml of milk , 0.1 ml of serum and 10100 mg of powdered dry and/or minced wet foodstuffs were suspended in 95% methanol and ultrasonicated for 5 min . after filtration through ekicrodisc 13cr or 25 fiter , the filtrate was dried under a stream of nitrogen gas . the dried methanol extract was dissolved in 1 ml of distilled water , and 0.015 ml of 2.25 m naoh was added to the dissolved free biotin in water . after 0.06 ml of 1.40 m phosphoric acid was added and mixed with the extract ( ph of approximately 5.4 ) , activated charcoal ( ca . 4 mg ) was added to adsorb the biotin onto the surface of activated charcoal . the charcoal was then washed by centrifugation with pbs ( phosphate - buffered saline ) and distilled water 3 times each . the free biotin adsorbed by the charcoal was extracted with 1 ml of 5% ammonia - ethanol , and the extracted solution was dried under a stream of nitrogen gas . the dried free biotin was dissolved by adding 0.1 ml of methanol and was derivatized by adding 0.08 ml of 0.1% adam solution as described previously ( 1 ) . since the numbers of foodstuff and biological samples were insufficient for estimating the distribution pattern , non - parametric analysis was applied in this text . commercially available foodstuffs in tokyo were analyzed using a short column ( 3.3 cm long ) . using the improved timetable shown in table 1 , the time required for analysis of biotin was reduced to 80 min from the previously reported analysis time of 92 min ( 1 ) . a representative example of analysis of foodstuffs ( carrot , onion and natural cheese ) is shown in fig . second chromatogram from the top : 0.002 ml of 10-fold diluted carrot sample ( from 34.4 mg wet weight ) was injected . the total biotin content of the carrot was 4.03 g / g wet weight . third chromatogram from the top : 0.002 ml of 10-fold diluted onion sample ( from 33.3 mg wet weight ) was injected . the total biotin content of the onion was 3.60 g / g wet weight . bottom chromatogram : 0.002 ml of 10-fold diluted natural cheese sample ( from 5.1 mg of weight ) was injected . the total biotin content of the natural cheese ( mimolette vieille ) was 28.8 g / g .. typical examples of total biotin analyses of foodstuffs . second chromatogram from the top : 0.002 ml of 10-fold diluted carrot sample ( from 34.4 mg wet weight ) was injected . the total biotin content of the carrot was 4.03 g / g wet weight . third chromatogram from the top : 0.002 ml of 10-fold diluted onion sample ( from 33.3 mg wet weight ) was injected . the total biotin content of the onion was 3.60 g / g wet weight . bottom chromatogram : 0.002 ml of 10-fold diluted natural cheese sample ( from 5.1 mg of weight ) was injected . the total biotin content of the natural cheese ( mimolette vieille ) was 28.8 g / g . the biotin concentration , as assessed by the median value , was highest in the royal jelly product from okayama . the difference in the values for royal jelly may be due to the production methods of the different prducers . natto ( a japanese food made from soybeans fermented with bacillus natto ) also possessed a high median value of biotin and a wide range in distribution . plant species may be important , since the ranges of peanut butter , root crops , banana and coffee were relatively narrow . geographical differences may also be observed ; i.e. , biotin content is higher in the philippines than in formosa for bananas and is higher in brazil than in indonesia and columbia for coffee . the distributions of biotin in various drinks and beverages are summarized in table 3table 3 . total biotin distributions in various drinks available in tokyo. it is apparent that fermented and fermented drinks have wider ranges in their distributions ( tables 2 and 3 ) . this may be due to differences in the microbe strains used in their fermentation and production methods . in our previous study ( 1 ) , we confirmed the seasonal differences in biotin content of bovine milk ( first reported by dr . umetaro suzuki ) ; i.e. , milk in summer contains a biotin concentration that is approximately 3 times that in winter . a similar range in total biotin content was also observed for ordinary milk powder ( table 3 ) . this may due to the seasons in which the raw materials ( bovine milk ) were obtained by the producers . free biotin is the important nutrition , since free biotin should be liberated and obtained from the usual bound - form biotin via hydrolysis by the amidase ( biotinidase ) in animals and in some bacteria and fungi , which are not able to synthesize biotin . typical foodstuffs that contain high ratio of free biotin are summarized in table 4table 4 . typical examples of high free - biotin containing foodstuffs and possible protection mechanisms against potential pathogens ( bacteria , fungi , etc). sera from healthy humans , microbes and plant vegetable cells usually contain less than 10% free biotn ( lower part of table 4 ) . foodstuffs containing high amounts of free biotin included good nutritional materials such as natto , chicken egg - yolk and milk . these foodstuffs seem to have their own mechanisms against invasion of microbes ( upper part of table 4 ) . milk and milk powder are very important foodstuffs for infants , who receive nutrition for several months after birth from only milk . the biotin contents of milk and milk powders are summarized in table 5table 5 . summary of the biotin contents of milk , milk powders and special milk powders for milk - related conditions. we found that all the tested milk and milk powders , except for gsd ( glycogen storage disease ) formulas , contained sufficient amounts of biotin ( table 5 ) . milk powder c ( follow - up milk ) contains relatively lower amounts of total and free biotin ; however , infants who are 9 mo of age or older can consume nutrients from ordinary foodstuffs . although soy milk is a foodstuff intended for adults , both soy milk and soy baby formula contained a high concentrations of total biotin ( table 5 ) . furthermore , soy formula also contained a high concentration of free biotin ( table 5 ) . biotin deficiency may occur in babies consuming milks with low levels of free biotin and low free biotin ratios . in fact , two babies receiving milfy ( meiji ) and one baby receiving new ma-1 ( morinaga ) were found to be biotin deficient at our hospital ( 6 ) . elemental formula ( meiji ) may also cause biotin deficiency in babies with normal biotinidase ( 7 ) . one 3-yr - old female gsd patient ( apparently normal biotinidase with heat labile km ; michaelis constant ) receiving gsd formulas d and n produced by meiji has also been found to be biotin deficient ( dr . babies and adults exhibiting unstable ( heat labile ) biotinidase may have a tendency to become biotin deficient ( table 6table 6 . thermal instability of serum biotinidase in biotin - deficient patients ) . the two biotin deficient patients showed increased km values ( decreased affinity ) compared with fresh controls after treatment at 37c for 4 h. the biotin - deficient baby received milfy ( meiji ) and was biotin deficient . this baby was then administerd 10 mg / day of biotin for 13 wk , and his biotin status improved ( serum total biotin 3.89 g / ml , free biotin 0.052 g / ml ) and dermatitis disappeared . the biotin - deficient adult in table 6 took ebios ( dried yeast ) and yakult ( lactobacillus casei , shirota ) for 4 mo , and her biotin status improved slightly ( serum total biotin 2.04 g / ml , free biotin 0.077 g / ml ) . this patient seems to have improved slightly ; however , the precise neurological tests may be necessary to be studied on this patient . in conclusion , precise knowledge of the biotin contents of foodstuffs is expected to be useful in improving the health and development of babies and adults .	the biotin ( vitamin h ) contents of various foodstuffs were determined by using a newly developed high - performance affinity chromatography with a trypsin - treated avidin - bound column . biotin was derivatized with 9-anthryldiazomethane ( adam ) to fluorescent biotin - adam ester . a wide range of biotin contents were found in various foodstuffs depending upon the species ( strain ) , season , organ ( of plants and animals ) , geography , freshness , preparation method and storage method . among the foodstuffs and fermented foods tested , it was found that wide distributions of biotin content were observed in powdered milk , natto , sake ( rice wine ) , beer , edible oil and sea weed . since powdered milk is important for child health and development , 14 kinds of powdered and special milks for use in children s diseases were intensively measured . we found that several special milk powders for children with allergies contained low levels of free biotin . use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase , and administration of free biotin improved their symptoms dramatically . therefore , it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people .
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Proceed to summarize the following text: blume and rojas2 ) first published in the 1980s a modified unilateral approach of posterior lumbar interbody fusion ( plif ) , named transforaminal lumbar interbody fusion ( tlif ) . harms and jeszensky12 ) confirmed its treatment outcome and contributed to the popularization of tlif during the 1990s . humphreys et al.18 ) and hankenberg et al.11 ) reported that tlif led to minimal loss of normal structure compared with plif , and that it showed no differences in blood loss , operation time , and hospital day . recently , tlif has shown a high success rate and a low complication rate , and it has been widely used as an effective treatment for various degenerative lumbar spinal disorders1114202526 ) . when initially performing tlif , two cages were usually inserted through bilateral approaches212 ) . as there have been several reports that unilateral cage showed no significant difference in treatment outcome and complications compared with bilateral cage and that it is biomechanically stable , tlif with a single cage ( unilateral tlif ) has been performed widely in recent years38 ) . in the unilateral tlif , on the other hand , contralateral foramen can be indirectly decompressed by increased disc height with a single cage inserted in the ipsilateral side1720 ) . however , hunt et al.19 ) recently reported a case showing postoperative contralateral side leg pain as a complication of a unilateral tlif . although the authors did not present precise data , they hypothesized that an increase of lordosis during surgery might have effect on contralateral foraminal stenosis and contralateral leg symptoms . despite the possibility of developing contralateral radicular symptom after a unilateral tlif , there have been no studies analyzing causes of this phenomenon . we tried to investigate risk factors to find out causes of contralateral radiculopathy after a unilateral tlif in this study . we did a retrospective study with patients who received tlif in the l1-s1 area between january 2005 and december 2013 . we performed unilateral tlif for a total of 592 patients , complaining low back pain and unilateral radicular symptoms , under diagnosis of degenerative stenosis with ipsilateral foraminal stenosis , spondylolisthesis , and repeated postoperative herniated nucleus pulposus ( hnp ) . we performed bilateral tlif and decompression for the patients with bilateral radicular symptoms , who were not included in this study either . a total of 546 patients were enrolled in this study excluding 46 patients who received the operation upon a diagnosis of infectious spondylitis , spine tumor , or fracture at the index level . mri was performed for all patients who had radicular symptoms which did not solved with medication and nerve blocks for more than 1 month . ct was prescribed for the patients who were indicated for surgery according to the mri findings . foraminal stenosis was diagnosed as root compression and loss of perineural fat layer at the neural foramen on mri . there was no foraminal stenosis on the contralateral side even though some patients had smaller contralateral foraminal area comparing to those of other asymptomatic levels . the patients were divided into two groups : one group comprised those who presented with new leg symptoms including pain , hypoesthesia , paresthesia , and motor weakness , in the side contralateral to the original symptom side within 1 week after surgery ( symptomatic group ) , and the other group comprised those without contralateral symptom after surgery ( asymptomatic group ) . the patient was positioned with lumbar extension by placing pillows at the chest and pelvis . contralateral side decompression was not performed and the contralateral ligamentum flavum was saved in unilateral tlif . after a meticulous endplate preparation for upper and lower endplates , disc space was filled with autologous bone chips from lamina and facet , and the capstoner peek cage ( medtronic sofamor danek inc . , memphis , tn , usa ) filled with the autologous bone chips was inserted obliquely . rod compression was not performed routinely due to prevent contralateral foraminal stenosis where the decompression was not performed . simple radiograph and ct were taken for all the patients with postoperative contralateral radicular symptoms . however , revision surgery was immediately performed when root compression was definitive on image studies or motor weakness was accompanied . revision surgeries , such as facetectomy , discectomy , screw reposition , and hematoma removal , were done for the contralateral pathologies . the causes of contralateral symptoms in the symptomatic group were categorized into five , contralateral foraminal stenosis , newly developed hnp , screw malposition , hematoma , and unknown origin . contralateral foraminal stenosis was defined as a decreased postoperative foraminal area ( fa ) compared with the preoperative value ; hnp as a newly developed hnp on the side contralateral to the tlif side ; and screw malposition as invasion of contralateral pedicle screw into central canal or neural foramen . to assess the difference in clinical outcomes between the symptomatic and asymptomatic groups , visual analogue scale ( vas ) scores and oswestry disability index ( odi ) were measured immediately before surgery and at 3 months after surgery . disc height ( dh ) , lumbar lordosis ( ll ) , and segmental angle ( sa ) were measured with plain radiographs before and after the surgery . dh was defined as the distance between the upper and lower endplates at the center of the disc space at the index level . ll was measured as the angle between the two upper endplates of l1 and s1 . sa was measured as the angle between the upper and lower endplates at the disc level where surgery was performed ( fig . postoperative changes of ll and sa were calculated by subtracting the preoperative angle from the postoperative angle in each group . dh , ll , and sa values were compared in each group , and postoperative changes of ll and sa were also compared . fa ( mm ) was defined as the area of bony neural foramen at the center of pedicle from the sagittal view of ct . postoperative change of fa was measured as the difference between preoperative and postoperative fa 's . all the factors were measured 3 times blindly for patient information , and the mean values were used for this study . data obtained through the measured vas and odi scores and radiological analysis of ll , sa , and fa were analyzed for statistical significance by using student 's t - test , and a p - value of < 0.05 was considered to be statistically significant . we did a retrospective study with patients who received tlif in the l1-s1 area between january 2005 and december 2013 . we performed unilateral tlif for a total of 592 patients , complaining low back pain and unilateral radicular symptoms , under diagnosis of degenerative stenosis with ipsilateral foraminal stenosis , spondylolisthesis , and repeated postoperative herniated nucleus pulposus ( hnp ) . we performed bilateral tlif and decompression for the patients with bilateral radicular symptoms , who were not included in this study either . a total of 546 patients were enrolled in this study excluding 46 patients who received the operation upon a diagnosis of infectious spondylitis , spine tumor , or fracture at the index level . mri was performed for all patients who had radicular symptoms which did not solved with medication and nerve blocks for more than 1 month . ct was prescribed for the patients who were indicated for surgery according to the mri findings . foraminal stenosis was diagnosed as root compression and loss of perineural fat layer at the neural foramen on mri . there was no foraminal stenosis on the contralateral side even though some patients had smaller contralateral foraminal area comparing to those of other asymptomatic levels . the patients were divided into two groups : one group comprised those who presented with new leg symptoms including pain , hypoesthesia , paresthesia , and motor weakness , in the side contralateral to the original symptom side within 1 week after surgery ( symptomatic group ) , and the other group comprised those without contralateral symptom after surgery ( asymptomatic group ) . the patient was positioned with lumbar extension by placing pillows at the chest and pelvis . contralateral side decompression was not performed and the contralateral ligamentum flavum was saved in unilateral tlif . after a meticulous endplate preparation for upper and lower endplates , disc space was filled with autologous bone chips from lamina and facet , and the capstoner peek cage ( medtronic sofamor danek inc . , memphis , tn , usa ) filled with the autologous bone chips was inserted obliquely . rod compression was not performed routinely due to prevent contralateral foraminal stenosis where the decompression was not performed . simple radiograph and ct were taken for all the patients with postoperative contralateral radicular symptoms . however , revision surgery was immediately performed when root compression was definitive on image studies or motor weakness was accompanied . revision surgeries , such as facetectomy , discectomy , screw reposition , and hematoma removal , were done for the contralateral pathologies . the causes of contralateral symptoms in the symptomatic group were categorized into five , contralateral foraminal stenosis , newly developed hnp , screw malposition , hematoma , and unknown origin . contralateral foraminal stenosis was defined as a decreased postoperative foraminal area ( fa ) compared with the preoperative value ; hnp as a newly developed hnp on the side contralateral to the tlif side ; and screw malposition as invasion of contralateral pedicle screw into central canal or neural foramen . to assess the difference in clinical outcomes between the symptomatic and asymptomatic groups , visual analogue scale ( vas ) scores and oswestry disability index ( odi ) were measured immediately before surgery and at 3 months after surgery . disc height ( dh ) , lumbar lordosis ( ll ) , and segmental angle ( sa ) were measured with plain radiographs before and after the surgery . dh was defined as the distance between the upper and lower endplates at the center of the disc space at the index level . ll was measured as the angle between the two upper endplates of l1 and s1 . sa was measured as the angle between the upper and lower endplates at the disc level where surgery was performed ( fig . postoperative changes of ll and sa were calculated by subtracting the preoperative angle from the postoperative angle in each group . preoperative and postoperative dh , ll , and sa values were compared in each group , and postoperative changes of ll and sa were also compared . fa ( mm ) was defined as the area of bony neural foramen at the center of pedicle from the sagittal view of ct . postoperative change of fa was measured as the difference between preoperative and postoperative fa 's . all the factors were measured 3 times blindly for patient information , and the mean values were used for this study . data obtained through the measured vas and odi scores and radiological analysis of ll , sa , and fa were analyzed for statistical significance by using student 's t - test , and a p - value of < 0.05 was considered to be statistically significant . the incidence of contralateral radiculopathy after a unilateral tlif surgery was 5.9% ( 32/546 ) . there was no statistically significant difference in mean age , sex ratio , bone mineral density t - score , and the number of operated levels between symptomatic and asymptomatic groups ( table 1 ) . the most common cause was contralateral foraminal stenosis ( 22 patients , 68.8% ) , followed by screw malposition ( 4 patients , 12.5% ) , hnp ( 3 patients , 9.3% ) , hematoma ( 1 patients , 3.1% ) , and unknown origin ( 2 patients , 6.3% ) ( table 2 ) . revision surgery was performed for 16 ( 50.0% ) of the 32 patients in the symptomatic group . revision surgery was performed for 10 cases of contralateral foraminal stenosis ( 45.5% ) , 4 cases of screw malposition ( 100% ) , 1 case of hnp ( 33.3% ) , and 1 case of hematoma ( 100% ) . the changes of disc height after surgery were similar in both groups ( table 3 ) . preoperative ll 's were 35.810.3 and 37.211.8 , and postoperative ll 's were 39.212.8 and 38.713.6 in symptomatic and asymptomatic groups , respectively , showing no significant difference between the two groups . the change of ll was higher in symptomatic group ( 3.03.5 ) than asymptomatic group ( 2.71.8 ) , but there was no statistical significance ( table 3 ) . pre- and postoperative sa 's of asymptomatic group were 14.89.1 and 15.910.2 , which showed no significant difference between the two angles . on the contrary , pre- and postoperative sa 's of symptomatic group were 13.78.8 and 19.612.8 , which showed a significant increase of the angle after surgery ( p<0.05 ) . the amount of change in sa 's after surgery were 6.02.7 and 1.52.0 in symptomatic and asymptomatic groups , respectively , revealing a significantly large increase in symptomatic group ( p<0.01 ) ( table 4 ) . in conclusion , sa and change of sa were seem to be an important factor for occurrence of contralateral radiculopathy after unilateral tlif , whereas la was not . preoperative contralateral fa value of symptomatic group ( 56.213.3 mm ) was significantly smaller than that of asymptomatic group ( 69.219.3 mm ) ( p<0.01 ) . postoperative contralateral fa 's were 38.89.3 mm and 78.218.3 mm in symptomatic and asymptomatic groups , indicating marked decrease in the symptomatic group ( p<0.01 ) . furthermore , postoperative changes of fa were -16.46.3 mm and 10.65.9 mm in symptomatic and asymptomatic groups , showing significant increase of the area after surgery in symptomatic group ( p<0.01 ) ( table 4 ) . preoperative vas scores were 5.83.2 and 6.02.9 in symptomatic and asymptomatic groups , and vas scores at 3 months after surgery were 1.81.1 and 1.61.2 , respectively , without significant difference between the two groups ( table 4 ) . preoperative odi 's were 66.821.7% and 61.023.5% in symptomatic and asymptomatic groups , and odi 's at 3 months after surgery were 18.610.5% and 20.212.8% , respectively , showing no significant difference between the two groups ( table 5 ) . the most common cause was contralateral foraminal stenosis ( 22 patients , 68.8% ) , followed by screw malposition ( 4 patients , 12.5% ) , hnp ( 3 patients , 9.3% ) , hematoma ( 1 patients , 3.1% ) , and unknown origin ( 2 patients , 6.3% ) ( table 2 ) . revision surgery was performed for 16 ( 50.0% ) of the 32 patients in the symptomatic group . revision surgery was performed for 10 cases of contralateral foraminal stenosis ( 45.5% ) , 4 cases of screw malposition ( 100% ) , 1 case of hnp ( 33.3% ) , and 1 case of hematoma ( 100% ) . the changes of disc height after surgery were similar in both groups ( table 3 ) . preoperative ll 's were 35.810.3 and 37.211.8 , and postoperative ll 's were 39.212.8 and 38.713.6 in symptomatic and asymptomatic groups , respectively , showing no significant difference between the two groups . the change of ll was higher in symptomatic group ( 3.03.5 ) than asymptomatic group ( 2.71.8 ) , but there was no statistical significance ( table 3 ) . pre- and postoperative sa 's of asymptomatic group were 14.89.1 and 15.910.2 , which showed no significant difference between the two angles . on the contrary , pre- and postoperative sa 's of symptomatic group were 13.78.8 and 19.612.8 , which showed a significant increase of the angle after surgery ( p<0.05 ) . the amount of change in sa 's after surgery were 6.02.7 and 1.52.0 in symptomatic and asymptomatic groups , respectively , revealing a significantly large increase in symptomatic group ( p<0.01 ) ( table 4 ) . in conclusion , sa and change of sa were seem to be an important factor for occurrence of contralateral radiculopathy after unilateral tlif , whereas la was not . preoperative contralateral fa value of symptomatic group ( 56.213.3 mm ) was significantly smaller than that of asymptomatic group ( 69.219.3 mm ) ( p<0.01 ) . postoperative contralateral fa 's were 38.89.3 mm and 78.218.3 mm in symptomatic and asymptomatic groups , indicating marked decrease in the symptomatic group ( p<0.01 ) . furthermore , postoperative changes of fa were -16.46.3 mm and 10.65.9 mm in symptomatic and asymptomatic groups , showing significant increase of the area after surgery in symptomatic group ( p<0.01 ) ( table 4 ) . preoperative vas scores were 5.83.2 and 6.02.9 in symptomatic and asymptomatic groups , and vas scores at 3 months after surgery were 1.81.1 and 1.61.2 , respectively , without significant difference between the two groups ( table 4 ) . preoperative odi 's were 66.821.7% and 61.023.5% in symptomatic and asymptomatic groups , and odi 's at 3 months after surgery were 18.610.5% and 20.212.8% , respectively , showing no significant difference between the two groups ( table 5 ) . tlif operation has been widely used due to its excellent surgical outcome , relatively few complications , and high fusion rate112226 ) . additionally , the tlif technique can preserves normal structures such as the facet joint and muscles at the contralateral side by using the unilateral approach4111529 ) . although its causes have been studied through case reports , to our best knowledge , there has been no detailed study analyzing the cause . the incidence of contralateral radiculopathy after a unilateral tlif was 5.9% based on our results . it seems to be closely related with contralateral foraminal stenosis , newly developed hnp , screw malposition , hematoma , among which contralateral foraminal stenosis was the most common cause . the contralateral complication itself did n't seem to affect the clinical results , which , we think , might be related with early diagnosis and proper management . although there have been many studies on various postoperative complications of lumbar spinal surgery through a number of literatures1561027 ) , there have been no study on the incidence of contralateral radiculopathy after a unilateral tlif . thus , it is difficult to directly compare the degree of incidence shown in this study . however , the incidence rate of 5.9% is not likely to be small , considering the high frequency of tlif use28 ) . the reasons of unexpectedly high incidence seem to be the sporadic development of the complication , mild and transient symptom which can be controlled well with conservative management . therefore , the postoperative contralateral radiculopathy seems to be an important complication of unilateral tlif surgeons should be cautious in the sense of not only its high incidence but also its unpredictability . so , analyzing its causes might be helpful in reducing or sometimes predicting the complication in the future . hunt et al.19 ) indicated contralateral foraminal stenosis after a unilateral tlif might be the cause of contralateral symptoms . but their report did n't seem to have an objective evidence because of a lack of quantitative analysis with a large number of cases . we were able to confirm the causes in a relatively objective manner by analyzing various factors in a large number of patients in this study . iwata et al.20 ) reported the morphological change in the contralateral lumbar foramen after surgery in 58 patients who underwent unilateral tlif . they proved the contralateral foraminal size was significantly increased with unilateral tlif , which showed the possibility of indirect decompression of the contralateral side by unilateral insertion of a single cage . however , they reported that contralateral decompression was not highly effective when preoperative contralateral foraminal stenosis was present . this is comparable with the previous report of hunt et al.19 ) that preexisting foraminal stenosis on the contralateral side might be the cause of contralateral symptoms . in our study , the contralateral foraminal area was already significantly smaller preoperatively and its size decreased significantly after surgery in the patients of symptomatic group . it seems to be ideal that establishing proper indications of unilateral or bilateral tlif according to contralateral foraminal size , which must be investigated in the future study . appropriate restoration of lordosis during lumbar spine surgery is important for improving clinical outcomes and preventing failed back surgery syndrome with an iatrogenic flat back7916213031 ) . on the other hand , increase in segmental lordotic angle of the operative level can cause contralateral foraminal stenosis1920 ) . our study showed similar results , both postoperative segmental angle and its change after surgery at the index level were found to be significant risk factors for contralateral radiculopathy after unilateral tlif . however , the lordotic angle of whole lumbar spine did not have any effect on postoperative contralateral symptoms . therefore , it seems that contralateral foraminal decompression or inserting bilateral cages might be safer when increase of segmental lordosis by surgery is predicted especially in the patients who already have narrow contralateral foramen preoperatively . lumbar foraminal stenosis can be caused by superior and ventral subluxations of the superior articular process of the inferior vertebra1324 ) . the same changes were also observed in some of our patients of symptomatic group ( fig . 2 ) . the superior or ventral displacement of superior articular process in the neural foramen seem to be related with inappropriately high grade rod compression at the contralateral side after the cage insertion or inappropriately high grade lordosis at the index level , respectively . the effect of superior articular process subluxation on the postoperative contralateral foraminal stenosis must be greater in the patients whose contralateral foraminal area was already smaller preoperatively so , the size and insertion angle of cage seem to be risk factors for contralateral foraminal stenosis after unilateral tlif . the curved cage was reported to be superior to the straight cage for making lumbar segmental angle23 ) . but we used the straight cage for all cases in this study because of surgeons ' preference . so , further study probing the mechanism or risk factors of contralateral foraminal stenosis related with cage seems to be necessary in the future . it was difficult to understand the mechanism of the newly developed hnp contralateral to the operation side at first . but we now are understanding that the unilaterally inserted cage might push the disc material to the contralateral side and result in contralateral disc herniation . it seems to be related with the insufficient disc removal remaining large amount of disc material in the disc space . because this was conducted as a retrospective study , a prospective study will be needed in the future for a controlled study on more diverse factors . although we were able to find several causes by analyzing various factors before and after surgery , we believe that there must be more risk factors which occur during surgery . ct was mainly used as a postoperative examination to avoid mri artifacts caused by screw and rod . however , the disadvantage was that changes in soft tissues such as ligament and annulus in the foramen were not able to be counted properly by ct . the preoperative contralateral neural foraminal size seems to be an important risk factor , but unfortunately we did not assess the critical foraminal size which may provoke contralateral symptom after surgery . the contralateral symptoms after unilateral tlif may be reduced by being cautious to the risk factors . but we think another surgical techniques : bilateral tlif , bilateral decompression with unilateral tlif , should be needed to prevent the complication . postoperative contralateral radiculopathy is a complication of unilateral tlif which can be caused by contralateral foraminal stenosis , screw malposition , newly developed hnp , and hematoma . the contralateral foraminal stenosis seems to be related with preoperative foraminal stenosis and postoperative increase of segmental angle . considering its relatively high incidence and unpredictability , finding risk factors and mechanisms will be helpful to prevent or solve the problem .	objectivecases of contralateral radiculopathy after a transforaminal lumbar interbody fusion with a single cage ( unilateral tlif ) had been reported , but the phenomenon has not been explained satisfactorily . the purpose of this study was to determine its incidence , causes , and risk factors.methodswe did retrospective study with 546 patients who underwent a unilateral tlif , and used ct and mri to study the causes of contralateral radicular symptoms that appeared within a week postoperatively . clinical and radiological results were compared by dividing the patients into the symptomatic group and asymptomatic group.resultscontralateral symptoms occurred in 32 ( 5.9% ) of the patients underwent unilateral tlif . the most common cause of contralateral symptoms was a contralateral foraminal stenosis in 22 ( 68.8% ) , screw malposition in 4 ( 12.5% ) , newly developed herniated nucleus pulposus in 3 ( 9.3% ) , hematoma in 1 ( 3.1% ) , and unknown origin in 2 patients ( 6.3% ) . 16 ( 50.0% ) of the 32 patients received revision surgery . there was no difference in visual analogue scale and oswestry disability index between the two groups at discharge . both preoperative and postoperative contralateral foraminal areas were significantly smaller , and postoperative segmental angle was significantly greater in the symptomatic group comparing to those of the asymptomatic group ( p<0.05).conclusionthe incidence rate is not likely to be small ( 5.9% ) . if unilateral tlif is performed for cases when preoperative contralateral foraminal stenosis already exists or when a large restoration of segmental lordosis is required , the probability of developing contralateral radiculopathy is increased and caution from the surgeon is needed .
You are an expert at summarizing long articles. Proceed to summarize the following text: the first phase of the present longitudinal study was conducted at the beginning of the mass vaccination campaign to assess the determinants of parents intention to have their child vaccinated with the 4cmenb vaccine ( or adolescents intention to receive the 4cmenb vaccine ) . a professional research and polling firm ( som recherches et sondages ) handled recruitment and data collection . computer - assisted telephone interviews were performed from may 9 to may 17 , 2014 . the sample was constituted using random - digit dialling methodology ( 8) . the households and potential respondent in the household were both randomly selected . when the selected person was < 16 years of age , the person in charge of health decisions for the selected child responded to the survey questionnaire , while participants 16 years of age answered for themselves . eligibility criteria were : to be either the main caregiver of at least one child between two months and 15 years of age or between 16 and 20 years of age ; and to live or study in saguenay the target was to enroll 875 respondents to provide a precision of 5% for the most conservative variable estimation . the study was evaluated positively with regard to its methodology by the ethics review board ( erb ) of the chu de qubec , but was exempted from complete evaluation by the erb due to article 2.5 of the tri - council policy statement on ethical conduct for research involving humans in canada ( 9 ) . before beginning the interview , respondents were informed about the objectives and the sponsors of the survey , and verbal consent was obtained . the survey instrument was developed only in french and included questions to measure the respondents knowledge and attitudes about imd and the 4cmenb vaccine as well as the respondents intention to have their child vaccinated ( or , for adolescents , to be vaccinated themselves ) and main reasons for intending or not intending to receive the 4cmenb vaccine . most of the questions used a four - point likert scale ranging from totally agree to totally disagree . the survey questionnaire was developed based on questions used in similar studies ( 1012 ) . before initiation of the study , the survey was reviewed by public health practitioners , physicians and nurses involved in the mass campaign for content validity . the survey questionnaire was also pretested with 10 respondents and additional clarifications were made in the wording of some questions . standard sociodemographic variables were collected ( for parents : age , level of education as well as age and sex of the child ; for adolescents : main occupation , level of education ) . expansion weights were assigned to ensure that the results were representative of the target population by adjusting for disproportionate sampling and nonresponse bias . weighting included a calibration that was applied to each respondent in the sample , based on sociodemographic characteristics drawn from the data contained in the quebec immunization registry developed specifically for the campaign as well as from census data . comparison between respondent groups according to demographic characteristics ( age and sex of the child , level of parents education , etc ) were performed using or fisher s exact tests as appropriate . beliefs and attitudes about meningococcal b vaccination were analyzed to explore the associated factors with vaccination intention . percent of agreement was dichotomized in agree ( totally agree , somewhat agree ) versus disagree ( somewhat disagree and totally disagree ) . a multivariate logistic regression model was used to determine variables independently associated with the adolescent s intention not to be vaccinated with the 4cmenb . the verbatim wording of the open - ended questions was transcribed by the interviewers and submitted to content analysis . qualitative data were organized into main coded themes and concepts belonging to a similar theme were regrouped . these themes were updated and revised until no new properties , dimensions or relationships emerged during analysis . this content analysis was first performed using word processing software and then imported into sas version 9.3 ( sas institute , usa ) when completed . all tests were considered to be statistically significant at the threshold of 5% ( p<0.05 ) . the first phase of the present longitudinal study was conducted at the beginning of the mass vaccination campaign to assess the determinants of parents intention to have their child vaccinated with the 4cmenb vaccine ( or adolescents intention to receive the 4cmenb vaccine ) . a professional research and polling firm ( som recherches et sondages ) handled recruitment and data collection . computer - assisted telephone interviews were performed from may 9 to may 17 , 2014 . the sample was constituted using random - digit dialling methodology ( 8) . the households and potential respondent in the household were both randomly selected . when the selected person was < 16 years of age , the person in charge of health decisions for the selected child responded to the survey questionnaire , while participants 16 years of age answered for themselves . eligibility criteria were : to be either the main caregiver of at least one child between two months and 15 years of age or between 16 and 20 years of age ; and to live or study in saguenay the target was to enroll 875 respondents to provide a precision of 5% for the most conservative variable estimation . the study was evaluated positively with regard to its methodology by the ethics review board ( erb ) of the chu de qubec , but was exempted from complete evaluation by the erb due to article 2.5 of the tri - council policy statement on ethical conduct for research involving humans in canada ( 9 ) . before beginning the interview , respondents were informed about the objectives and the sponsors of the survey , and verbal consent was obtained . the survey instrument was developed only in french and included questions to measure the respondents knowledge and attitudes about imd and the 4cmenb vaccine as well as the respondents intention to have their child vaccinated ( or , for adolescents , to be vaccinated themselves ) and main reasons for intending or not intending to receive the 4cmenb vaccine . most of the questions used a four - point likert scale ranging from totally agree to totally disagree . the survey questionnaire was developed based on questions used in similar studies ( 1012 ) . before initiation of the study , the survey was reviewed by public health practitioners , physicians and nurses involved in the mass campaign for content validity . the survey questionnaire was also pretested with 10 respondents and additional clarifications were made in the wording of some questions . standard sociodemographic variables were collected ( for parents : age , level of education as well as age and sex of the child ; for adolescents : main occupation , level of education ) . expansion weights were assigned to ensure that the results were representative of the target population by adjusting for disproportionate sampling and nonresponse bias . weighting included a calibration that was applied to each respondent in the sample , based on sociodemographic characteristics drawn from the data contained in the quebec immunization registry developed specifically for the campaign as well as from census data . comparison between respondent groups according to demographic characteristics ( age and sex of the child , level of parents education , etc ) were performed using or fisher s exact tests as appropriate . beliefs and attitudes about meningococcal b vaccination were analyzed to explore the associated factors with vaccination intention . percent of agreement was dichotomized in agree ( totally agree , somewhat agree ) versus disagree ( somewhat disagree and totally disagree ) . a multivariate logistic regression model was used to determine variables independently associated with the adolescent s intention not to be vaccinated with the 4cmenb . the verbatim wording of the open - ended questions was transcribed by the interviewers and submitted to content analysis . qualitative data were organized into main coded themes and concepts belonging to a similar theme were regrouped . these themes were updated and revised until no new properties , dimensions or relationships emerged during analysis . this content analysis was first performed using word processing software and then imported into sas version 9.3 ( sas institute , usa ) when completed . all tests were considered to be statistically significant at the threshold of 5% ( p<0.05 ) . the response rate was 72% and 887 interviews were completed ( 703 interviews with parents of children between two months and 16 years of age and 184 with adolescents 16 years of age ) . more than one - half of parents were between 30 and 39 years of age ( 54% ) . almost two - thirds of parents had a university ( 32% ) or collegial degree ( 31% ) . the majority of parents reported that their child had received all recommended vaccines ( 92% ) , whereas 66% of adolescents reported this . less than 1% of parents and adolescents mentioned not receiving any vaccine ( table 1 ) . a higher proportion of parents of children five to 12 years of age reported that their child was fully vaccinated compared with parents of children < 5 years of age or > 12 years of age . overall , most parents ( 99% ) and adolescents ( 90% ) knew that a vaccination campaign against meningitis was launched in their region . among adolescents , more girls than boys were aware of the campaign ( 98% versus 83% ; the majority of parents ( 93% ) intended to have their child vaccinated with the 4cmenb vaccine or had already done so ( table 2).a higher proportion of parents of children < 5 years of age did not know whether they would have their child vaccinated with the 4cmenb vaccine . approximately 75% of adolescents also intended to be vaccinated or had received the 4cmenb vaccine ( table 2 ) . a positive association between self - reported complete vaccination status for other recommended vaccines and intention to receive the 4cmenb vaccine ( or being already vaccinated ) was observed , for both parents and adolescents . the main reason cited by parents for intending to have or having had their child vaccinated with the 4cmenb vaccine was to protect him / her against meningitis . the desire to be protected against meningitis was also the main reason stated by adolescents who intended to receive or had received the vaccine . general trust in vaccines , the perception that the benefits of the 4cmenb outweighed the risks , and knowing someone who suffered from meningitis were others reasons for vaccination reported by parents and adolescents . in addition , some adolescents indicated ease of access to the 4cmenb vaccine and that it was free as reasons for intending to be or being vaccinated . the most frequent reason mentioned by parents who did not intend to have their child vaccinated was opposition to vaccination in general . for adolescents , lack of interest , time or information about the vaccine and low perceived susceptibility to infection and perceived low disease severity were the main reasons cited by adolescents who did not intend to be vaccinated . the results of questions assessing respondents knowledge , beliefs and attitudes regarding imd and the 4cmenb vaccine are illustrated in figure 1 . generally , the majority of respondents considered imd to be a severe disease and were worried about their own or their child s susceptibility to it . more than 90% of respondents also considered that it was their duty to receive the vaccine or to vaccinate their child to prevent the transmission of meningitis to others in their community . parents who were less educated ( high school diploma or less ) were more worried that their child could contract meningitis when compared with parents with a college or university degree ( 78% , 62% and 57% , respectively , p<0.0001 ) . in addition , among adolescents , more girls than boys were worried about the risk of contracting meningitis ( 57% versus 29% , p=0.0002 ) . because almost all parents intended to have or had their child vaccinated , it was not possible to perform multivariate analyses . however , a multivariate regression analysis was performed to identify the factors associated with adolescents intention not to be vaccinated . not being aware of the vaccination campaign , believing that the 4cmenb was not safe and not believing that it was a duty to be vaccinated to prevent the spread of infection in the community were associated with the intention not to be vaccinated with the 4cmenb vaccine . results of the first phase of our study indicated that the majority of respondents were aware of the mass vaccination campaign against meningococcal serogroup b disease that was going on in saguenay lac - st - jean . this is not surprising given that the survey was conducted two weeks after the official announcement of the campaign , which was highly publicized in local media ( 1316 ) . our results illustrated a high willingness to receive the new 4cmenb in the context of a targeted mass campaign . more than nine of 10 parents indicated an intention to have their child vaccinated or had already done so . almost three of four adolescents surveyed indicated an intention to receive the vaccine or had received it . as in other studies , our results indicate a strong association between having received all recommended vaccines and intending to receive the new vaccine ( 17,18 ) . our results mirror findings from prelicensure trials that showed a high level of acceptability for the vaccine ( 1726 ) . because it can arise in an unpredictable manner , develop rapidly and lead to serious consequences , meningitis has characteristics that increase the population s perception of risk ( 27 ) . meningitis was perceived as being a dangerous disease by the majority of parents and adolescents surveyed . the death due to serogroup b meningitis of a 16-year - old adolescent living in saguenay lac - st - jean in spring 2014 , before the launch of the campaign , was covered extensively by the media and has probably contributed to the perceived risk of the disease in the public ( 2830 ) . in fact , approximately two - thirds of parents reported that they were worried about the risk for their child contracting the disease . although adolescents between 15 and 20 years of age have high rates of imd ( 31 ) , fewer adolescents were concerned about their own risk for contracting meningitis . different studies have shown that new vaccines are likely to engender doubts and concerns ( 17,32,33 ) . for instance , in a recent pan - canadian survey , one - half of the parents were concerned that new vaccines are not as safe as older vaccines , and one - third believed that children today receive too many vaccines ( 34 ) . the fact that the 4cmenb vaccine is new did not have an important influence on its acceptability by parents and adolescents in our study . a minority of respondents have cited the novelty of the vaccine as a reason for not intending to be vaccinated . in the particular context of an endemic situation , the perceived threat of the disease may outweigh the perceived risks associated with new vaccines ( 35,36 ) . clinical trials showed the 4cmenb vaccine to be more reactogenic than vaccines routinely used in quebec , which we hypothesized could have a negative impact on its acceptability ( 37,38 ) . we anticipated that ( real or perceived ) side effects after the first dose , such as pain and fever , could compromise the acceptability of the subsequent doses and even have a negative impact on the acceptability of other routine vaccines ( 3942 ) . for instance , the results of one study on vaccination against seasonal influenza highlighted the reduced acceptability of a second dose of the vaccine on the basis of the severity of side effects perceived by parents after the first dose ( 39 ) . the response rate of 72% is well above rates typically obtained with telephone surveys ( 43,44 ) . other strengths of the present study were the use of random - digit dialling methodology for data collection and the use of case - weights to adjust for disproportionate sampling and non - response bias . however , similar to most surveys , we can not exclude the potential of socially desirable responses , which is the tendency of respondents to reply in a manner that will be viewed favourably by others . the survey was conducted only in french ; however , < 1% of the contacted households did not participate due to a language barrier . we used a telephone survey , which resulted in a recruitment bias toward more educated individuals and against the young and new residents of a community with no household telephone number ( 43 ) . however , it is known that in quebec most families with young children are residents of the same community for several years and most of them have a household telephone number . in 2013 , 89% of quebecers households with children finally , because of the gap between intention and behaviour ( 46,47 ) , our findings are limited by the fact that our survey was conducted at the beginning of the campaign . to conclude , the first phase of the present study has indicated high acceptance of the new 4cmenb vaccine at the beginning of the mass campaign in saguenay intention to receive the vaccine was high , as was perceived severity and susceptibility of the disease . the second phase of the study , which will be conducted in march and april 2015 , after the end of the campaign , will provide additional information on the determinants of acceptance of the vaccine . it will then be possible to describe the determinants for having received one , all or none of the recommended doses of the 4cmenb vaccine . we will also be able to assess the potential impact of adverse events after the first dose ( real or perceived ) on the acceptability of the subsequent doses and of other scheduled vaccines .	since the implementation of the meningococcal c conjugate vaccine as a component of the routine vaccination schedule of children in quebec , the incidence of meningitis caused by serogroup c neisseria meningitidis has declined significantly . currently , serogroup b causes the majority of cases of invasive meningococcal disease in quebec . a vaccine against serogroup b became available in 2013 ; accordingly , a mass vaccination campaign was launched in saguenay lac - st - jean , quebec , in 2014 . at the beginning of the campaign , a telephone survey was conducted to assess opinions on the vaccine , and willingness to be vaccinated among adolescents ( 16 to 18 years of age ) and parents of children < 16 years of age .
You are an expert at summarizing long articles. Proceed to summarize the following text: puromycin aminonucleoside ( pan)-induced nephrotic syndrome is one of the most extensively studied models of glomerulonephritis in rats . this experimental model mimics the minimal change disease glomerulopathy found in human pathology . in vivo micropuncture studies in the unilateral model of pan - induced nephrotic syndrome have shown that sodium reabsorption is specifically increased in the collecting duct and not in the proximal tubule and distal nephron11 ) . micropuncture of the accessible distal convoluted tubule revealed that the tubular sodium load was similar in pan - treated kidney and control kidney of the same rat , and the final urine sodium excretion was threefold lower in urine collected from the pan - treated kidney compared to that in the untreated control kidney in the same animal11 ) . thus , this study directly point to a role of increased sodium reabsorption in the collecting duct and , hence , it may be hypothesized that dysregulation of the key sodium channels and transporters in the collecting duct may be responsible for this . a few studies have subsequently demonstrated that sodium retention in pan nephrotic rats is correlated with increased na , k - atpase activity and expression in the cortical collecting duct12 , 13 ) . we demonstrated that pan - induced nephrotic syndrome is associated with 1 ) sodium retention , decreased urinary excretion of sodium , a marked ascites , and increased plasma aldosterone level , 2 ) upregulation of protein abundance of specific enac subunits in cortex , outer medulla and inner medulla , and 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments observed as increased immunolabeling in apical plasma membrane domains14 ) . in contrast , the protein levels of other major sodium transporters expressed in nephron segments at a site proximal to the connecting tubule ( i.e. , nhe3 , nkcc2 , na , k - atpase and ncc ) were significantly reduced in these segments14 ) . indeed , there was no downregulation of the na , k - atpase expression in the collecting duct . taken together , these observations therefore strongly support the view that the renal sodium retention associated with pan - induced nephrotic syndrome is caused by increased sodium reabsorption in connecting tubule and collecting duct14 ) . thus , this is likely to represent a key molecular basis for the sodium retention associated with pan - induced nephrotic syndrome combined with the previously demonstrated increase in collecting duct na , katpase activity and protein abundance12 , 13 ) . moreover , our results extend this study by demonstrating the segmental specific upregulation of all three enac subunits , and most importantly provide evidence for a significantly increased apical targeting by immunoelectron microscopy . the latter is essential since it is important to document that the increased expression is at the site of function i.e. in the plasma membrane . finally , we also confirm that there is a uniform downregulation of all investigated sodium transporters in the thick ascending limb of henle ( tal ) and proximal tubule . this together with observed increased expression and increased targeting of enac subunits in combination with the previous functional micropuncture studies strongly support the view that the sodium retention in the nephrotic syndrome may occur in the connecting tubule and collecting duct15 ) . nephrotic syndrome may develop as a result of primary diseases such as minimal change disease or immune glomerulonephritis . membranous glomerulonephritis ( mgn ) remains the most common cause of primary nephrotic syndrome in adult . a further reason for its importance is that approximately 25 to 50% of patients progress to end - stage kidney disease over 10 years . thus , mgn has a different and more progressive clinical course as compared to the non - progressive benign character of minimal change nephrotic syndrome16 ) . mercury chloride has been known to induce a systemic autoimmune disease including membranous nephropathy with igg deposits . this nephropathy is responsible for the development of high - range proteinuria and a full - blown nephrotic syndrome associated with generalized edema and ascites17 ) . to elucidate whether the changes in expression and plasma membrane targeting of enac subunits in the pan - induced minimal change nephrotic syndrome ( caused by podocyte injury , not immune complex mediated ) is unique to the pan model or may be a more general characteristic of nephrotic syndrome including immune complex mediated glomerulonephritis , and to investigate the physiologic role of changes in subcellular distribution of enac subunits in the abnormal sodium retention and ascites formation during disease states , we examined the changes of enac abundance and/or trafficking and 11hsd2 in hgcl2 induced nephritic syndrome in brown norway rats . the results demonstrated that hgcl2-induced immune complex glomerulonephritis was associated with 1 ) sodium retention , decreased urinary sodium excretion , development of ascites , and increased plasma aldosterone level ; 2 ) increased abundance of enac ( fig . 1 ) 3 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments ; and 4 ) decreased protein abundance of 11hsd2 ( fig . these observations therefore strongly support the view that the renal sodium retention associated with hgcl2 induced nephrotic syndrome is caused by increased sodium reabsorption in the distal nephron including the connecting tubule and collecting duct . these results are consistent with the results of our previous studies of nephrotic syndrome induced by pan14 ) . thus , a straightforward interpretation of these observations would be that increased apical targeting of enac subunits plays a role in the development of sodium retention in hgcl2 nephrotic syndrome as well , and it could be argued that this mechanism in fact could be a general characteristic of the nephrotic syndrome including both minimal change disease and immune complex mediated glomerulonephritis . apical targeting of enac subunits and protein abundance of enac can be regulated by aldosterone7,8 ) . indeed , plasma aldosterone levels were significantly increased in hgcl2 nephropathy , and thus , it can be speculated that aldosterone stimulate the sodium reabsorption in hgcl2 nephropathy . however , substantial evidence argue against a major role of aldosterone in nephrotic syndrome : 1 ) ascites and edema occur in rats with nephrosis attributable to rabbit anti - rat kidney serum injection , without elevated aldosterone levels19 ) . 2 ) inhibition of angiotensin converting enzyme by captopril failed to induce natriuresis in rats with pan nephrosis despite decreased aldosterone levels20 ) . and 3 ) sodium retention was observed only in the affected proteinuria kidney in rats unilateral models of pan nephrosis11 ) . these observations suggest that aldosterone may not alone be involved in the sodium retention and presumably in the enac regulation in the nephrotic syndrome . it has been suggested that the activity of type 2 11hydroxysteroid dehydrogenase ( 11hsd2 ) can regulate the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid induced activation of mineralocorticoid receptor ( mr)21 ) . we also demonstrate that there is a downregulation of 11hsd2 protein expression in the kidney in hgcl2 nephrotic syndrome as well as in pan nephrosis . this finding is in line with recent studies in nephrotic patients22 ) and in experimental nephrotic syndrome induced by pan or adriamycin23 ) . the investigators observed a rise in the plasma ratio of corticosterone/11-dehydrocorticosterone as well as in the urinary ratio of ( thb+5-thb)/tha in nephritic rats23 ) , and interpreted this change as a result of a decreased activity of 11-hydroxysteroid dehydrogenase . we here propose that this may occur via a downregulated protein expression of 11hsd2 in nephrotic syndrome . these findings suggest that downregulation of 11hsd2 provides access of glucocorticoids to the mr , resulting in increased activity of mr and collecting duct sodium retention in rats with nephrotic syndrome . systemic and local factors are known to regulate enac abundance , targeting and activity as well as na , k - atpase activity . these include aldosterone7 , 8) , vasopressin6 ) and angiotensin ii ( ang ii)24 ) . aldosterone is known to increase na , k - atpase activity in the cortical collecting duct and to increase enac redistribution to the apical membrane of connecting tubule and collecting duct principal cells and to promote the expression of a 70 kda form of the gamma enac subunit , thought to be linked to activation of the channel by proteolytic cleavage7 , 8) . it is therefore tempting to link this hormone with the findings observed in pan nephrosis . moreover levels of vasopressin , ang ii and aldosterone are increased in the initial phase of nephrosis14 , 25 , 26 ) , leading to the hypothesis that they may play significant roles in the development of sodium retention . as the time course and magnitude of sodium retention is indistinguishable in nephrotic brattleboro rats ( vasopressin deficient ) compared to normal nephrotic rats12 ) , vasopressin is not considered to be a major factor for the development of sodium retention in nephrotic syndrome . we have also demonstrated that ang ii at1 receptor blockade , candesartan , did not improve the sodium retention associated with pan - induced nephrotic syndrome nor the time course of its development27 ) . there have been contradictory studies on the impact of aldosterone absence on the development of edema in nephrotic syndrome28 , 29 ) . furthermore , even though pharmacologic blockade of aldosterone has been proven to be unsuccessful regarding sodium retention in experimental nephrosis15 ) , this can not rule out aldosterone effect as pharmacologic mineralocorticoid receptor blockade has been demonstrated to be useless against enac trafficking30 ) and would not avoid aldosterone action through the glucocorticoid receptor31 ) . finally , na , k - atpase in the collecting duct of nephrotic rats is still activated in the absence of aldosterone29 ) . it is therefore not known whether the enhanced enac targeting observed in pan nephrosis is a primary dysregulation in nephrotic syndrome or is secondary to the high aldosterone levels observed in this condition . we therefore studied the development of nephrotic syndrome in adrenalectomized rats , aiming at defining the role of aldosterone on sodium retention and enac targeting observed in this pathologic condition . puromycin treatment of adrenalectomized rats supplemented with dexamethasone induced sodium retention in spite of the absence of aldosterone . immunocytochemical analyses revealed absence of enhanced apical targeting of enac subunits in pan treated adrenalectomized ( adx - pan ) rats with distribution of labelling similar to adrenalectomized dexamethasone - treated control rats ( adx ) . key sodium transporters were downregulated as previously observed in non - adrenalectomized puromycin - treated rats , whereas the global expression of the alpha-1 subunit of the na , k - atpase was unchanged . taken together , pan treatment in the absence of aldosterone induced sodium retention , increased enac expression but did not change the subcellular distribution of enac . this indicates that the previously observed enhanced apical targeting of enac in pan induced neprotic syndrome is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in nephrotic syndrome . however , it is not entirely clear whether whether puromycin - treatment results in an increased sensitivity of kidneys to systemic hormonal changes and which local factors might influence the sodium retention and enac activity . renal sodium and water retention has been shown to be responsible for the development of ascites not only in patients with liver cirrhosis but also in experimental cirrhotic rats . atrial natriuretic peptide resistance , arginine vasopressin ( avp ) , renin - angiotensin - aldosterone , and sympathetic nerve overactivity have been shown to modulate renal tubular functions and have been considered to be mainly involved in sodium and water retention in liver cirrhosis . in kidneys of cirrhotic rat , increased reabsorption of sodium and water in the distal nephron and collecting duct has been suggested to be one of the most important renal tubular dysfuctions involved in the pathogenesis of ascites . however , the underlying molecular and cellular mechanisms for the sodium retention are still incompletely understood . in particular , the role of aldosterone in sodium retention and ascites formation in liver cirrhosis is still unclear . previous studies demonstrated that plasma aldosterone levels were usually elevated in liver cirrhosis with ascites33 ) and that spironolactone , a mineralocorticoid receptor antagonist , increased sodium excretion in these patients34 ) . there has been some controversy as to the role of regulation of the renal sodium transporters and channels in the development of edema in animal models of cirrhosis . rats are treated with carbon tetrachloride ( ccl4 ) in combination with phenobarbital supplied in the drinking water , which results in liver toxicity . in the second , rats have their common bile duct ligated , and increased circulating levels of bile acids ( cholestasis ) results in hepatotoxicity and eventually cirrhosis . differences in the severity , reproducibility , and rapidity of the manifestation of ascites in the two models probably play a role in some discrepancies35 ) . the renal responses for sodium retention displayed wide variations among the rats with ccl4-induced liver cirrhosis . some rats showed markedly decreased urinary sodium excretion ( sodium retaining stage , group a ) whereas the others exhibited unchanged urinary sodium excretion ( maintenance stage , group b ) compared with controls , even though all ccl4-treated rats had significant amount of ascites ( fig . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . previous studies demonstrated that aldosterone causes a mobility shift of enac from an 85 kda band to 70 kda band without a change in total enac protein abundance7 ) . the same changes are observed in chronically sodium - restricted rats in addition to a significant downregulation of the enac subunit7 ) . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation . it is noteworthy that at 8 weeks of liver cirrhosis we did not see any evidence of positive sodium balance even though all rats showed marked ascites39 ) . these findings may suggest that there are dynamic changes in sodium retention at different stages ( i.e. , a sodium retaining stage and a compensatory stage ) of liver cirrhosis . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . recognition of this apparent biphasic renal response to cirrhosis , with progression from the early to the late phase of decompensation , may help to clarify apparent discrepancies in a variety of earlier studies . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . 5 ) , but not in 8 week liver cirrhotic rats without positive sodium balance39 ) . since increased enac plasma membrane targeting is strongly associated with increased sodium reabsorption , this finding very strongly suggests that increased enac targetings contribute significantly to the increased renal tubular sodium reabsorption . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . since there was a markedly enhanced apical plasma membrane expression of all enac subunits at 6 wk liver cirrhosis associated with maintained plasma aldosterone levels but not at 8 wk liver cirrhosis where aldosterone levels were markedly reduced , it is likely that aldosterone plays an essential role . have demonstrated that the mr has an equal affinity for aldosterone and glucocorticoids , yet in vivo it displays specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids . this specificity is conferred by 11hsd2 , an enzyme that converts glucocorticoids to inactive 11-ketosteroid derivatives that have weak or no affinity for the mr in mineralocorticoid sensitive tissues including the distal nephron21 ) . loss of function mutation of 11hsd2 or inhibition of 11hsd2 activity allows glucocortiocids to promote renal sodium retention and potassium excretion in the cortical collecting tubule40 , 41 ) . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . we demonstrate in the present studies that there is downregulation of 11hsd2 abundance in the kidney in decompensated liver cirrhosis rats . this finding is in line with recent studies in compensated liver cirrhosis reported by quattropani et al.42 ) and escher et al.43 ) . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . plasma aldosterone levels , at least in humans and rats without liver cirrhosis , have been shown to be reduced in conditions with low expression of 11hsd240 , 41 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in contrast , in response to liver cirrhosis for 8 weeks enac abundance was decreased ( fig . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . it can be speculated that 11hsd2 is still functioning but not at full power ( thus aldosterone may play a major role ) , or it may be speculated that differences in enac trafficking between 6 and 8 weeks are independent of aldosterone and/or 11hsd2 . in normal rats both the apical targeting and protein abundance of enac however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . it may be speculated that glucocorticoids may be involved in this regulation , and if this is the case the observations suggest that regulation of enac abundance is not as sensitive , as is the regulation of changes in the subcellular localization of enac , to mr activation by glucocorticoids . whether the differential regulation of enac subunit targeting and expression may be related to differences in the kinetics of mr - ligand interaction remains unknown ; aldosterone dissociates more slowly from mr than cortisol does , indicating that the stability of the aldosterone - mr complex is higher than that of the cortisol - mr complex45 , 46 ) . in addition , the mr activity can be regulated by at the level of transcription activation or repression by cell specific co - factors47 ) . it is also interesting to note that the rats with liver cirrhosis apparently exhibited dynamic changes in sodium retention and enac trafficking . after 6 weeks , liver cirrhosis animals exhibited sodium retention , potassium loss and decreased urine na / k ratio indicating increased aldosterone - like activity . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . on the other hand , at 8 weeks the rats with liver cirrhosis did not exhibit any changes in urinary sodium and potassium excretion even though they all had marked ascites . the underlying mechanism for the time course changes in circulating aldosterone levels and changes in enac subunits trafficking remains undefined . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. in this model after 6 weeks of cbdl ) . the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . this pahse may represent active and successful escape from the sodium retention by means of down - regulation of the activity of the enac . the renal responses for sodium retention displayed wide variations among the rats with ccl4-induced liver cirrhosis . some rats showed markedly decreased urinary sodium excretion ( sodium retaining stage , group a ) whereas the others exhibited unchanged urinary sodium excretion ( maintenance stage , group b ) compared with controls , even though all ccl4-treated rats had significant amount of ascites ( fig . the results demonstrated that ccl4-induced sodium retaining stage ( group a ) of liver cirrhosis was associated with 1 ) decreased urinary sodium excretion , and increased or maintained plasma aldosterone levels ; 2 ) increased apical targeting of enac subunits in dct2 , cnt and collecting duct segments ; and 3 ) decreased protein abundance of 11hsd2 . in contrast , maintenance stage ( group b ) of liver cirrhosis was associated with no changes in the urinary sodium excretion , trafficking and abundance of enac subunits and the abundance of 11hsd236 ) . the most important finding is the striking increase in targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining stage ( group a ) , but not in the maintenance stage ( group b ) of liver cirrhosis . since an increased targeting of all enac subunits to the apical plasma membrane is associated with increased sodium reabsorption , the present finding of an increased enac targeting in the sodium - retaining stage of liver cirrhosis could contribute significantly to the increased renal tubular sodium reabsorption . these observations therefore strongly support the view that the renal sodium retention in the decompensated liver cirrhosis is mainly caused by an increased sodium reabsorption in distal nephron including the collecting duct37 ) . we demonstrated that the abundance of enac was decreased or unchanged , and the abundance of the 70-kda form of enac was increased while the 85-kda band was markedly decreased in the sodium - retaining stage ( group a ) of cirrhotic rats ( fig . previous studies demonstrated that aldosterone causes a mobility shift of enac from an 85 kda band to 70 kda band without a change in total enac protein abundance7 ) . the same changes are observed in chronically sodium - restricted rats in addition to a significant downregulation of the enac subunit7 ) . thus , the observed increased apical targeting and altered expression of - and enac subunits in group a could be caused by the stimulation of mineralocorticoid receptor ( mr ) in the aldosterone - responsive epithelium . long - term bile duct ligation in rats is a well known experimental model of liver cirrhosis and portal hypertension , and has been widely used to study the renal mechanisms of sodium retention , as well as systemic and splanchnic hemodynamics38 ) . cbdl rats showed decreased fractional excretion of sodium and hence , positive sodium balance in the sodium retaining stage of liver cirrhosis ( at 6 wks ) . it is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation . it is noteworthy that at 8 weeks of liver cirrhosis we did not see any evidence of positive sodium balance even though all rats showed marked ascites39 ) . these findings may suggest that there are dynamic changes in sodium retention at different stages ( i.e. , a sodium retaining stage and a compensatory stage ) of liver cirrhosis . it should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial of full compensation . recognition of this apparent biphasic renal response to cirrhosis , with progression from the early to the late phase of decompensation , may help to clarify apparent discrepancies in a variety of earlier studies . the most important finding is the increased targeting of all enac subunits to the apical plasma membrane domain in dct2 , cnt and collecting duct in the sodium retaining cirrhotic rats ( fig . 5 ) , but not in 8 week liver cirrhotic rats without positive sodium balance39 ) . since increased enac plasma membrane targeting is strongly associated with increased sodium reabsorption , this finding very strongly suggests that increased enac targetings contribute significantly to the increased renal tubular sodium reabsorption . the increased apical targeting of enac subunits in cbdl - induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of mineralocorticoid receptor ( mr ) in the aldosterone responsive epithelium . since there was a markedly enhanced apical plasma membrane expression of all enac subunits at 6 wk liver cirrhosis associated with maintained plasma aldosterone levels but not at 8 wk liver cirrhosis where aldosterone levels were markedly reduced , it is likely that aldosterone plays an essential role . have demonstrated that the mr has an equal affinity for aldosterone and glucocorticoids , yet in vivo it displays specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids . this specificity is conferred by 11hsd2 , an enzyme that converts glucocorticoids to inactive 11-ketosteroid derivatives that have weak or no affinity for the mr in mineralocorticoid sensitive tissues including the distal nephron21 ) . loss of function mutation of 11hsd2 or inhibition of 11hsd2 activity allows glucocortiocids to promote renal sodium retention and potassium excretion in the cortical collecting tubule40 , 41 ) . these findings suggest that the 11hsd2 regulates the sodium reabsorption in the aldosterone responsive renal tubules by glucocorticoid - induced activation of the mr . we demonstrate in the present studies that there is downregulation of 11hsd2 abundance in the kidney in decompensated liver cirrhosis rats . this finding is in line with recent studies in compensated liver cirrhosis reported by quattropani et al.42 ) and escher et al.43 ) . they demonstrated that 11 hsd2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation42 , 43 ) . these findings together suggest that reduced expression and activity provides basis for access of glucocorticoids to the mr , thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis . this notion was strongly supported by the increased apical targeting of enac subunits in rats with glycyrrhizic acid - induced inhibition of 11hsd2 activity44 ) . plasma aldosterone levels , at least in humans and rats without liver cirrhosis , have been shown to be reduced in conditions with low expression of 11hsd240 , 41 ) . it is noteworthy that plasma aldosterone levels were not reduced in 6 wk liver cirrhosis rats , despite that the protein abundance of 11hsd2 was significantly decreased . thus , coordinated activation of mr by both glucocorticoid as a consequence of reduced activity of 11hsd2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss , and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in cbdl - induced liver cirrhosis . in contrast , in response to liver cirrhosis for 8 weeks enac abundance was decreased ( fig . in addition , the increased apical targeting of enac subunits was retrieved in spite of decreased abundance of 11hsd2 . it can be speculated that 11hsd2 is still functioning but not at full power ( thus aldosterone may play a major role ) , or it may be speculated that differences in enac trafficking between 6 and 8 weeks are independent of aldosterone and/or 11hsd2 . in normal rats both the apical targeting and protein abundance of enac are increased by high dose aldosterone treatment7 ) . however , in the present study we did not observe an increase in enac protein expression in the cortex in kidneys from rats with cbdl - induced liver cirrhosis for 6 weeks although increased abundance of enac was seen in inner stripe of the outer medulla . it may be speculated that glucocorticoids may be involved in this regulation , and if this is the case the observations suggest that regulation of enac abundance is not as sensitive , as is the regulation of changes in the subcellular localization of enac , to mr activation by glucocorticoids . whether the differential regulation of enac subunit targeting and expression may be related to differences in the kinetics of mr - ligand interaction remains unknown ; aldosterone dissociates more slowly from mr than cortisol does , indicating that the stability of the aldosterone - mr complex is higher than that of the cortisol - mr complex45 , 46 ) . in addition , the mr activity can be regulated by at the level of transcription activation or repression by cell specific co - factors47 ) . it is also interesting to note that the rats with liver cirrhosis apparently exhibited dynamic changes in sodium retention and enac trafficking . after 6 weeks , liver cirrhosis animals exhibited sodium retention , potassium loss and decreased urine na / k ratio indicating increased aldosterone - like activity . these findings can be explained in part by decreased abundance of 11hsd2 , which permits the stimulation of mr by glucocorticoid resulting in increased apical targeting of enac subunits . on the other hand , at 8 weeks the rats with liver cirrhosis did not exhibit any changes in urinary sodium and potassium excretion even though they all had marked ascites . the underlying mechanism for the time course changes in circulating aldosterone levels and changes in enac subunits trafficking remains undefined . however , it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptaide ( anp ) contribute to the decrease in plasma aldosterone levels and , hence , limit enac abundance / trafficking since anp has been known to directly inhibit aldosterone secretion in the adrenal glands through specific anp receptors in the zona glomerulosa48 ) . the results strongly suggests that the marked increase in apical enac subunit targeting combined with diminished abundance of 11hsd2 in the dct2 , connecting tubule and collecting duct is likely to play key roles for the sodium retention associated with cbdl - induced liver cirrhosis in rats in the sodium retaining stage ( i.e. in this model after 6 weeks of cbdl ) . the subsequent decrease in the expression of enac subunits and the reduced targeting after 8 weeks of cbdl - induced liver cirrhosis may contribute to promote sodium excretion at later stages of liver cirrhosis . this pahse may represent active and successful escape from the sodium retention by means of down - regulation of the activity of the enac . increased abundance and/or apical targeting of enac subunits in the connecting tubule and collecting duct play an important role in the pathogenesis of sodium retention in nephrotic syndrome and liver cirrhosis .	nephrotic syndrome and liver cirrhosis are common clinical manifestations , and are associated with avid sodium retention leading to the development of edema and ascites . however , the mechanism for the sodium retention is still incompletely understood and the molecular basis remains undefined . we examined the changes of sodium ( co)transporters and epithelial sodium channels ( enacs ) in the kidneys of experimental nephrotic syndrome and liver cirrhosis . the results demonstrated that puromycin- or hgcl2-induced nephrotic syndrome was associated with 1 ) sodium retention , decreased urinary sodium excretion , development of ascites , and increased plasma aldosterone level ; 2 ) increased apical targeting of enac subunits in connecting tubule and collecting duct segments ; and 3 ) decreased protein abundance of type 2 11-hydroxysteroid dehydrogenase ( 11hsd2 ) . experimental liver cirrhosis was induced in rats by ccl4 treatment or common bile duct ligation . an increased apical targeting of alpha- , beta- , and gamma - enac subunits in connecting tubule , and cortical and medullary collecting duct segments in sodium retaining phase of liver cirhosis but not in escape phase of sodium retention . immunolabeling intensity of 11hsd2 in the connecting tubule and cortical collecting duct was significantly reduced in sodium retaining phase of liver cirrhosis , and this was confirmed by immunoblotting . these observations therefore strongly support the view that the renal sodium retention associated with nephrotic syndrome and liver cirrhosis is caused by increased sodium reabsorption in the aldosterone sensitive distal nephron including the connecting tubule and collecting duct , and increased apical targeting of enac subunits plays a role in the development of sodium retention in nephrotic syndrome and liver cirrhosis .
You are an expert at summarizing long articles. Proceed to summarize the following text: public health agencies have long relied on data and information produced by the health care sector.1,2 notifiable disease reporting , community health assessments , outbreak detection , intervention planning , and other activities can benefit from data and information generated in clinical contexts by physicians , hospitals , and other health care organizations . the good news for public health is that more opportunities exist now to access these data . currently , the health care sector is undergoing a health information technology and informatics revolution with an unprecedented volume of health information being created . furthermore , us health policy supports the sharing of this information between different organizations by encouraging the adoption of standards and interoperable technology.3,4 one avenue to increase access to health information and data , particularly for local health departments ( lhds ) , is participation in electronic exchange of health information ( eehi ) . eehi is the process of electronically sharing ( patient / health ) information between different entities.5 in this study , we use eehi as the main focus , rather than health information exchange , another commonly used term that refers to an entity for data exchange . various organizations across the united states provide the technical infrastructure and organizational governance necessary for eehi to occur . these eehi - facilitating organizations are variously referred to as health information organizations , regional health information organizations , or health information exchanges ( hies ) . the laboratory encounters , demographics , diagnoses , and imaging data available through the hies have a wide range of potential benefits to public health , including disease reporting , outbreak surveillance , and population health monitoring , among others.6,7 a small number of state health agencies and lhds have realized that they produce higher quality as well as more accurate , complete , and timely data from participation in exchange relationships with hie organizations . also notable are epidemiologic , disease surveillance , and/or community assessment gains , and improved disaster response capabilities from this participation . although eehi is promising , evidence suggests the public health system is underleveraging the potential benefits of eehi . advances in availability and timeliness of health information can offer public health agencies a wide range of potential avenues for improving public health service provision.6 health departments may benefit from better availability of more accurate and complete data supported by electronic data exchange ( edt).810 public health agencies with robust informatics capacities may benefit from more thorough health monitoring , disease investigation , and assessment.11 real time data exchange between public health agencies and health care providers can allow continual surveillance and prediction of disease occurrences.2,1215 evidence from the health departments using hie suggests that these benefits are being realized in some settings.8,15 lhds ' capacity to electronically exchange data with health care providers can be significantly reduced if they are not connected to hies . compared with other forms of public health informatics including immunization registries , electronic disease reporting systems , electronic laboratory reporting , and electronic health records ( ehrs)hies are the least commonly used information system by lhds.16 national surveys indicate increasing but still very low levels of lhds ' adoption of the hie . in 2013 , just 1 in 10 lhds reported hie adoption , up from 7% in 2010.16,17 even considering the 19% of lhds who reported plans to implement hie in 2013,16 it still leaves the majority of lhds without hie participation , or without any plans to leverage the potential benefits of hie in the near future . considering the broad scope of potential benefits of lhds ' eehi to public health practice , it is critical to understand the current national landscape of practices of eehi . it is also imperative to understand the types of entities with which lhds exchange data on an ongoing basis and the variation in frequency of exchange by partner types . understanding whether there are certain patterns or predictors of eehi may help to inform future directions or policy priorities in ensuring that interested lhds are able to participate in eehi . lhds ' low overall level of informatics capacity may imply that they face significant challenges in use of informatics , including eehi . the purpose of this study is to utilize a novel , nationwide data to explore the current capacity for electronically exchanging information at lhds , and the barriers to such an exchange . data were drawn from the 2015 informatics capacity and needs assessment survey , conducted by the jiann - ping hsu college of public health at georgia southern university in collaboration with the national association of county and city health officials ( naccho ) . this web - based survey had a target population of all lhds in the united states . a representative sample of 650 lhds was drawn using a stratified random sampling design on the basis of 7 population strata : less than 25 000 , 25 000 to 49 999 , 50 000 to 99 999 , 100 000 to 249 999 , 250 000 to 499 999 , 500 000 to 999 999 , and 1 000 000 and more . lhds with larger population were systematically oversampled to ensure inclusion of a sufficient number of large lhds in the completed surveys . respondents were informatics or information systems staff identified by lhds before the main survey . a structured questionnaire that included measures to examine lhds ' current informatics capacity and needs was constructed and pretested with 20 informatics staff . the completed questionnaire was administered to the sample of 650 lhds via qualtrics survey software . statistical weights were developed to account for 3 factors : ( a ) disproportionate response rate by population size ( using 7 population strata , typically used in naccho surveys ) , ( b ) oversampling of lhds with larger population sizes , and ( c ) sampling rather than a census approach . two primary domains related to hie were included in this study : ( 1 ) connectivity with exchange partners by type of partners and ( 2 ) challenges experienced . exchange partners and the direction of exchange were captured concurrently . respondents were asked whether the lhd was able to electronically send or receive health information from a listing of 10 exchange partner organizations ( table 1 ) . challenges to electronically sending and receiving information were identified through a series of yes / no questions . the list of challenges was developed from a review of the literature and expanded with the help of the project advisory committee , consisting of more than 12 subject matter experts from the industry . p values for the population categories are based on somer 's d , and for governance category , based on . italicized values show significance of subgroup differences at p .05 . we described the proportion of lhds ' participation in electronic exchange of information for all lhds by types of exchange partners , and challenges , using frequencies and percentage . we used for examining the difference in hie participation and challenges to information exchange by lhd governance category . to assess the differences in hie participation and challenges by jurisdiction size , we used somer 's d. we performed all analyses for this study using spss version 23.0 . data were drawn from the 2015 informatics capacity and needs assessment survey , conducted by the jiann - ping hsu college of public health at georgia southern university in collaboration with the national association of county and city health officials ( naccho ) . this web - based survey had a target population of all lhds in the united states . a representative sample of 650 lhds was drawn using a stratified random sampling design on the basis of 7 population strata : less than 25 000 , 25 000 to 49 999 , 50 000 to 99 999 , 100 000 to 249 999 , 250 000 to 499 999 , 500 000 to 999 999 , and 1 000 000 and more . lhds with larger population were systematically oversampled to ensure inclusion of a sufficient number of large lhds in the completed surveys . respondents were informatics or information systems staff identified by lhds before the main survey . a structured questionnaire that included measures to examine lhds ' current informatics capacity and needs was constructed and pretested with 20 informatics staff . the completed questionnaire was administered to the sample of 650 lhds via qualtrics survey software . statistical weights were developed to account for 3 factors : ( a ) disproportionate response rate by population size ( using 7 population strata , typically used in naccho surveys ) , ( b ) oversampling of lhds with larger population sizes , and ( c ) sampling rather than a census approach . two primary domains related to hie were included in this study : ( 1 ) connectivity with exchange partners by type of partners and ( 2 ) challenges experienced . exchange partners and the direction of exchange were captured concurrently . respondents were asked whether the lhd was able to electronically send or receive health information from a listing of 10 exchange partner organizations ( table 1 ) . challenges to electronically sending and receiving information were identified through a series of yes / no questions . the list of challenges was developed from a review of the literature and expanded with the help of the project advisory committee , consisting of more than 12 subject matter experts from the industry . p values for the population categories are based on somer 's d , and for governance category , based on . italicized values show significance of subgroup differences at p .05 . we described the proportion of lhds ' participation in electronic exchange of information for all lhds by types of exchange partners , and challenges , using frequencies and percentage . we used for examining the difference in hie participation and challenges to information exchange by lhd governance category . to assess the differences in hie participation and challenges by jurisdiction size , we used somer 's d. we performed all analyses for this study using spss version 23.0 . substantial variation existed in the level of lhd participation in eehi by type of exchange partners ( table 1 ) . lhds most commonly exchange health information electronically with the state department of health and/or human services ( 71% ) . the next most common exchange partners included laboratories , hospitals , and other county / city departments . least common exchange partners included jail / correctional health , followed by health or county - based purchasing plans and home health agencies . there was a significant variation in lhds ' eehi with all other partners by jurisdiction size with the exception of primary care clinics , and health and county - based purchasing plans . small lhds with a jurisdiction population of less than 50 000 were more likely to participate in eehi with state health departments and home health agencies than large lhds ( population size 500 000 people ) . variation also existed in eehi with other partners by the type of lhd - state health agency governance relationship , with one exception exchange with other county / city health departments . with the exception of exchange with other county / city lhds , lhds with shared and local governance were more likely to participate than state - governed lhds in eehi . lhds reported a number of challenges to exchanging health information electronically ( table 2 ) . forty percent of lhds reported health insurance portability and accountability act ( hipaa ) , privacy or legal concerns affected their electronic exchange of health information with outside organizations . thirty - seven percent reported lack of access to technical support or expertise , and 36% reported that there were other competing priorities . over a quarter of lhds ( 28% ) reported that they were unclear about the value of the return on investment of the development of information systems . an equal proportion of lhds ( 28% ) indicated that lack of knowledge about exchange partners ' ability to electronically exchange health information presented a challenge . high subscription rates for exchange services , insufficient information on exchange options such as exchange partners , transport mechanisms , and message formats , and lack of support from lhd , state , and local board of health leaders were each reported by 22% of the lhds . one fifth of lhds reported lack of capacity on the part of potential exchange partners and 9% reported that their lhd had limited broadband / internet access . abbreviations : ehr , electronic health record ; hipaa , health insurance portability and accountability act ; lboh , local board of health ; lhds , local health department ; roi , return on investment . n = 277 ; p values for the population categories are based on somer 's d , and for governance category , based on . italicized values show significance of subgroup differences at p .05 . there was some variation in the challenges lhds experienced by jurisdiction size and governance category . more lhds in jurisdictions of fewer than 50 000 people than in jurisdictions of larger sizes reported challenges related to lack of resources , including lack of access to technical support or expertise , prohibitively high subscription rates , and lack of leadership support . a significantly higher proportion of lhds with larger jurisdiction populations reported competing priorities , and exchange partners without the ability , interest , or incentive to electronically exchange health information . although not having broadband was a challenge for 10% of lhds in small and medium - sized lhds , none of the lhds in jurisdiction populations of 500 000 or more people reported broadband challenges to information exchanges . challenges comparatively more common among state - governed lhds than in lhds in local or shared governance arrangements included hipaa , privacy or legal concerns ; lack of or access to technical support or expertise ; and exchange partners not having the ability , interest , or incentive to electronically exchange health information . in contrast , a higher proportion of lhds with local governance compared with those with state or shared governance arrangements experienced challenges in information exchange concerning lack of clarity about the development of information systems ' return on investment ; cost prohibitive ? subscription rates for exchange services ; and inability of the lhd 's ehr system to generate or receive electronic messages or transactions in standardized format . lhds in shared governance systems had more common exchange challenges with respect to insufficient information on exchange options available such as exchange partners , transport mechanisms ; lack of support from leadership ; and limited broadband / internet access . this less - than - ubiquitous participation by public health agencies has already been noted in evaluation of eehi efforts.18,19 as a result of low levels of participation , a large proportion of the public health system does not have the opportunity to realize the benefits of eehi that can accrue to both clinical and nonclinical services and functions universally , organizational size ( of which jurisdictional size is a proxy ) is associated with innovation adoption , and particularly information technology innovations : smaller organizations do not adopt information technology at the same rate as larger organizations.16,20 in this instance , however , lhds serving small jurisdictions may be doubly challenged . these lhds reported internal capacity needs ( eg , broadband connection ) more commonly than those serving large jurisdictions . at the same time , lhds serving small jurisdictions may also face external capacity needs as small and rural jurisdictions are less likely to have an active hie organization with which to participate.16 as further described later , options exist for supporting small and medium - sized lhds , but such options will not create exchange partners . in areas where no hie organization exists the challenges to participation in eehi identified in our survey can be subdivided into 2 broad themes : technological and organizational / interorganizational . the most common technical challenges included lack of technical expertise , privacy concerns , and use of an ehr system that could not exchange information . although locally governed and small lhds most frequently reported a lack of technical expertise as a barrier , our survey suggests that lhds across the board would benefit from an infusion of health information technology capacity and resources . hie organizations have staff and expertise in sharing information , but connecting to and utilizing any new health information system can be a daunting task and requires organizational support and staffing . prior survey work has indicated that increasing the numbers of information technology staff is associated with more public health information system usage.22 lhds with state - level governance also were significantly more likely to report hipaa , privacy or legal concerns . nevertheless , the challenge in exchanging information with health organizations is in some ways not surprising . public health agencies have historically been challenged sharing information among themselves.9,20,23 we also identified the largest challenges related to eehi with outside organizations . those challenges varied according to governance type , suggesting that there are both advantages and disadvantages of having centralized public health governance . overall , lhds with shared governance had the highest levels of eehi , followed by local and state . the low levels of usage for lhds with state - level governance may point to a need for tailored interventions and policies for the lhds governed by state health agencies . a corollary of this finding is that lhds with local governance were significantly more likely to report lacking access to technical support or expertise as a challenge to eehi . solutions targeting increased adoption of eehi should therefore be targeted to a specific audience , such as lhds serving smaller jurisdictions , those with local governance , etc . our findings about the partners with which lhds are most likely to connect to electronically exchange information have important implications . lhds ' knowledge of the most common exchange partners will be important in making decisions about resource allocation and building interoperability with other partners . for instance , when strategically prioritizing the resources to develop fully interoperable systems , it may be advantageous ( at least in the short term ) to build capacity for interoperability with laboratory systems rather than jails or correctional facilities , given the relative priority for these 2 systems shown by our results . however , longer term prioritization of information system interoperability should also be driven by the relative impact of that partner on an lhd 's operational and strategic priorities . our findings , that lhds of different sizes differ in nature of their challenges , have clear implications for policy and advocacy efforts . lhds serving larger jurisdictions ( ie , 500 000 ) report challenges such as competing priorities rather than a lack of resources . this may be indicative of their relatively broader scope and scale as well as their informatics needs and resources . efforts to incentivize hie may benefit from prioritizing training and educating lhd staff about the importance of data and information exchange rather than providing technical or physical resources such as broadband . lhds in jurisdictions of fewer than 50 000 people , on the other hand , need more resources such as technical support and expertise . our findings suggest that leadership support is among important factors for improving data exchange capacity of lhds with smaller jurisdiction and those in shared governance . therefore , before sending out the survey , the project team asked naccho 's contact persons for the lhds in the study sample ( mostly top executives ) to identify the most relevant informatics staff . because roughly one fourth of the lhds provided the informatics staff contacts , the mixed perspective of lhds , informatics and the leadership staff may have implications for interpretation for our results . we could not stratify the use of data exchange by the type of services provided by lhds because the survey did not collect data on types of services provided . this less - than - ubiquitous participation by public health agencies has already been noted in evaluation of eehi efforts.18,19 as a result of low levels of participation , a large proportion of the public health system does not have the opportunity to realize the benefits of eehi that can accrue to both clinical and nonclinical services and functions universally , organizational size ( of which jurisdictional size is a proxy ) is associated with innovation adoption , and particularly information technology innovations : smaller organizations do not adopt information technology at the same rate as larger organizations.16,20 in this instance , however , lhds serving small jurisdictions may be doubly challenged . these lhds reported internal capacity needs ( eg , broadband connection ) more commonly than those serving large jurisdictions . at the same time , lhds serving small jurisdictions may also face external capacity needs as small and rural jurisdictions are less likely to have an active hie organization with which to participate.16 as further described later , options exist for supporting small and medium - sized lhds , but such options will not create exchange partners . in areas where no hie organization exists the challenges to participation in eehi identified in our survey can be subdivided into 2 broad themes : technological and organizational / interorganizational . the most common technical challenges included lack of technical expertise , privacy concerns , and use of an ehr system that could not exchange information . although locally governed and small lhds most frequently reported a lack of technical expertise as a barrier , our survey suggests that lhds across the board would benefit from an infusion of health information technology capacity and resources . hie organizations have staff and expertise in sharing information , but connecting to and utilizing any new health information system can be a daunting task and requires organizational support and staffing . prior survey work has indicated that increasing the numbers of information technology staff is associated with more public health information system usage.22 lhds with state - level governance also were significantly more likely to report hipaa , privacy or legal concerns . nevertheless , the challenge in exchanging information with health organizations is in some ways not surprising . public health agencies have historically been challenged sharing information among themselves.9,20,23 we also identified the largest challenges related to eehi with outside organizations . those challenges varied according to governance type , suggesting that there are both advantages and disadvantages of having centralized public health governance . overall , lhds with shared governance had the highest levels of eehi , followed by local and state . the low levels of usage for lhds with state - level governance may point to a need for tailored interventions and policies for the lhds governed by state health agencies . a corollary of this finding is that lhds with local governance were significantly more likely to report lacking access to technical support or expertise as a challenge to eehi . solutions targeting increased adoption of eehi should therefore be targeted to a specific audience , such as lhds serving smaller jurisdictions , those with local governance , etc . our findings about the partners with which lhds are most likely to connect to electronically exchange information have important implications . lhds ' knowledge of the most common exchange partners will be important in making decisions about resource allocation and building interoperability with other partners . for instance , when strategically prioritizing the resources to develop fully interoperable systems , it may be advantageous ( at least in the short term ) to build capacity for interoperability with laboratory systems rather than jails or correctional facilities , given the relative priority for these 2 systems shown by our results . however , longer term prioritization of information system interoperability should also be driven by the relative impact of that partner on an lhd 's operational and strategic priorities . our findings , that lhds of different sizes differ in nature of their challenges , have clear implications for policy and advocacy efforts . lhds serving larger jurisdictions ( ie , 500 000 ) report challenges such as competing priorities rather than a lack of resources . this may be indicative of their relatively broader scope and scale as well as their informatics needs and resources . efforts to incentivize hie may benefit from prioritizing training and educating lhd staff about the importance of data and information exchange rather than providing technical or physical resources such as broadband . lhds in jurisdictions of fewer than 50 000 people , on the other hand , need more resources such as technical support and expertise . our findings suggest that leadership support is among important factors for improving data exchange capacity of lhds with smaller jurisdiction and those in shared governance . therefore , before sending out the survey , the project team asked naccho 's contact persons for the lhds in the study sample ( mostly top executives ) to identify the most relevant informatics staff . because roughly one fourth of the lhds provided the informatics staff contacts , the mixed perspective of lhds , informatics and the leadership staff may have implications for interpretation for our results . we could not stratify the use of data exchange by the type of services provided by lhds because the survey did not collect data on types of services provided . hies have become more common throughout many parts of the health care sector over the past couple of decades and have shown some promise in improving our health care delivery system . yet , lhds have had relatively low participation in hies and thus may not fully benefit from these crucial data resources . unlike health care , where market forces and federal programs such as payments associated with meaningful use of ehrs incentivize infrastructure development,24 infrastructure development for public health informatics tends to be driven by political forces.13 these forces may present substantial barriers to the development and use of public health information systems.13 although our findings show a large proportion of lhds participating in eehi with some exchange partners , a small proportion participates in exchanges with many other partners . lhds commonly exchange information with state departments of health , public health laboratories , and hospitals . exchange with other partners was , however , less common , indicating opportunities to expand the exchange relationships with other partners , including pharmacies , long - term care facilities , home health agencies , health- and county - based purchasing plans , and jail / correctional health . regardless of lhd size , hipaa , privacy or legal concern was the most common challenge , indicating a clear need for concerted effort to improve lhd staff 's knowledge about this important aspect of data exchange . training on hipaa may cover at least the basic elements of the privacy rule , more specifically knowledge about who is covered under hipaa regulations , the type of information that is protected , and ways to protect information . challenges pertaining to electronic exchange of information differ for lhds of different sizes and different governance relationships with states . developing and managing robust information systems that allow sending , receiving , and integrating data and information with minimal human intervention is essential . for the local , state , and federal public health enterprise to continue to take advantage of health informatics , lhds will need to address barriers to connectivity , including political ones ; better coordinate across programs ; look at new ways to implement systems ; and place a greater focus on their informatics workforce.25	the health care sector is undergoing a health information technology and informatics revolution with an unprecedented volume of health information being created , presenting opportunities for local health departments to access these data . this study utilizes novel , nationwide data to explore the current capacity for electronically exchanging information at local health departments , and the barriers to such an exchange .
You are an expert at summarizing long articles. Proceed to summarize the following text: of the 35 patients with cutaneous or mucocutaneous / mucosal leishmaniasis , 30 were german tourists ( table 1 ) . ten had contracted cutaneous or mucocutaneous / mucosal leishmaniasis in europe , 11 in central and south america , 6 in asia , and 3 in africa . two persons had been infected during work stays of 1 to 4 months in french guyana , one each in peru and libya , and one patient had immigrated from afghanistan . the median duration of lesions until the diagnosis of leishmaniasis was made was 4 months ( range 3 weeks to 2 years ) . sixteen patients had more than one lesion ( median 2 , range 16 lesions ) . seventeen lesions were located in the face , including mouth and nose , 28 on the upper extremities and 21 on the lower extremities . lesions were ulcerated in 39 cases , papular - nodular in 24 , and plaque - like in 3 . parasites were detected in 13 of 20 smears , in 9 of 10 cultures , and in 14 of 16 histologic sections ; by using pcr , leishmania - specific dna was detected in 16 of 16 biopsy specimens . patient 2 had received methotrexate and steroids for treatment of systemic collagenosis for several weeks . both patients were tested for leishmanial infection in the blood ; in both patients , the leishmania - specific pcr of the buffy coat of the blood was positive . she had been treated for a skin lesion caused by l. braziliensis 3 years earlier . a total of 18 of the 23 visceral leishmaniasis patients were german tourists ; 3 were immigrants from angola , iran , and togo ; and 2 were visitors from italy and portugal ( table 2 ) . the median time between symptom onset and the correct diagnosis was 4 months ( range 116 months ) . all case - patients had fever , 17 ( 74% ) had splenomegaly , 11 ( 48% ) hepatomegaly , 20 ( 87% ) anemia , 17 ( 74% ) leukopenia , and 8 ( 35% ) thrombocytopenia . bone marrow smears indicated leishmania in 18 of 20 , bone marrow culture in 6 of 7 , bone marrow histologic sections in 7 of 8 , pcr of the bone marrow in 8 of 9 , and pcr of the buffy coat of the blood in 7 of 7 cases . additionally , antibodies were detected in medium to high concentration by an immunofluorescence test , enzyme - linked immunosorbent assay ( elisa ) , or both , in 14 of 15 cases . species was identified in 7 of 18 visceral cases contracted in southern europe and indicated leishmania belonging to the l. donovani complex , which implicated infection with l. infantum . six cases of visceral leishmaniasis occurred in children 2 months of age to 11 years of age . four german tourists and two immigrants had long - known hiv infection ( median duration 3 years , range 8 months6 years ) . all hiv co - infected patients had cd4-cell counts below 200/l ( median 108 , range 23185 cd4 cells/l ) when the diagnosis of visceral leishmaniasis was made . of the remaining 11 patients , 1 had a thymoma with impaired t - helper-1 cell function , 2 had received intermittent immunosuppressive therapy ( methotrexate and steroids ) for rheumatologic disease , and 2 patients had their spleens removed . three patients were in an impaired general condition because of combinations of diabetes , hypertonus , hypercholesterolemia , and emphysema . in the remaining three patients ( 5368 years of age ) , information on single cases and a small case series of imported leishmaniases in germany is available , but systematic reporting on frequency , type , and origin of leishmanial infections in germany did not exist until 2000 ( 57 ) . our recent surveillance is dependent on passive consultation and reporting and therefore may have selection bias because if visceral leishmaniasis , a potentially fatal disease that requires hospitalization , is suspected , advice on diagnosis and treatment is sought more often than for the skin infection . we assume that our system captures approximately half of the visceral leishmaniasis cases and approximately one third of the classical cutaneous cases imported to germany . a total of 47% of all cases , but 78% of the visceral cases were contracted in the european mediterranean area and portugal , and most of the infections indicated a species of the l. donovani complex , most probably l. infantum , as the probable causative agent . thirteen infections ( 22% ) were acquired on the mediterranean islands of ibiza , ischia , majorca , malta , korfu , or sicily . this distribution reflects the fact , that the mediterranean countries , spain , italy , and the mediterranean islands , in particular , are the favorite vacation areas for germans . annually , germans take 18 million vacations to the european mediterranean area ( including 8 million to spain and 6 million to italy ) with a median duration of 2 weeks . sixty percent of travel to italy and 90% of travel to spain are to leishmania - endemic areas . while leishmaniasis has always been endemic in the mediterranean countries , the maximum northern latitude for sandfly survival is speculated to move further to the north , beyond germany ( 1 ) because of global warming . if this scenario is correct , the imported cases may serve as a potential substrate for the sandfly vector . dogs that are imported as pets from the disease - endemic areas of southwestern europe or that contract the infection when accompanying their owners for vacation are another potential substrate ( 8) . infections with l. infantum in a child , as well as in a horse who had never left germany , have recently been described and have led to speculations about an autochthonous focus ( 9,10 ) . also recently , the first sandfly species , phlebotomus mascittii grassi , 1908 , was detected in southern germany , although its potential as a vector of leishmania remains to be demonstrated ( 11 ) . as expected , visceral leishmaniasis is often manifested in persons with impaired immunocompetence because of young age , hiv infection , immunosuppressive therapy and , in our analysis , in older persons with concomitant diseases . notably , 12 ( 67% ) of 18 of the visceral cases contracted in the european mediterranean area were in adults , thus confirming a change in age groups affected . formerly , visceral leishmaniasis was known mainly as a disease of children ( 1,2 ) . this change may partly be explained by the increased proportion of leishmania and hiv co - infected persons and partly by increased travel activities of otherwise immunocompromised persons , including elderly persons . furthermore , even in patients with cutaneous leishmaniasis , dissemination of parasites has to be excluded in case of impaired immunocompetence ( e.g. , immunosuppressive treatment ) . in these cases , leishmania - specific pcr of the buffy coat of the peripheral blood is a sensitive method for detecting parasite spread beyond the skin . parents of small children and persons with reduced immunocompetence should be informed about their increased susceptibility to infection with leishmania when traveling to disease - endemic areas . measures to reduce the exposure to sandflies , such as clothes , repellents , and mosquito nets as well as collars impregnated with repellents for accompanying dogs , should be recommended .	in 2000 , a reference center was created to systematically record leishmaniases in germany . we analyzed 58 cases of leishmaniases imported during a 2-year period . these findings will serve as a baseline for the sandfly vector s anticipated northward move because of global warming and as an advisory for immunocompromised persons traveling to leishmaniasis - endemic areas .
You are an expert at summarizing long articles. Proceed to summarize the following text: respiratory failure is common in the advanced stages of amyotrophic lateral sclerosis ( als ) and is the major cause of morbidity . in contrast , it is rare that als presents with respiratory failure as the sole initial manifestation [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ] . the majority of patients in previous reports are men , and the interval between respiratory failure and limb weakness is between 0 and 3 months . we here present the case of a male patient with als who had respiratory failure 13 months prior to the appearance of limb weakness . a 72-year - old male gradually developed exertional dyspnea 13 months before his admission to our hospital . he had a history of a lacunar stroke ( at the thalamus with mild right leg weakness and numbness without difficulty , which remained unchanged before his admission ) . he had an occasional cough because of his mild chronic obstructive pulmonary disease ( copd ) , but he never had dyspnea before . he was first suspected of suffering from heart failure , but his chest x - ray and serum brain natriuretic peptide level were normal . the patient was then suspected to have a worsening of his copd , and medication for copd was started in the respiratory disease department , without any benefits . when he was admitted to the neurology department 13 months after symptom onset , he had lost 10 kg . he had no atrophy or fasciculation of the tongue and had no dysarthria or dysphagia but a pre - existing mild right leg weakness . his pinprick sensation had mildly decreased in the right leg . however , on examination , he had a mild fasciculation in his shoulder , the pectoralis major , his arm and his platysma muscles bilaterally . the patient 's deep tendon reflexes were exaggerated bilaterally , and his plantar reflexes were extensor . a spirometer showed a normal forced expiratory volume in 1 second of 81.3% ( normal > 71% ) , while he had a markedly low percent vital capacity of 41.3% ( normal > 90% ) . the blood gas analysis ( in room air ) showed hypercapnia ( po2 75.8 mm hg , pco2 54.4 mm hg ) , and his chest x - ray indicated bilateral phrenic nerve palsy ( fig . 1 ) . electromyography revealed an acute denervation and fibrillation potentials in the diaphragm , tongue , limbs and paraspinal muscles with almost the same severity . three days after his admission , he suddenly felt drowsy , and a second blood gas analysis revealed anoxia and co2 narcosis . the patient was started on non - invasive ventilatory support and was discharged from the hospital . it is rare that als presents with respiratory failure as the sole initial manifestation [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ] . in such cases , patients often visit pulmonary disease departments first and diagnosing als is extremely difficult . in the previous reports , the interval between respiratory failure and limb weakness is between 0 and 3 months ( table 1 ) . the only exception is case no . 5 by chen et al . who presented with limb weakness 12 months after respiratory failure . clinical features of als cases who present with respiratory failure as the sole initial manifestation [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ] , including ours , are unique because ( 1 ) all patients , except for one case ( by parhad et al . ) , are men , with the rate being higher than in the total als cohort , ( 2 ) motor weakness and atrophy following respiratory failure are present in the limbs , which is in contrast to the observation of bulbar palsy often preceding or occurring together with respiratory failure , and ( 3 ) preceding pulmonary diseases ( copd , pneumothorax , atelectasis , etc . ) occurred in half of the patients a rate which is higher than that in the total als population . why respiratory failure becomes the sole initial manifestation in these patients remains uncertain . however , it is reported that denervation by electromyography was more severe in the diaphragm than in any other skeletal muscles [ 7 , 8 ] , and , in these patients , a cell loss of the anterior horn was most severe in the cervical spinal cord along the neuraxis . therefore , in these patients , phrenic anterior horn cells might have been involved early . the mechanisms accounting for male preponderance remain unsolved . as for the comorbidity of preceding pulmonary diseases and als , experimental studies suggest that comorbid muscle weakness [ by orthopedic ( limbs ) , pulmonary ( diaphragm ) etiologies , etc . ] might hasten anterior horn neurodegeneration since fast fatigable motor units are more vulnerable to degenerative disease processes in als mice . in conclusion , although rare , als can present with respiratory failure as the sole initial manifestation more than 1 year prior to limb weakness .	it is rare that amyotrophic lateral sclerosis ( als ) presents with respiratory failure as the sole initial manifestation . a 72-year - old man with mild chronic obstructive pulmonary disease developed exertional dyspnea for 13 months . he then progressed to limb weakness that led to the diagnosis of als . although rare , als can present with respiratory failure as the sole initial manifestation more than 1 year prior to limb weakness .
You are an expert at summarizing long articles. Proceed to summarize the following text: pure titanium and titanium alloys are the most used biomaterials for fabrication of surgical implants due to their excellent mechanical properties , biocompatibility15 and resistance to corrosion16 . they are considered ideal materials because they have shown better acceptability by human tissues than other metals under diverse circumstances16 . the high biocompatibility of titanium derives partially from the stable and protective oxide layer , which apparently aids in connecting extracellular matrix to the implant surface16 . the knowledge of the biomaterial - bone tissue interface is extremely important to define which material would promote a better tissue response and which kind of surface would be more adequate for the proliferation of bone cells21 . after placement of an implant in the surgical cavity , ideally , these events should lead to wound healing by intimate apposition of the bone to the biomaterial , i.e. , osseointegration6 . osseointegration is a direct structural and functional connection between living bone and the surface of an implant5 . regardless of their external shape , microscopically they can present smooth , porous or textured surfaces1,4,5,10,19,26 . several studies have shown that the success or failure of surgical implants can be related to chemical3,12,19 and biological12 properties of their surfaces as well as to their micromorphology13,16,26 . the differences in the microstructure of implant surfaces seem to influence stress distribution , bone retention , cellular response on its surface and consequently the osseointegration3,12,23 . the porous surface has been considered as a good alternative to rough coating11 . biomaterials with porous structure are aimed at optimizing the interfacial resistance between the material and the bone , leading to a more effective fixation of the implant . porous implants allow an interdigitation of the bone tissue to the implant , thus increasing the interfacial resistance18 . additionally , a porous surface provides a more efficient fixation , shorter initial healing time19 and increased cellular adhesion potential . pilliar , et al.19 ( 1998 ) reviewed and compared several designs and surfaces of surgical implants and concluded that the surface with pores of 100 m in diameter would be ideal for the neoformation of a three - dimensional osseointegration net inside the implant . oliveira , et al.18 ( 2002 ) determined that , although the fabrication process parameters have been optimized , the ideal porous requirements for surgical implants have not yet been reached . these authors reported that some changes are necessary in order to increase porosity and advocated that an analysis of pore size distribution along the sample has been performed to indicate more efficiently which porous fraction would better meet implant requirements17 . therefore , the purpose of this study was to analyze , by histological and histomorphometric methods , the bone repair around cylindrical grade-2 commercially pure titanium implants with rough and porous surface , fabricated using powder metallurgy technique , after their insertion in tibiae of rabbits . seven male new zealand albino rabbits aged 6 to 8 months and weighing 3.5 kg on average were used in this study . the animals were provided by the vivarium of so jose dos campos dental school and were kept in individual cages and fed a commercial pet chow ( coelhil r ; socil , belo horizonte , mg , brazil ) and water ad libitum . the commercially pure titanium implants with either rough or porous surface were fabricated at the department of materials of the air and space institute at cta ( brazilian air and space technical center , so jos dos campos , sp , brazil ) in an association with the national institute of technology of rio de janeiro ( rio de janeiro , rj , brazil ) . the implants had 3.0 mm in diameter and pore diameters ranging from 250 and 350 m . the implants were cleaned , wrapped and sterilized in autoclave at 121c for 15 min . prior to surgery , the animals were weighed and injected intramuscularly with 13 mg / kg of aqueous solution of 2% hydrochloride of 2-(2,6-xylidine)-5,6-dihydro-4h-1,3-thiazin ( rompum ; bayer , so paulo , sp , brazil ) , which is an analgesic , sedative and muscular relaxant substance . general anesthesia was obtained with administration of 33 mg / kg of ketamine ( dopalen ; agribrands do brasil ltda , paulinia , sp , brazil ) . ) . a local anesthetic composed of 3% octapressin combined with prilocaine hydrochloride and felypressin ( 3%citanest dentsply ) was also used . the implant was removed from the wrap , placed in the perforation and pressed into the surgical cavity until it was fixed to the cortical bone . in order to standardize the procedure , the right tibia received a rough surface implant ( control ) group , while the left tibia received a porous - surface implant . the muscular tissue was sutured with absorbable thread and the skin with mononylon 4 - 0 surgical thread . after that , all animals were given penicillin and were monitored until sacrifice 30 days after surgery . after euthanasia , the surgical segments with the implants were removed and the implants were tested for mobility using a clinical clamp . the segments were placed in 10% formalin solution for at least 48 h. next , the specimens were dehydrated in an increasing ethanol series ( 50% , 75% , 90% and 100% ) , embedded in polyester resin ( orto cristal t 208 ; valglass com . so paulo , sp , brazil ) and sectioned longitudinally in a sectioning machine for hard tissues ( labcut 1010 ; extec technologies , inc . , enfield , ct , usa ) providing 80-m - thick serial sections on average . the sections were ground in a polishing machine ( labpol 8 - 12 ; extec technologies , inc . ) using sandpapers of decreasing abrasiveness ( # 400 , # 600 and # 1200 ) until reaching a thickness of 30 - 40 m . images were taken with a digital camera ( dsc - s85 , cyber - shot , sony ) coupled to the optical microscope and evaluated on a television monitor ( panasonic ) . three sections of each implant were evaluated for the percentage of bone neoformation at implant - bone interface . two areas of each section were digitized ( x100 ) , representing the medial and distal interfaces of the implant . bone neoformation rate and bone ingrowth into the pores were calculated using the image j software ( nih , usa ) . the final data were submitted to statistical analysis using the minitab software version 12.3 ( minitab inc , usa ) and student 's t - test at 5% significance level . all animals presented satisfactory postoperative results , without any evidence of inflammation or infection of the surgical site . no adverse reaction was observed during the procedure . during the clinical evaluation , performed with a clinical clamp , none of the implants presented mobility . in both groups ( rough- and porous - surface implants ) , there was bone neoformation around the implant leading to osseointegration ( figures 1 and 2 ) . this new bone formation was similar in both groups and was constituted by mature bone trabeculae that presented lamellar arrangement and by medullar spaces of different sizes . in several specimens , regardless of the analyzed group , bone neoformation above the implants and at their bottom surface was observed , sometimes contacting the internal cortical face on the opposite side . a clear delimitation between the new bone formation and the preexistent cortical bone was also noticed ( figures 3 , 4 and 5 ) . the results showed that the types of implant used presented statistically significant difference to each other regarding the amount of new bone formation on the implant - bone interface , the porous - surface implants showing a larger amount neoformed bone . table 1 shows the means of bone neoformation ( % ) at bone - implant interface of rough- vs. porous - surface implants . statistically significant difference . in our study , cylindrical implants with porous surface were compared to cylindrical implants with rough surface regarding the quantity and quality of new bone formation on the implant - bone interface after implantation in rabbit tibiae . however , when the quantity of bone neoformation at implant - bone interface was evaluated , a larger formation of bone tissue was observed for the porous - surface implants , and this difference was statistically significant . the most important factors to dental implant osseointegration are related to the characteristics of its surfaces , which include topography and chemical and electric properties of the material19 , since bone - implant interaction is mainly related to the most external layers of the implants13 . important factors to a more successful osseointegration are : implant material , implant shape , surgical technique20 , quantity of bone tissue20,25 , load6 and implant resistance4 . however , some other factors such as surface energy , sterilization techniques and chemical and topographic properties of the implant surface are extremely important for the final outcome of osseointegration2,8,12,16 . in the present study , the results showed that all implants were well tolerated when placed in rabbit tibiae , thus corroborating the findings of previous studies that indicated titanium as the best biomaterial for surgical implants15,16 . bone growth is also dependent on factors such as percentage of surface porosity , stability and degree of micromovement of the implant and the presence of gaps between the implant and the bone at the time of placement . implants placed under pressure inside the surgical cavity help minimizing the gaps and implant micromovement8 . therefore , in order to obtain osseointegration , the surgical cavity must be prepared with the least injury possible15,20 . in order to cause minimal damage to the surrounding bone tissues , in the present study , bone perforation was performed using burs of increasingly larger diameters , and under constant saline irrigation . after that , the implants were gently pressed into the surgical cavity , which diminished the gap between the implant and the bone and promoted efficient stability . additionally , all implants were evaluated with a clinical clamp at the moment of sacrifice , 30 days after surgery , and were stable . some previous studies used a 4-week healing period to evaluate the biocompatibility of metal materials7,14,17,22 . deporter , et al.10 ( 1986 ) reported that healing periods ( i.e. , between implant placement and euthanasia of the animals ) longer than 4 weeks added no benefits to increase the quantity of bone tissue ingrowth into porous - surface implants , and observed that only bone tissue maturation took place after this period . thus , in the present study , a 4-week period was used to evaluate the biocompatibility of porous - surface grade 2 commercially pure titanium implants fabricated by means of powder metallurgy technique . the purpose of studying and developing porous - surface implants is to promote a more stable and biocompatible fixation of titanium implants . the creation of a porous geometry surface aims not only at increasing contact area but also at allowing bone ingrowth into the pores , including those located more centrally . such ingrowth is due to pore intercommunication , which produces a three - dimensional net and allows a mechanical entanglement7,8,9,18 . the porous - surface implant have an ideal diameter for the proliferation of bone cells2,12 , and most studies report that pore diameter ranges from 100 to 400 m . in addition , the pores have been shown to be interconnected , so that bone interdigitation into the porous structure may occur , thus reaching maximum interfacial resistance18,24 . however , nguyen , et al.17 ( 2004 ) reported that small pores measuring only 45 m in diameter also allow bone ingrowth . furthermore frosch , et al.14 ( 2002 ) found that 300 to 600 m pores showed a three - dimensional , reticular osteoblast - like cell development within 4 weeks , and that in 1000 m pores cell ingrowth was incomplete . in the present study , the diameter of the pores varied between 250 and 350 m , corroborating the findings of other studies9,19,23 . svehla , et al.23 ( 2000 ) compared five different implant surfaces and verified that porous surfaces promoted an excellent substrate for the ingrowth of bone tissue and consequent implant fixation , whereas smooth surfaces resulted in the formation of a fibrous tissue and improper fixation . in the present study , when the cylindrical porous - surface implants were compared to the cylindrical rough - surfaces implants , osseointegration was observed in both groups . deporter , et al.11 ( 1990 ) concluded that a smaller segment of porous - surface implants allowed a more effective contact with the bone , due to a bigger contact area , determined by its topography . similar findings were observed in the present study in which a statistically significant difference was observed between the groups in relation to the quantity of bone neoformation at titanium - bone interface . therefore , smaller porous - surface implants could be developed to be used in complex clinical situations such as regions with low - quality bone9,19 . the results of this study showed that because of the larger contact surface promoted by the presence of pores , there was more bone ingrowth on the implant - bone interface . such results are consistent with those of cook and rust - dawicki7 ( 1995 ) , deporter , et al.9 ( 2002 ) , deporter , et al.10 ( 1986 ) , deporter , et al.11 ( 1990 ) , frosch , et al.14 ( 2002 ) , karabuda , et al.15 ( 1999 ) , story , et al.22 ( 1998 ) , svehla , et al.23 ( 2000 ) , vidigal junior , et al.25 ( 1999 ) , zinger , et al.26 ( 2005 ) who also observed more effectiveness of the porous - surface implants compared to other types of implants . based on the results of this study , it may be concluded that the cylindrical porous - surface implants yielded greater bone neoformation than the cylindrical rough - surface implants because of their larger area in contact with the bone tissue and the presence of an intercommunicating porous structure that allowed the formation of a three - dimensional osseointegration network .	the purpose of this study was to analyze the bone repair around commercially pure titanium implants with rough and porous surface , fabricated using powder metallurgy technique , after their insertion in tibiae of rabbits . seven male rabbits were used . each animal received 3 porous - surface implants in the left tibia and 3 rough - surface implants in the right tibia . the rabbits were sacrificed 4 weeks after surgery and fragments of the tibiae containing the implants were submitted to histological and histomorphometric analyses to evaluate new bone formation at the implant - bone interface . means ( % ) of bone neoformation obtained in the histomorphometric analysis were compared by student 's t - test for paired samples at 5% significance level .. the results of the histological analysis showed that osseointegration occurred for both types of implants with similar quality of bone tissue . the histomorphometric analysis revealed means of new bone formation at implant - bone interface of 79.69 1.00% and 65.05 1.23% for the porous- and rough - surface implants , respectively . statistically significant difference was observed between the two types of implants with respect to the amount new bone formation ( p<0.05 ) . in conclusion , the porous - surface implants contributed to the osseointegration because they provide a larger contact area at implant - bone interface .
You are an expert at summarizing long articles. Proceed to summarize the following text: to investigate the impact of molecular crowding on gene expression , we used genetic components from phage t7 due to their well - characterized kinetics and functions in synthetic systems ( supplementary fig . we quantified interactions between t7 rna polymerase ( rnap ) and dna in the presence of crowding agents because molecular crowding can increase excluded volumes , which would affect large molecules more significantly than small molecules . to visualize the response , we constructed a red fluorescent protein ( rfp)-t7rnap fusion rna polymerase that transcribed a cyan fluorescent protein ( cfp ) from a pt7 promoter ( supplementary fig . b and supplementary information ( si ) ) . since transcriptional activities are sensitive to the diffusion of macromolecules , we first focused on diffusion dynamics of t7 rnap in different crowding environments outside of the artificial cell environment . we modulated crowding molecular size because a previous work has suggested that natural cells could use crowding molecules as molecular sieves to filter molecules and modulate system dynamics . specifically , we performed fluorescence recovery after photobleaching ( frap , supplementary fig . 3 , supplementary movie 1 ) using either small ( 6x10g / mol , dex - small ) or big ( 2x10g / mol , dex - big ) dextran polymers ( fig . these molecules are commonly used as inert molecules that mimic crowded intracellular environments , but do not react with the systems of interest . we found that initial recovery rates of mobile rfp - t7rnap were significantly reduced by increasing densities of both dex - small and dex - big ( fig . this result suggests that diffusion of the mobile fraction of rfp - t7rnap were affected by the crowding molecules . both dex - small and dex - big also resulted in higher immobile fractions of rfp - t7rnap with increasing crowding densities ( fig . based on existing diffusion models , the increased immobile fractions are likely due to increased sub - diffusion fractions of rfp - t7rnap in crowded environments , in which the polymerase explores a smaller space than predicted by classical brownian dynamics . therefore , our findings suggest that both dex - small and dex - big reduced the diffusion of rfp - t7rnap . in addition to diffusion of macromolecules , gene transcription is sensitive to the binding between transcription factors and promoters , which was requisite to understand the impact of molecular crowding on gene expression . to measure the binding between rfp - t7rnap and a pt7 promoter , we built on our frap experiments and conjugated biotinylated - cy3-pt7 dna molecules onto peg - surfaces through avidin - biotin linkages ( supplementary fig . 4 ) . when cy3-pt7 was immobilized on the surface , rfp - t7rnap would freely diffuse in the chamber and bind to pt7 on the surface . we used this approach to measure co - localization of rfp - t7rnap with cy3-pt7 , which would suggest that there was binding between them ( fig . 1d , supplementary fig . 5a , supplementary movie 2 ) . we expected that a decreased dissociation rate constant would shift the mean of bound time distribution to a higher bound time and an increased association rate constant would increase detected binding events at low bound time . with cy3-pt7 , 10% dex - big significantly increased the number of binding events by ~100% compared to the control ( 0.2% dex - big ) ( fig . 10 % dex - big also increased the mean of the bound time distribution by ~100ms compared to the control ( 0.2% dex - big ) , suggesting a decreased dissociation rate constant . in contrast , 10% dex - small only increased the number of binding events by ~25% compared to the control ( 0.2% dex - small ) . these observations suggest that dex - big decreased dissociation rate constants and increased association rate constants between t7 rnap and pt7 more significantly than dex - small , which is consistent with a general theory of molecular crowding that suggests a larger enhancement of molecular binding by large crowding molecules than small crowding molecules . using a control dna without the pt7 promoter , 5b ) , suggesting that the observation was not due to non - specific interactions between t7 rnap and either the peg surface or dna . to gain insight into connections between microscopic dynamics observed using single molecule imaging and gene expression dynamics , we created a parsimonious model based on previous models of molecular crowding and qualitative results obtained from the frap and single molecule experiments ( si , fig . 6 ) . based on the model , we first predicted expression dynamics of a genetic module with a native pt7 promoter that regulates the expression of a cyan fluorescent protein ( cfp ) as a reporter of gene expression ( fig . our model predicts that a small crowding molecule would result in a biphasic response , where gene expression rates would first increase with increasing crowding densities , but then would decrease after a specific crowding density ( fig . the biphasic response arises because dex - small only increased the number of binding events by ~25% ( fig . the predicted biphasic dynamic response was indeed observed with our cell - free expression system , which consisted of an s12 bacterial extract , a s12 supplement , and dex - small ( fig . the cell - free expression system is a synthetic system that couples both transcription and translation and has been used to create artificial cells . in contrast , both our model and experiments show that dex - big caused a monotonic increase of gene expression rates with increasing crowding densities ( fig . 7 ) because dex - big significantly increased the association rate constant and reduced the dissociation rate constant between t7rnap and pt7 promoter ( fig . additional tests using alternative crowding agents , including ficoll 400 ( 400x10g / mol ) , peg-100k ( 100x10g / mol ) , and peg8000 ( 8x10g / mol ) , suggested that the size - dependent impact of molecule crowding on gene expression rates is likely a generic phenomenon ( supplementary fig . specifically , the size of ficoll-400 is approximately the same as the size of dex - big and resulted in monotonically increasing expression rates ; peg-8000 , a small crowding molecule , caused biphasic expression rates ; and peg-100k , a crowding molecule with an intermediate size , significantly increased gene expression rates as compared to peg8000 . similar to the observed impact of molecular crowding on dynamics of nuclear proteins in heterochromatin , our results suggest that molecular composition of genetic microenvironments can distinctly modulate gene expression rates . to respond to changing environments , natural cells could couple crowding densities with both promoters and ribosomal binding sites ( rbs ) to modulate gene expression rates . to investigate this link between genetic components and molecular crowding , we constructed two additional genetic modules , with either a weak pt7 promoter ( pt7,weak ) or a weak rbs ( rbsweak ) ( fig . previous studies have also demonstrated that these genetic components exhibit higher dissociation rate constants between t7 rnap and pt7 and ribosome and rbs as compared to the components used in the original module ( fig . , mathematical modeling predicts that these modules would result in higher fold changes in gene expression rates with increasing crowding densities as compared to the base module ( fig . 2d ) , which arises due to faster decrease in dissociation constants of macromolecules and their respective binding sites with increasing crowding densities . consistent with our model predictions , gene expression rates of both pt7,weak and rbsweak increased more than those of the base module with increasing crowding densities ( fig . molecular crowding is a cellular feature that has been maintained throughout evolution , suggesting that molecular crowding could be essential in maintaining robustness of system dynamics , which is a hallmark of biological systems . to allow for a more systematic assessment of this hypothesis than would be possible experimentally , we first created two parameter sets using the same mathematical model , each representing either a low or a high crowding environment . next , we used computer simulations to emulate perturbations of these base models by randomizing their kinetic constants within 10-fold of their base values ( si ) . the changes in gene expression rates of perturbed models as compared to the base models are quantified as in contrast , in a low crowding environment , fold perturbations are non - zero , showing that the system is more sensitive to the parameter perturbations . these simulation results suggest that molecular crowding may support the robustness of gene expression in natural cells . furthermore , these results are consistent with simulation results of a stochastic model that includes intrinsic noise ( si & supplementary fig . we tested the impact of molecular crowding on system robustness experimentally by changing concentrations of potassium , magnesium , ammonium , spermidine , and folinic acid ( fig . our experimental results show that a low crowding environment ( 0.2% dex - big ) resulted in higher perturbations of gene expression relative to a highly crowded environment ( 10% dex - big ) for magnesium , ammonium , and spermidine . furthermore , we tested if the robustness of gene expression could be fine - tuned using intermediate crowding densities . we observed that ammonium indeed caused a monotonic decrease in fold perturbations with increasing crowding densities ( supplementary fig . in addition to genetic elements , gene circuit dynamics are known to be modulated by negative feedback loops , which are the most ubiquitous motif in natural gene circuits , likely due to their noise reduction and auto - regulatory roles . here , we tested if molecular crowding can modulate gene expression of a negative feedback loop . to gain insight into interactions between feedback loops and molecular crowding , we simulated expression dynamics of a negative feedback loop that consisted of a t7 lysozyme that binds to t7 rna polymerase and we assume that binding between t7 rnap and pt7 would be enhanced at a smaller crowding density relative to the binding between t7 rnap and t7 lysozyme , which is based on existing theories of molecular crowding . as a result , our model predicts that the negative feedback loop would cause biphasic expression rates in environments with large crowding molecules ( fig . our experimental results support the model prediction as expression rates were maximized at 8% crowding density ( fig . we note that this was not due to increased metabolic burden in the expression systems ( supplementary fig . 10a ) . to further corroborate our model , we investigated the impact of a reduced t7 rnap concentration on gene expression rates . based on our model , we expected similar biphasic gene expression rates with a negligible shift in the crowding density that maximizes gene expression rates ( supplementary fig . 10b , black line ) . to test the prediction , we modulated the amount of t7rnap by controlling the ratio of cell free expression systems that either contains t7 rnap or does not contain t7 rnap , which resulted in a 75% reduction of gene expression rates . based on this system , we observed the same biphasic gene expression rates with a maximum rate at 8% crowding density ( supplementary fig . 10b , black squares ) . in natural cells , compartmentalization is the hallmark for creating unique functional units such as in golgi , nucleus , and mitochondria . based on our results with cell free systems , we hypothesized that crowding could similarly affect gene expression in physiologically - relevant volumes . to test this hypothesis , we constructed artificial cells that consist of phospholipid membranes , synthetic expression systems , and a genetic construct ( fig . we created artificial cells of different radii ranging from 100nm-10m that expressed green fluorescent proteins ( gfp ) using a genetic construct . to ensure that gfp was expressed inside artificial cells , we added rnase to inhibit rna synthesis outside artificial cells ( fig . 4b ) ; with liposomes that protect the enclosed volumes , rnase did not inhibit gfp expression . next , we investigated the influence of cell volume on the impact of molecular crowding . specifically , we used the integrated intensity of a cy5 fluorescent dye inside artificial cells as a surrogate of encapsulation volume ( supplementary fig . 12b d ) . based on our results using cell - free expression systems that essentially resulted in semi - infinite reaction volumes , we expect that molecular crowding would exert a larger impact on gene expression in large volumes as compared to small volumes . indeed , we observed that dex - big increased gfp expression in artificial cells of large volumes as compared to artificial cells without dex - big ( fig . this is likely due to reduced reaction rates in large volumes without molecular crowding . as the size of artificial cells decreased , gfp levels in artificial cells with dex - big approached gfp levels of artificial cells without dex - big . these findings indicate that in our synthetic cellular systems , compartmentalization and crowding can play essential roles in controlling gene expression , factors that need to be considered in both mimicking genetic systems for applications in synthetic biology and accurately modeling cellular biochemistry for systems biology . in this study , we demonstrated that molecular crowding increases robustness of gene expression and that molecular crowding can be harnessed for the control of a basic genetic construct in artificial cells . we have also shown the impact of molecular crowding on the dynamics of t7 rnap and how the influence of molecular crowding on gene expression rates can be modulated using genetic components , a negative feedback loop , and the size of crowding molecules . our results suggest that the construction of both in vitro circuits and programmable artificial cells would have to take into account molecular crowding effects , which would be important in modulating system dynamics . our findings underscore how scientists could harness molecular crowding for developing advantages in engineered cell functions such as gene expression , metabolic pathways , and cellular computing . for natural systems , our conclusions with respect to the impact of molecular crowding sizes , strength of genetic components , and network architectures could potentially explain how gene circuit dynamics are modulated by changing environments . our studies would also impact in vitro studies of gene expression and their extension to in vivo environments that are crowded with molecules . as we move towards a bottom up and a priori approach of constructing either artificial cells or cells with completely designed genomes , it is critical to understand the impact of crowded environments on system robustness and then exploit these design principles to our advantage just as natural cells have already accomplished . furthermore , systems biology has begun to take advantage of synthetic cellular systems as high throughput methods to characterize interactions between cellular components . in this context , it is critical to understand how molecular crowding can fundamentally impact interactions between cellular components and generate emergent dynamics that can not be predicted or controlled without considering molecular crowding .	summarythe integration of synthetic and cell - free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution - based chemistry : only the concentrations of reacting species modulate gene expression rates . however , it is known that macromolecular crowding , a key feature of natural cells , can dramatically influence biochemical kinetics by volume exclusion effects that reduce diffusion rates and enhance binding rates of macromolecules . here , we demonstrate that macromolecular crowding can increase the robustness of gene expression through integrating synthetic cellular components of biological circuits and artificial cellular nanosystems . in addition , we reveal how ubiquitous cellular modules , including genetic components , a negative feedback loop , and the size of crowding molecules , can fine tune gene circuit response to molecular crowding . by bridging a key gap between artificial and living cells , our work has implications for efficient and robust control of both synthetic and natural cellular circuits .
You are an expert at summarizing long articles. Proceed to summarize the following text: most gp skin cancer training courses in australia encourage beginners to use dermatoscopy algorithms when deciding if a lesion requires biopsy to exclude skin cancer . indeed , the ability to recognise dermatoscopic criteria correctly and apply a dermatoscopic algorithm is often seen as the sine qua non of excellence in primary care skin cancer practice . the benefits of using a scored dermatoscopy - only algorithm are well documented . however , this approach does not score often useful clinical information such as history of lesion change , the ugly duckling diagnostic approach , where these clinical aspects are also scored , may reduce the chances of the student missing less obvious skin cancers . we performed a retrospective analytical trial to see if a new algorithm , incorporating clinical as well as dermatoscopic criteria , was any different to the three methods commonly used in australia primary care practice to diagnose skin cancer . the new algorithm , blinck , was developed and compared to two dermatoscopy - only algorithms , ( the 3-point checklist and the menzies method ) , and clinical assessment alone . the blinck algorithm was designed as an assessment tool for primary care skin cancer clinicians and does not require the user to be an in contrast to most existing algorithms , the distinction between melanocytic and non - melanocytic lesions is not necessary and both pigmented and non - pigmented lesions may be assessed . the acronym , blinck , refers to six questions that should be asked when assessing a skin lesion and includes both clinical and dermatoscopic features . b benign : is the lesion immediately recognisable as a common benign tumour , on clinical and dermatoscopic examination , with other similar lesions being present on that part of the body , e.g. , typical solar lentigo , seborrheic keratosis , haemangioma or dermatofibroma ? if lonely : is this lesion , clinically and dermatoscopically , the only one of its type on that region of the body , i.e. , an outlier or irregular : for pigmented lesions , is the lesion dermatoscopically irregular , that is , does it have an asymmetrical pigmentation pattern and more than one colour ? for non - pigmented lesions , is there an irregular vascular pattern ? n nervous : is the patient nervous or concerned that this particular lesion may be a skin cancer ? c change : does the patient , or another observer , feel that the lesion is changing ? ( note that only a total score of 1 can be given if either or both of these last two questions are answered yes. ) k known clues : does the lesion definitely have any one of the following dermatoscopic clues to malignancy ? unmistakable variation in thickness of network line pseudopods or streaks segmental black dots , globules or clods irregular and peripheral eccentric structureless zone blue or grey colour irregular distribution vessels1 . a total score of 2 or more out of 4 requires biopsy . to compare blinck with the other diagnostic methods a pilot trial was conducted using images of skin lesions typically seen in australian primary care skin cancer practice . from june 1 to july 6 , 2009 , all skin lesions consecutively excised to exclude skin cancer were recorded by an experienced skin cancer doctor , ( a.c . ) , working in a dedicated skin cancer practice in brisbane , australia . clinically obvious basal cell carcinomas which could be easily diagnosed without dermoscopy were not included in the collection set . high quality clinical and dermatoscopic photographs of 50 skin lesions were obtained , ( non - polarised dermatoscopic images taken with a non - polarised dermlite foto attachment , or dermlite fluid dermatoscope , ( 3gen , llc ) , and canon d40 digital camera , ( tokyo , japan ) . written patient consent was obtained in every case and any history of lesion change or patient concern was documented , as well as whether the lesion was thought to be an ugly duckling by the original examiner . as common lesions such as the dermatofibroma , seborrhoeic keratosis and congenital naevus sometimes pose a diagnostic challenge for inexperienced clinicians , an example of each , seen during the collection period , was included in the set of 50 . as well , a flat naevus that was unchanged on sequential digital monitoring was included in the set without biopsy . histopathological examination of the other 46 lesions revealed 19 to be skin cancers with nine being melanomas ( eight in situ and one invasive ) . four primary care clinicians , ( three gps and a clinical nurse ) , with varying levels of dermatoscopic experience , were asked to review the photographs and select which lesions were suspicious for malignancy , hence requiring biopsy . this assessment was done on four occasions , each time using a different diagnostic approach . dermatoscopic and clinical images were supplied as well as information regarding reported lesion change , ugly duckling sign or patient concern as recorded by original examiner . the clinicians received prior instruction on the use of the three algorithms , and excel answer sheets for each method listed the various criteria used in that algorithm . the clinicians were asked to decide if these criteria were present or not and the spreadsheet was used to calculate the results . the clinician s response for each case was compared to the correct diagnosis and graded as true positive , false negative , false positive or true negative . the number of cancers and melanomas correctly detected and the number of biopsies indicated for each clinician and each method were also recorded ( table 1 ) . as two of the methods related only to pigmented lesions , ( 3-point and menzies ) , the five non - pigmented specimens in the set of 50 were excluded from the contingency tables for these methods . analysis of variance ( anova ) was used with the lsd test , ( least significant difference test ) , to detect differences between clinicians and methods . a p - value of < 0.05 indicated statistical significance . the means for specificity , sensitivity and diagnostic accuracy are shown with their 95% confidence interval , ( 95% ci ) . there were no differences between the clinicians regarding sensitivity , specificity , diagnostic accuracy , number of cancers detected , number of melanomas found or biopsies indicated , however , there were significant differences between the four methods ( table 2 ) . blinck had higher sensitivity and found significantly more melanomas than the other three methods . however , the menzies method and clinical only approach had higher specificity and resulted in fewer biopsies than blinck . diagnostic accuracy was the same for blinck , menzies and clinical only , and all were better than the 3-point checklist . blinck had higher sensitivity , diagnostic accuracy , number of cancers found and number of melanomas found than the 3-point checklist , but had similar specificity and number of biopsies required . the 50 lesions used in the trial were sourced from 46 patients , 22 male and 24 female , with ages varying between 30 and 60 years ( average 58 years ) . four clinically benign lesions were included in the set without a histological diagnosis , ( dermatofibroma , seborrhoeic keratosis , congenital naevus and monitored flat naevus ) , and the remainder were subjected to histological examination ( table 4 ) . it is the third most common cancer in australia in both men and in women . approximately two out of every three australians will be diagnosed with skin cancer before the age of 70 and roughly a million gp visits are made annually for skin cancer . this makes skin cancer the most expensive of all cancers for the australian health system [ 9 , 10 ] . more skin cancers are diagnosed and treated in australia by primary care doctors than by medical specialists . these generalists require a simple yet accurate screening tool that will allow the detection of melanoma and other skin cancers at an early stage when complete cure is possible . currently , most introductory skin cancer courses in australia endorse dermatoscopic evaluation of suspicious skin lesions as the preferred screening method , commonly using the 3-point checklist or the menzies method . other clinical features that may assist with the diagnosis of skin cancer have been previously studied . the ugly duckling sign has been shown to be of possible use in melanoma screening . importantly , the ability to assess whether a lesion is different from surrounding lesions does not appear to require advanced training . hence , it would seem sensible that primary care clinicians seek out ugly duckling lesions when performing a skin examination , ( lonely in the blinck algorithm ) . lesion change is also known to be associated with malignancy , particularly in patients over 50 years of age , and consideration of this clinical feature would also seem prudent . a disproportionate amount of concern for a lesion by the patient , ( nervous ) , may have significance for two reasons . firstly , patients may be uncertain if their lesion has changed or perhaps they may simply fail to volunteer the history of change , bleeding , itch or soreness . they suspect that it is a cancer but assume the doctor is able to make the diagnosis by mere inspection without needing any clinical history . however , this history may be the only clue to malignancy in dermatoscopically bland lesions , and a false negative diagnosis may be made using dermatoscopic assessment alone . secondly , dismissing a lesion about which the patient is quite concerned may have medico - legal consequences should it prove later to be malignant . in this small trial , the blinck algorithm , which scored these extra clinical features along with basic dermatoscopic assessment , found more skin cancers and melanomas than the commonly endorsed methods in australia . this raises the question as to what is the best measure of accuracy in melanoma diagnosis . argenziano has suggested that nne , ( number of melanocytic lesions needed to be excised in order to find one melanoma ) , may be useful for measuring accuracy in melanoma detection and compared the nne in specialised and non - specialised clinical settings . in his study the nne reduced over time from 12.8 to 6.8 with specialised clinics but remained unchanged at 29.4 in non - specialised centres . this would seem to suggest that a level around 6.8 may be an appropriate goal for skin cancer clinicians . in our study , the overall nne for melanoma by all clinicians using the blinck algorithm was 6 , with the 3-point checklist 11 , the menzies method 13 and clinical assessment only 22 , suggesting that blinck may have value as a skin cancer screening tool . as this trial was limited by the small number of skin cancers and melanomas , and by the fact that it was a virtual study without direct assessment of patients lesions , it is difficult to draw definite conclusions regarding the benefits of combining clinical with dermatoscopic features in one diagnostic algorithm . indeed , it could be said that clinicians using dermatoscopic - only algorithms implicitly incorporate relevant clinical aspects of the case in their decision making process . however , knowing how much weight to give these clinical aspects is often difficult for novices . being forced to score the clinical with the dermatoscopic findings may help develop a more structured and holistic approach when deciding if a lesion requires biopsy .	background : deciding whether a skin lesion requires biopsy to exclude skin cancer is often challenging for primary care clinicians in australia . there are several published algorithms designed to assist with the diagnosis of skin cancer but apart from the clinical abcd rule , these algorithms only evaluate the dermatoscopic features of a lesion.objectives:the blinck algorithm explores the effect of combining clinical history and examination with fundamental dermatoscopic assessment in primary care skin cancer practice.patients/methods:clinical and dermatoscopic images of 50 skin lesions were collected and shown to four primary care practitioners . the cases were assessed by each participant and lesions requiring biopsy were determined on separate occasions using the 3-point checklist , the menzies method , clinical assessment alone and the blinck algorithm.results:the blinck algorithm had the highest sensitivity and found more melanomas than any of the other methods . however , blinck required more biopsies than the other methods . when comparing diagnostic accuracy , there was no difference between blinck , menzies method and clinical assessment but all were better than the 3-point checklist.conclusions:these results suggest that the blink algorithm may be a useful skin cancer screening tool for australian primary care practice .
You are an expert at summarizing long articles. Proceed to summarize the following text: the gram - positive genus microbacterium belongs to the family microbacteriaceae , within the phylum actinobacteria . strain ( bh-3 - 3 - 3 ) was isolated from the leaf surface of lettuce ( lactuca sativa ) originating from a conventional field in vestfold , norway . genomic dna was extracted using genomic - tip 500/g kit ( qiagen gmbh , hilden , germany ) , a library was created using pacbio ( pacific biosciences , california , usa ) 20 kb library preparation protocol and whole genome sequencing was performed using pacbio rs ii . the library was sequenced using p6-c4 chemistry with 360 min movie time on one single - molecule real - time ( smrt ) cell . the reads were assembled using hgap v3 ( pacific biosciences , smrt analysis software v2.3.0 ) . the minimus2 software of the amos package was used to circularize the contig , which was confirmed by a dot plot to contain the same sequence at the beginning and end of the contig . rs_resequencing.1 software ( smrt analysis version v2.3.0 ) was used to map reads back to the assembled and circularized sequence in order to correct the sequence after circularization . the sequencing service was provided by the norwegian sequencing centre ( www.sequencing.uio.no ) , a national technology platform hosted by the university of oslo and supported by the functional genomics and infrastructure programs of the research council of norway and the southeastern regional health authorities . the genome of microbacterium sp . bh 3 - 3 - 3 was annotated using ncbi prokaryotic genome annotation pipeline , genemarks + v 3.3 and the rapid annotation system technology ( rast ) server . 1 presents an overview of the count of each subsystem feature and the subsystem coverage . the circular chromosome has a gc content of 70.5% , consisted of 3,508,491 bp and contained 3113 coding sequences ( cdss ) , 9 rrna genes , 45 trnas , and 3 noncoding rna ( ncrna ) genes . this whole genome project has been deposited at ncbi genbank under the accession number cp017674 .	the genus microbacterium contains bacteria that are ubiquitously distributed in various environments and includes plant - associated bacteria that are able to colonize tissue of agricultural crop plants . here , we report the 3,508,491 bp complete genome sequence of microbacterium sp . strain bh-3 - 3 - 3 , isolated from conventionally grown lettuce ( lactuca sativa ) from a field in vestfold , norway . the nucleotide sequence of this genome was deposited into ncbi genbank under the accession cp017674 .
You are an expert at summarizing long articles. Proceed to summarize the following text: all biological processes depend critically on highly specific recognition between molecules with carefully tuned affinities . however , despite the universal nature of these interactions , our understanding of their molecular basis is limited . as a result , structure - based design of small molecules that modulate these interactions is seriously compromised , resulting in failure to capitalize on the wealth of data that are being generated from various structural genomics projects worldwide . the ability to design such molecules rapidly and efficiently at will ( for example , as drug candidates or biochemical tools ) using structural data as a starting point is arguably one of the most important unmet challenges in contemporary science . limited ability to predict affinity from structure is largely due to the complexity of the problem , whereby competing thermodynamic processes all contribute to binding affinity . the standard free energy of binding gb , which determines interaction strength ( eq 1 ) , not only is governed by structural terms but also involves dynamics ( eq 2 ) : where ka is the association constant , hb is the standard enthalpy of binding , sb is the standard entropy of binding , r is the gas constant , and t is the absolute temperature . the key to a full understanding of the binding process is a decomposition of the binding into enthalpic ( structural ) and entropic ( dynamic ) contributions from the protein , the cognate ligand and solvent water . in this manner we successfully rationalized the thermodynamics of binding of small - molecule ligands to a model hydrophobic binder , recombinant mouse major urinary protein ( rmup ) . in the present study we deconvolute the binding thermodynamics of a second member of the lipocalin family , recombinant histamine binding protein ( rrahbp2 ) , which , as its name suggests , is a we reasoned that the study of binding thermodynamics in a second protein with a fold similar to that of rmup would enable us to compare and contrast the entropic contributions to binding from protein , ligand , and solvent , in two systems with classically distinct binding signatures . plasmid pet23a(+ ) hbp2(d24r ) was transformed into e. coli bl21 ( de3 ) cells using the heat shock method . a 1 l aliquot of ligation product was transferred to 25 l of bl21 ( de3 ) cells and incubated on ice for 20 min . the cells were then heat shocked at 42 c for 90 s. next , 75 l of soc medium ( preheated to 42 c ) was added to the cells , and the culture was incubated with agitation at 37 c for 6090 min . aliquots were streaked on to lb plates containing100 g / ml carbenicillin and were grown at 37 c overnight . single colonies of fresh transformants were picked and used to inoculate 5 ml of liquid lb medium and incubated at 37 c overnight . one milliliter was taken from the overnight cultures and used to inoculate 2 1 l of single n - labeled or double c-,n - labeled silantes rich growth medium ( silantes , germany ) , which were incubated at 37 c until the culture density reached od600 0.4 . at this point after 30 min , expression was induced with 1 mm iptg , and the culture was incubated overnight . the cells were harvested by centrifugation at 5000 g for 10 min at 4 c . the cells were suspended in 5 ml / g 50 mm hepes at a ph of 6.8 containing 0.16 mg / ml lysozyme and agitated for 20 min followed by addition of 1 ml / g of deoxycholic acid in 20 mm sodium phosphate ( 4 mg / ml ) both at ph 7.0 . after 30 min of incubation at 37 c , 1 l / g dnasei ( 9 g/l ) was added , and the cells incubated at 37 c for 20 min with agitation . the supernatant was filtered with a 0.22 m filter prior to loading on an anion exchange column ( q - sepharose resin , sigma - aldrich ) . the protein was bound to the column and eluted using a gradient of 0 to 1 m nacl over 200 ml . the protein was concentrated to approximately 3 mg / ml ( 0.15 mm ) using vivaspin 20 centrifugal concentrators with a 5 kda cutoff ( sartorius , u.k . ) . the protein was then further purified by gel filtration with sephacryl s-100 resin ( sigma - aldrich ) . all chromatography steps were carried out on an kta fplc system ( ge healthcare , sweden ) . a 0.4 ml portion of 0.5 mm uniformly enriched c-,n - rra - hbp2(d24r ) in 50 mm potassium phosphate ph 7.4 was placed in a shigemi tube with sodium azide , and dss was added to final concentrations of 1 and 0.2 mm , respectively . all experiments were carried out at 298 k. for the complex with histamine , 1.1 molar equiv of ligand was added to the protein sample ( 0.55 mm ) . three - dimensional assignment experiments were carried out on varian unity spectrometers equipped with triple resonance probes at proton frequencies of 500 mhz ( cbca(co)nh ) and 750 mhz ( hnca and hncacb ) . experiments carried out at 750 mhz on the apo protein and the histamine - bound complex were acquired using a cryo - probe ; all other experiments were acquired using a room temperature probe . cbca(co)nh spectra were acquired with 32 scans , 2048 points , and sweep widths of 8510 , 7200 , and 1620 hz in the h , c , and n dimensions , respectively . hnca spectra were acquired with 16 scans , 1024 points , and sweep widths of 10474 , 4320 , and 2400 hz in the h , c , and n dimensions , respectively . hncacb spectra were acquired with 40 scans , 2048 points , and sweep widths of 15000 , 13000 , and 2400 hz in the h , c , and n dimensions , respectively . data were processed using nmrpipe.(5 ) the rance - kay macro was employed to generate pure absorptive line shapes from sensitivity enhanced spectra . in all cases the data were processed using a cosine - bell function with zero - filling followed by fourier transformation . baseline correction was carried out first in the h dimension then the c followed by the n dimensions and was achieved using the poly function in nmrpipe . backbone assignments were made using the ccpn analysis software package.(6 ) n relaxation - time measurements were carried out essentially according to farrow et al.(7 ) amide relaxation times for mup were obtained at 500 and 600 mhz with t1 relaxation delays of 10.9 , 54.3 , 108.6 , 217.3 , 434.6 , 651.8 , 923.4 , 1195.0 , and 1521.0 ms and t2 relaxation delays of 16.6 , 33.2 , 49.7 , 66.3 , 82.9 , 99.5 , 132.6 ms . delays of 217.3 and 923.4 ms for t1 experiments and 33.2 and 99.5 ms for t2 experiments were repeated to estimate the uncertainty in peak intensity . n steady - state noe spectra were acquired at a proton frequency of 600 mhz with 80 scans ; 2048 and 256 points were acquired with sweep widths of 8510.64 and 2500.00 hz in the h and n dimensions , respectively . all relaxation experiments were recorded in an interleaved manner so as to reduce the effects of any changes in state of the sample . peak intensities from n relaxation measurements were calculated from the center of the peaks ( to minimize peak overlap ) using nmrview software in combination with software written in - house.(8 ) relaxation rates were calculated by fitting the peak intensities as a function of the relaxation delays to a two - parameter exponential decay using nonlinear levenbergmarquardt least - squares fitting . errors were determined using 1000 bootstrap error simulations seeded from the difference between duplicate experiments . steady - state noe values were calculated from the ratios of the peak intensities with and without proton saturation . uncertainty in the steady - state values was determined from the standard deviation in background noise levels using nmrview . the hydrodynamic radius of the molecule was estimated by submitting the crystal structure of the protein to hydronmr.(9 ) viscosity values of h2o : d2o ( 90:10% ) were taken from data provided with hydronmr . all residues showing spectral overlap or steady - state noe values < 0.65 were omitted before the data were subjected to model free analysis . model free analysis of n was performed using the relax software package ( v1.0.3 ) assuming an isotropic diffusion tensor . values of 1.02 and 170 ppm were used for the nh bond length and csa tensor , respectively . twenty rounds of newton minimization were performed for optimization of the model - free parameters . each round consisted of fitting of the relaxation data to each of the model - free models 15 with c held fixed , followed by aic model selection and then refinement of c based on the model free results . changes in conformational entropy were determined from the results of the model free analysis using the relationship described by yang and kay.(12 ) optimal conditions for crystallization of 2.6 m ammonium sulfate , 100 mm bicine buffer ph 8.2 , 18 c were based on initial screening . drops containing 2 l of rrahbp2(d24r ) ( 10 mg / ml , containing 2 mm histamine ) and 2 l of reservoir solution were equilibrated against reservoir solution by vapor diffusion using the hanging drop method . crystals grew over a period of 37 days . using similar conditions , it was possible to obtain crystals of the apo form of rrahbp2(d24r ) , although they grew as multiple crystals and were unsuitable for x - ray analysis . in order to obtain a more single form of crystal , isopropanol ( 510% ) was added to the crystallization conditions . after soaking for 10 s in a cryoprotecting solution consisting of reservoir solution with the addition of 20% ( v / v ) glycerol , crystals were flash - frozen in liquid nitrogen . data from crystals of rrahbp2(d24r ) complexed with histamine were recorded at 100 k with an adsc q315 ccd detector with synchrotron radiation ( beamline i02 , wavelength 0.95 nm , diamond light source , u.k . ) . data from crystals of the apoprotein were recorded at 100 k with an r - axis iv++ image plate detector mounted on a rigaku ru - h3r rotating anode . the structure of wild - type rrahbp2 complexed with histamine ( pdb accession number 1qft ) with all water molecules and bound ligand removed was used as the starting model and rigid - body refined into the unit cell of the rrahbp2(d24r ) complex with histamine . structure refinement was carried out using refmac ( ccp4(15 ) ) , and subsequent model building and water placement was carried out in coot.(16 ) the protein structure was validated using procheck(17 ) and molprobity.(18 ) because of different crystal morphology , the structure of the apoprotein was solved by molecular replacement using the program amore(19 ) using 1qft as trial model . refinement and model building was carried out as before . crystal coordinates and structure factors have been deposited in the rcsb protein data bank with accession numbers 3g7x ( rrahbp2(d24r)-histamine complex ) and 3gaq ( rrahbp2(d24r ) ) . protein samples were dialyzed against 50 mm tris at ph 7.4 overnight , and the dialysate was used to prepare ligand solutions . protein concentration was confirmed by uv absorption ( hbp2 280 = 38 640 m cm calculated using the expasy proteomics server ) . protein concentrations of between 30 and 50 m were used with ligand concentrations of 500 to 700 m . the first injection was a volume of 2 l , which was discarded during fitting to allow for equilibration of ligand / receptor at the needle tip . fitting was performed using the one - site model present in the origin 5.0 software ( microcal inc . , usa ) for the interaction of histamine with rrahbp2(d24r ) or using the two - site model , present in the same software , to fit the data recorded for the interaction of histamine with wild - type rrahbp2 . the simulations involving rrahbp2(d24r ) were carried out using amber 8,(20 ) with the parm99sb force field , which was developed by cornell et al.(21 ) and modified by hornak et al.(22 ) simulations on free histamine were carried out using the gaff forcefield.(23 ) initial coordinates were based on the crystal structure reported herein . the structures were preprocessed with xleap , where the hydrogen atoms were added to the system . the histamine ligand [ ( c3h3n2(ch2)2nh3 ] was optimized using the ab initio rhf/6 - 31 g * basis set in gaussian98,(24 ) and resp charges(25 ) were subsequently generated and fitted . simulations of rrahbp2(d24r ) ( uncomplexed and in complex with histamine ) and free histamine were then subjected to 5000 cycles of energy minimization and afterward immersed in a periodic cubic box of tip3p water . after initial energy minimization ( 2500 cycles ) , md simulations were carried under npt conditions at a pressure of 1 atm and a temperature of 298 k. the particle mesh ewald technique(26 ) and shake constraints were used . the cutoff for nonbonded interactions was 12 , and the time step was 2 fs . translational and rotational center - of - mass motions were removed every 5 ps . during the initial phase of an equilibration period ( 20 ps ) , the protein backbone atoms and heavy atoms of histamine were harmonically restrained ( 25 kcal / mol a ) . the water molecules were removed , and the backbone atoms of mutant hbp were superimposed on the corresponding crystal structure . in order to check the stability and equilibration , rms deviation on heavy atoms and atomic fluctuations of these atoms were calculated along the trajectory . generalized order parameters(27 ) were calculated from the trajectory of individual backbone amide bond vectors as(28 ) where x , y , and z are components of a unit vector along the amide bond and angular brackets denote the time average over the trajectory . convergence of the dynamics of interest was tested using the approach described by best et al.(31 ) a cumulative time function s ( ) is defined using eq 3 , with the time averages taken from t = 0 to t = . this function was evaluated for 100 equally spaced time - points across the trajectory . the trajectory was deemed to have converged if the difference between the maximum and minimum values of this function over the final 50 time - points was less than 0.05 . the statistical error in the derived entropies was estimated by blocking the data into four equal parts and computing the entropies for each part , followed by estimation of the standard error . mean square displacement and correlation function analysis of solvent water molecules were computed by use of the diffusion and time correlation analysis functionality , respectively , in the ptraj module of amber . vibrational entropies for the ligand in the free and bound states were computed using the covariance matrix approach of schlitter.(32 ) the statistical error in the derived entropies was estimated as above by blocking into eight equal parts followed by estimation of the standard error . ligand free solvation energies were calculated using the conductor - like screening model ( cosmo).(33 ) prior to running calculations , the ligands were optimized using ab initio quantum mechanical calculations ( restricted hartreefock ) , with a 6 - 31 g * basis set . the optimized structures were subjected to cosmo calculations at three different temperature settings ( 270 , 300 , and 330 k ) in order to extract the enthalpic and entropic contributions to the free energy of solvation . the approximation used herein is based on the assumption that the heat capacity is constant over a certain range of temperatures near the target temperature , t. in the case of solute molecules solvated in water , this approximation ( called the finite - difference approach ) usually holds near room temperature for temperature ranges ( denoted as t ) as wide as 50 k. using the finite - difference approximation , the entropy can be approximated at the target temperature as(34 ) where s(t ) denotes entropy at target temperature , g(t ) is the free energy of solvation energy , and t is the temperature difference . for the calculations presented here t was 30 k. the crystal structure of the complex of rrahbp2 with histamine revealed two binding sites for the ligand , with one site ( h ) possessing a higher intrinsic affinity than the other ( l).(35 ) thus , in order to simplify the thermodynamic analysis of ligand binding , a d24r mutant of the protein was engineered , whereby the bulk and charge of r24 abolishes binding of ligand to the l site . binding stoichiometry of 1:1 was verified from isothermal titration calorimetry ( itc ) experiments ( see below ) . in order to enable a structure - based interpretation of the thermodynamic measurements that follow , the crystal structures of rrahbp2(d24r ) and the two structures superimpose well , and the backbone and positioning of many side chains remain consistent . data collection and processing statistics are shown in table 1 , and a stereo image detailing the binding site with bound histamine is shown in figure 1 . binding - site residues are colored blue , the ligand is colored red , and ordered water molecules are shown as green spheres . figure prepared using molmol(36 ) the ligand is bound in a conformation essentially identical to that in the wild - type protein.(35 ) there exists an extensive network of hydrogen bonds and ionic interactions involving the nitrogen atoms of the ligand and y36 , d39 , e82 , d110 , and e135 . in addition there is a number of ordered water molecules in the binding pocket , which appear to be stabilized in part by hydrogen bond interactions with n130 , and the imidazole ring of the histamine is embraced by w42 and f108 as observed by paesen et al.(35 ) to facilitate an overall assessment of the global thermodynamics of binding of histamine to rrahbp2 ( d24r ) , isothermal titration calorimetry experiments were performed at 278 , 288 and 298 k. typical isotherms are shown in figure 2 , and the resulting thermodynamic parameters are shown in table 2 . least - squares fitting of the standard enthalpy at all three temperature gives rise to the change in heat capacity for binding at constant pressure cp = 780 127 j / mol / k . typical itc isotherms for the binding of histamine to ( above ) rrahbp2(d24r ) and ( below ) wild - type rrahbp2 at 298 k. values are expressed as the mean of three measurements . it can be seen that histamine binds with an affinity in the nanomolar range and with 1:1 stoichiometry , in a process that is largely enthalpy driven . by contrast , histamine binds to the wild - type protein with 2:1 stoichiometry ( figure 2 ) . the binding affinity of histamine in the h site of the wild - type protein is similar to that for binding to the d24r mutant , but the binding enthalpies and entropies are somewhat different , which is a manifestation of the enthalpyentropy compensation phenomenon that is universally observed in biomolecular interactions in solution . errors in the observed entropies ( reported in table 2 ) are substantially larger than would typically be anticipated for itc measurements , which is a reflection of tight affinities that approach the limit of the method . in principle more accurate data could be obtained by use of displacement itc methods , but this would require a reference ligand with a suitable binding affinity . however , the affinity of histamine for the h site of wild - type rrahbp-2 derived from itc ( 7.0 3.6 nm , table 2 ) is in good agreement with that derived from h - histamine radioactive binding assays ( 2 nm).(35 ) the difference in affinity amounts to ca . 3 kj / mol , and given that hb will be faithfully reported by itc even for very tight binders , this lends confidence that the tsb values reported in table 2 are not erroneous . a further issue concerning the above data concerns the possibility of proton release or binding during the interaction , whereby the observed enthalpy change hb may contain contributions from the heat of ionization of the buffer ( hion ) . such effects must be accounted for to obtain the correct value for hb . to assess this effect , titrations between histamine and rra - hbp2(d24r ) tris has a hion of 47.45 kj / mol at ph 7.4 , whereas the relevant hion for phosphate is 3.6 kj / mol.(37 ) any contribution from proton exchange should be easily detectable on the basis of the differences in hb for the interaction of histamine with hbp2(d24r ) due to the large differences in ionization enthalpy for these buffers . in pbs at 298 k the hb for the hbp2(d24r)-histamine interaction is 58.3 1.2 kj / mol , whereas in tris it is 61.1 0.2 kj / mol , suggesting that there are no significant protonation effects for histamine binding to hbp2(d24r ) . in order to gain deeper insight into the entropic contribution to binding of histamine to rrahbp2(d24r ) , we utilized n nmr relaxation measurements to probe per - residue conformational entropies for backbone amides for the free protein and for the complex . backbone n longitudinal and transverse relaxation rates ( r1 = 1/t1 and r2 = 1/t2 , respectively ) were determined for the free protein and the complex with histamine using uniformly n , c ( > 97% ) enriched rrahbp2(d24r ) . amide n and h resonance assignments in the free protein ( apo ) and the complex were determined by use of conventional three - dimensional triple - resonance experiments . in total , n r1 , r2 , and noe data were obtained for 116 amide positions , subject to the requirement for nonoverlapping resonances in both the complex and the apoprotein . assignments and relaxation data are reported in supporting information . in common with earlier observations and in particular our own measurements on the binding of small pyrazine - derived ligands to a related lipocalin,(1 ) both positive and negative changes in local backbone entropy ( tsamide ) are observed . these are not restricted to the binding pocket but are dispersed over the protein ( figure 3 ) . , i.e. , an overall change in backbone entropy that is not statistically different from zero . stereo images of the rrahbp2(d24r)histamine complex , showing positive ( green ) and negative ( blue ) contributions or no contribution ( within the standard error , red ) to the overall binding entropy from amide bond vectors on binding histamine ( blue and yellow spheres ) . robust methods have been developed for the estimation of protein methyl - containing side - chain entropies by measurement and cross - validation of h nmr relaxation parameters for protein side chains . however , the dearth of methyl - containing residues in rrahbp2(d24r ) , especially in the binding pocket ( one valine ) , renders this approach untenable . in principle , nmr methods could be developed to probe c relaxation for a more complete set of binding pocket side chains , but as described by muhandiram et al,(40 ) the interpretation of such data is fraught with difficulty , particularly in the study of complexes where the relaxation of c may be influenced substantially by thus , in order to examine the contribution of changes in protein side - chain dynamics to overall binding thermodynamics , we resorted to all - atom molecular dynamics simulations of uncomplexed rrahbp2(d24r ) and the analogous complex with histamine with explicit inclusion of solvent water . side chain entropies for the terminal heavy atom bond of each residue ( tsside chain ) were computed from the simulation ( see materials and methods ) , together with tsside chain for only binding - site residues ( d110 , f108 , y100 , e82 , w42 , d39 , e135 , v41 , y36 , n130 ) . moreover , as a test of the robustness of these simulations , tsamide values were also computed from the simulation , resulting in the data presented in table 3 . the latter compare favorably with the value ( 12.4 9.8 kj / mol ) derived above from nmr measurements and taken together suggest an overall increase in entropy on ligand binding . while this result is at first sight counterintuitive f108 , y100 , e82 , w42 , d39 , e135 , v41 , y36 , n130 ) . since the principal thermodynamic parameters are state functions , the binding of a ligand - protein association can conveniently be interpreted using a thermodynamic cycle ( bornhaber cycle ) approach , leading to the following expression for the overall entropy of binding : where sb is the overall entropy of binding ( as determined from itc experiments for example ) , si is the intrinsic entropic contribution in the absence of solvent , and ssolvpl , ssolvp , and ssolvl are the standard entropies of solvation of the complex , free protein , and free ligand , respectively . turning first to si , this comprises changes in protein degrees of freedom on binding that are estimated from the nmr relaxation measurements and md simulations above , together with changes in ligand degrees of freedom . the latter involve not only changes in internal degrees of freedom but also the loss of translational and rotational entropy . assuming that internal degrees of freedom of the ligand are essentially frozen on binding , the corresponding unfavorable contribution from the three relevant internal degrees of freedom of histamine amounts to ca . 12 kj / mol.(48 ) moreover , the entropic contribution from loss of translational and rotational degrees of freedom of a ligand depends on the logarithm of the molecular mass , and on the basis of earlier work this represents an unfavorable contribution that can be estimated as ca . 25 kj / mol.(49 ) in addition to these contributions , there is also a contribution from the loss in vibrational degrees of freedom of the ligand on binding.(50 ) this was estimated from the covariance matrix approach of schlitter ( see materials and methods),(32 ) leading to an unfavorable contribution of ca . 22 2.4 kj / mol . in earlier work on the binding of small hydrophobic ligands to the major urinary protein ( mup ) , we were able to measure experimentally the solvation entropy of the ligand ssolvl by use of water / vapor partitioning experiments.(4 ) in the present case , the nonvolatility of the histamine ligand renders this approach untenable , and hence we resorted to conductor - like screening model ( cosmo ) calculations(33 ) to estimate the solvation thermodynamics of histamine ( see materials and methods ) . clearly it is very difficult to assess the accuracy of such calculations , but the experimental solvation thermodynamics of two related fragments of histamine have been reported , and these are given in table 4 together with the analogous cosmo - derived parameters . it can be seen that the solvation free energies are reproduced very well , and the solvation entropies and enthalpies reasonably well ( for n - propylamine ) compared with experiment , which lends some confidence in the computed values for histamine . taking together with the above entropic data for the binding of histamine to rrahbp-2(d24r ) , it is possible to estimate the entropic contributions to binding from ligand , protein , and solvent , as shown in table 5 . here , the desolvation contribution from the protein / complex tssolvpl ssolvp is effectively impossible to measure experimentally or to compute , but this can be determined arithmetically since it is the only unknown . for comparison , also shown in table 5 are the entropic contributions derived previously(4 ) for the binding of the small hydrophobic ligand 2-methoxy-3-isopropylpyrazine ( ipmp ) to mup . a comparison of the entropic contributions to binding in mup versus rrahbp2(d24r ) offers interesting insight into the binding process . remarkably , the overall entropy of binding tsb is the same within error for both proteins , yet the contributions from protein , ligand , and solvent are very different . in mup , a favorable contribution to binding entropy derives from ligand desolvation , which can not however overcome the unfavorable contribution from freezing ligand degrees of freedom on binding . the favorable entropic contribution from desolvation of the protein binding pocket that one would predict in a hydrophobic interaction is absent in mup , since the occluded binding pocket is substantially desolvated prior to binding.(2 ) this phenomenon has subsequently been observed in other proteins . in the case of rrahbp2(d24r ) , the dominant favorable entropic contribution to binding again derives from ligand desolvation , with a significant contribution from protein degrees of freedom . however , the overall entropic contribution to binding is still unfavorable , leading to the suggestion that the entropic contribution from desolvation of the protein binding pocket is strongly unfavorable . further insight into the possible source of this contribution can be had by examining the solvation of the binding pocket of rrahbp2(d24r ) and its reorganization on binding histamine during the course of the above md simulations . an average of five to six water molecules is observed in the binding pocket of the protein over the time - course of the simulation of rrahbp2(d24r ) in the absence of ligand . analysis of the diffusion and rotational correlation functions of these solvent molecules suggests that their dynamics are very similar to those of bulk water ( figure 4 ) . ( left ) typical mean square displacements of solvent water molecules in the binding pocket of uncomplexed rrahbp2(d24r ) ( red , green , and blue traces ) in comparison with bulk water ( black trace ) . ( right ) typical mean square displacements of solvent water molecules in the binding pocket of rrahbp2(d24r ) in complex with histamine ( red , green , blue , and gray traces ) . in both panels thus , the return of these water molecules to bulk solution on ligand binding would be expected to offer no significant contribution to the binding entropy . in contrast , four solvent water molecules are sequestered in the binding pocket in the complex , whose diffusion ( measured by mean square displacement ) is indicative of a degree of order substantially higher than bulk ; unlike solvent water molecules in the binding pocket of the uncomplexed protein , these water molecules have residence times extending to 20 ns or more during the simulation ( figure 4 ) . given that the translation entropy represents the dominant contribution to the overall entropy of water,(54 ) these data indicate that the presence of ligand results in the sequestration of water molecules into the binding pocket with significantly lower entropy than bulk water . this is also borne out by the rotational correlation times of the water molecules in the complex , which do not decay to zero on a time - scale of 50 ps or more , with one water molecule ( gray trace in figure 4 ) that appears to be very ordered indeed . these results are consistent with stabilization of waters in the bound state by a hydrogen - bonding network subtended by the combined presence of the polar side chains of glu 135 , asp 110 , and tyr 100 together with the charged amide group of the ligand . it is difficult to estimate the entropic contribution from these water molecules , but it is possible to set limits . recent theoretical considerations suggest that the total entropy of liquid water at 298 k is ca . waters.(54 ) on the other hand , on the basis of the experimental entropies of salt hydrates , dunitz estimated that the entropy cost of transferring a water molecule from bulk solvent to protein is in the region from zero to ca . 10 kj / mol , with the higher values corresponding to water molecules that are most firmly bound to metal centers or polar groups . thus , the unfavorable entropic contribution from sequestration of four ordered water molecules in the rrahbp2(d24r)-histamine complex can be estimated as ca . 30 to 40 kj / mol , which is not inconsistent with the data in table 5 considering sources of error . these observations are also qualitatively consistent with the observed sign of cp on binding ( see above ) . using the qualitative relationship derived by spolar et al.,(55 ) + 400 j / mol / k , i.e. , a contribution of 400 j / mol / k to cp on binding . in addition , the sequestration of water molecules into the binding pocket amounts to a net solvation of polar groups , which is known to make a negative contribution to cp.(56 ) our studies suggest that this contribution is on the order of 300 j / mol / k , but this value must be interpreted with caution since there are potentially a number of other contributions to cp that the present studies are unable to capture.(57 ) in reaching the conclusion that the sequestration of solvent water molecules in the complex contributes to the unfavorable entropic binding signature , we were struck with parallels with carbohydrateprotein interactions . these systems typically exhibit extreme enthalpyentropy compensation , with very large unfavorable entropies of binding.(58 ) over a decade ago , in seeking to rationalize this phenomenon , and on the basis of the extremely limited data that were available at the time , lemieux proposed(59 ) at nonpolar surfaces ... an organized layer of molecules is formed that , on being released to bulk , provides an increase in entropy . in contrast , the liberation of water molecules from polyamphiphilic surfaces causes important decreases in both h and ts .... given that this hypothesis has yet to find experimental support to the best of our knowledge , the present data suggest that an alternative explanation might entail the sequestration of solvent water molecules on complexation .	in the present study we characterize the thermodynamics of binding of histamine to recombinant histamine - binding protein ( rrahbp2 ) , a member of the lipocalin family isolated from the brown - ear tick rhipicephalus appendiculatus . the binding pocket of this protein contains a number of charged residues , consistent with histamine binding , and is thus a typical example of a hydrophilic binder . in contrast , a second member of the lipocalin family , the recombinant major urinary protein ( rmup ) , binds small hydrophobic ligands , with a similar overall entropy of binding in comparison with rrahbp2 . having extensively studied ligand binding thermodynamics for rmup previously , the data we obtained in the present study for hbp enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand , protein , and solvent . in the case of rrahbp2 , we find favorable entropic contributions to binding from desolvation of the ligand ; however , the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom , together with the sequestration of solvent water molecules into the binding pocket in the complex . this contrasts with binding in rmup where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding .
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Proceed to summarize the following text: potentially life - threatening systemic diseases like hematologic disorders can initially present with isolated ocular symptoms . therefore , accurate ophthalmologic assessment and prompt ancillary testing facilitates timely diagnosis and subsequent therapy in undefined cases . thus , we report of a patient presenting with isolated decreased monocular vision . ophthalmic and clinical workup led to the diagnosis of acute lymphoblastic leukemia ( all ) . only a sparse number of similar cases have been reported in the literature [ 1 , 2 , 3 , 4 , 5 ] . we would like to elucidate the striking correlation between the amount of subretinal fluid , total leukocyte count and visual acuity . the 28-year - old female patient presented with decreased central vision in her right eye since getting up that morning . the initial corrected visual acuities were 20/40 ( od ) and 20/20 ( os ) , but it dropped to 20/200 ( od ) on day 5 of clinical follow - up . fundus examination showed a vital optic nerve head , but the macula of the right eye presented with a prominent center - involving serous neurosensory retinal detachment and whitish - yellowish choroidal infiltrations . it was less prominent in the central macula of the left eye , but extensive serous detachment was present in the area of the papillomacular bundle ( fig . 1 ) at the inferotemporal vascular arcade . optical coherence tomography ( oct ) confirmed prominent subretinal fluid overlying an area of choroidal thickening . fluorescein angiography detected multifocal areas of leakage at the level of the retinal pigment epithelium ( fig . 1 ) . due to her initial findings , the primary care ophthalmologist considered central serous retinopathy or chorioretinitis of unknown origin as potential differential diagnoses . follow - up on day 3 revealed further deterioration of symptoms despite proper treatment with oral prednisone and ketorolac eyedrops . upon referral , the leukocyte count ( normal range in parentheses ) was found to be substantially increased to a total of 38.2 g / l ( 4.010.0 ) [ erythrocytes 4.65 g / l ( 3.84.2 ) , thrombocytes 30 g / l ( 150380 ) , blasts 87% ( 0.0 ) and increased ldh 645 u / l ( 100250 ) ] . considering this life - threatening situation , the patient was admitted to the department of hematology . upon further enquiry and more detailed medical history , on consecutive ophthalmic examinations , her vision dropped in both eyes as shown in figure 2 . the clinical findings and oct images demonstrated a striking relationship between visual acuity , amount of subretinal fluid and leukocyte count . to depict this significant correlation , the thickness of submacular fluid was measured and compared to vision and leukocyte count ( fig . leukemia is a potential life - threatening malignant disease characterized by uncontrolled proliferation and accumulation of mature and premature malignant leukocytes in the bone marrow with a subsequent peripheral distribution . different forms of leukemia can be classified by their cell of origin and by their time of duration . detailed subclasses are described by their genetic and immunochemical characteristics [ 7 , 8 ] . the overall incidence rate of all is 1.28/100,000 per year showing a bimodal distribution with higher rates in younger ( 014 years ) and older patients ( over 54 years ) . direct leukemic manifestations as infiltrates have been reported of the optic nerve , choroid , retina , iris , ciliary body and anterior chamber . indirect leukemic manifestations such as central serous chorioretinopathy overlaying choroidal infiltrations , retinal vascular sheathing and subconjunctival , anterior chamber , intraretinal , or intravitreal hemorrhages have been found . histopathology of postmortem specimens performed by kincaid and green at the wilmer ophthalmological institute between 1923 und 1980 found ocular involvement in 82% of patients with all . reddy et al . reported ocular findings in 35.4% of 288 newly diagnosed cases of leukemia in children and adults , but obvious clinical symptoms were only present in 10% . ocular involvement in hematologic disease entities may benefit from immediate systemic chemotherapy of all as they are usually not treated directly . the leukemic infiltrates were found in almost any location in the eye ; however , they are most frequent in the choroid with primary or compressive involvement of the adjacent choriocapillaris . the consecutive dysfunction of the retinal pigment epithelium and the outer blood - retina barrier may ultimately lead to serous retinal detachment as observed in our patient [ 12 , 13 ] . these findings correlate well with the multifocal areas of leakage at the level of the retinal pigment epithelium shown by fluorescein angiography in our case . they suggested that an increased osmotic gradient caused by subretinal accumulation of high - molecular - weight immunoglobulins induces exudation . due to immediate induction of chemotherapy by the department of oncology and hematology , the leukocyte count of the patient declined adequately . upon her last visit to the department of ophthalmology her visual acuity improved to 20/40 ( od ) and 20/20 ( os ) , and the reduction of subretinal fluid showed an evidently positive correlation with regressing leukocyte counts ( fig . , her ocular biomicroscopic examination , oct and fluorescein angiography had resolved back to normal ( fig . this case report was conducted in accordance with good clinical practices . the authors state to have full control of all primary data and have no ethical conflicts to disclose .	this case illustrates that hematologic disorders must be considered as a potentially life - threatening cause for vision loss . proper laboratory workup and timely interdisciplinary approach are essential to ensure the best possible care for ophthalmic patients . historically , before the use of bone marrow biopsy , the ophthalmologist was often asked to assist in the diagnosis of leukemia . since ophthalmological symptoms may be the initial presenting signs of leukemia as highlighted in this case , the ophthalmogist is still of crucial importance .
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Proceed to summarize the following text: gastric carcinoids are of increasing clinical importance . whereas , in older studies these composed only 2 - 3.8% of all carcinoids , more recently investigators have suggested that the incidence may be significantly higher-11 - 30% of all carcinoids . in addition , to the increased frequency of gastric carcinoids , they are receiving more attention because of recognition that they occur not only sporadically ( type - iii ) , but also with increased frequency in chronic hypergastrenemic states ( atrophic gastritis type - i ) and zollinger - ellison syndrome , type - ii . recognition of gastric carcinoids is important because each type can , on occasion , become malignant and metastasize to lymph nodes or the liver ( type - i , 5% ; type - ii , 30% ; and type iii , 71% ) . in particular the type - iii carcinoid tumors are sporadic , large solitary tumors not associated with a hypergastrenemic state , are highly proliferating due to intense over expression of a mutated p53 gene , have a high propensity to ulcerate and are more likely to be invasive with metastasis . they appear with striking predominance in men ; 80% patients diagnosed with type - iii carcinoids are men . accurate pre - therapy staging with other non - invasive imaging modalities is therefore mandatory to select the appropriate mode of therapy . in this context , we report a case of a patient having gastric carcinoid with liver metastases ( type - iii ) with classic textbook description except that the tumor was non - functional and the patient did not have any of the clinical syndromes . accurate pre - therapy localization was carried out by positron emission tomography using two different radiotracers . the diagnosis was confirmed by fine needle aspiration cytology ( fnac ) from a liver space occupying lesion ( sol ) and a gastric biopsy . a 32-year - old man presented with features of abdominal pain and enlargement of 6 months duration . clinical examination revealed a massively enlarged liver reaching up to the umbilicus with no other positive clinical findings . biochemical examination , liver function tests , and viral markers were within normal limits except for an elevation of serum alkaline phosphatase . a preliminary high - resolution dual phase computed tomography ( ct ) revealed a grossly enlarged liver with multiple enhancing hypodense lesions suggestive of hypervascular secondaries and thickening of the greater curvature of the stomach [ figure 1 ] . an upper gastrointestinal ( ugi ) endoscopy done during the same time revealed a 3 cm 2 cm ulcer with rolled up edges along the greater curvature of the stomach . fnac from one of the liver sol revealed features of neuroendocrine tumor ( net ) with positive immunohistochemistry and a ki-67 index of 40 - 50% , following which the patient underwent positron emission tomography / ct ( pet / ct ) using two different radiotracers with differing imaging perspectives : f - fluorodeoxyglucose ( f - fdg ) ( a metabolic tracer ) and gallium - dota - noc ( somatostatin receptor expressing tumor seeking tracer ) . non - contrast computed tomography ( a ) and dual phase contrast enhanced computed tomography ( b and c ) images of abdomen showing multiple hypodense lesions in an enlarged liver with significant marginal contrast enhancement and rapid washout on the venous phase suggestive of hypervascular metastasis . thickening along the greater curvature of the stomach is also evident ( arrow ) f - fdg pet / ct revealed multiple hypodense lesions in liver with focally increased radiotracer uptake , suggestive of poorly differentiated secondaries with high metabolic activity , which was earlier proven on fnac from a liver sol [ figure 2a ] while foci of increased tracer uptake in a soft - tissue mass at the greater curvature of the stomach was seen on a ga - dota - noc pet / ct scan suggesting a well - differentiated primary somatostatin receptor expressing net in addition to regional lymph node involvement while the liver lesions showed no tracer uptake [ figure 2b ] . a gastric biopsy later confirmed this finding ( well - differentiated net ; ki-67 index-2% ) [ figure 3 ] . based on a combination of these findings the patient was deferred from surgery and instead underwent chemotherapy protocol with etoposide and cisplatin , following which he went into a near total clinical and radiological remission [ figure 4 ] . the patient however had recurrence later on and despite aggressive treatment even including a bone marrow transplant , he succumbed to his disease . ( a ) f - fluorodeoxyglucose positron emission tomography / computed tomography ( pet / ct ) images showing multiple focal areas of increased radiotracer uptake in both lobes of the liver , implicating metabolically active lesions . no significant tracer uptake was noted in the greater curvature of the stomach ( arrow ) ( b ) ga - dota - noc pet / ct showing increased radiotracer uptake in the mass at the greater curvature ( arrow ) while the liver lesions show only minimal radiotracer uptake gastric biopsy showing superficial mucosal fragments with tumor cells arranged as nests and as acini ( h and e , 200 ) had a small rim of cytoplasm and had a stippled nuclear chromatin ( a ) . they were strongly immunopositive for chromogranin and synaptophysin , two of the common markers used for detection and demonstration of neuroendocrine differentiation ( b and c ) fnac from the liver mass ( d ) showed sheets and fragments of tumor cells with cells having moderately pleomorphic nuclei and coarse chromatin ( papanicolaou , 200 ) . the tumor was more poorly differentiated as compared to the gastric primary six months after , chemotherapy ( a ) f - fluorodeoxyglucose positron emission tomography / computed tomography ( pet / ct ) showing normal physiological distribution the radiotracer . the multiple liver lesions , which previously had intense tracer uptake , have disappeared ( b ) ga - dota - noc pet / ct showing that the greater curvature of the stomach has normalized and no lesion is visible on the post - therapy image among the gastrointestinal carcinoids , small intestine is the most frequent site ( 28% ) , followed by rectum ( 19% ) and colon ( 8% ) . the average age at diagnosis of patients with gastric carcinoids is 62 years with an equal male to female distribution . patients are often asymptomatic ; carcinoids being found incidentally at the time of ugi endoscopy . when symptomatology occurs they are usually dyspeptic . it has been reported that almost all patients with gastric carcinoids have elevations of plasma chromogranin - a , a peptide secreted by all neuroendocrine cells , although prospective studies for its use as a tumor marker for carcinoids have not yet been proposed . the management of gastric carcinoids is controversial ; sporadic carcinoids have a higher rate of regional lymph node involvement and thus surgical resection with lymph node sampling is generally recommended . the ct appearance of these lesions is similar to other hepatic metastasis : multiple , low - density lesions that are rarely calcified and with variable density after intravenous contrast enhancement . gastric carcinoids , similar to other carcinoids have a high density of somatostatin receptors ( sstr ) . with recent studies showing that somatostatin receptor scintigraphy ( srs ) are highly sensitive and specific in localizing carcinoids in other locations recently ga coupled to octreotide via a bifunctional chelate dota ( 1 , 4 , 7 , 10 tetraazacyclododecane-1 , 4,7,10 tetra acetic acid ) have been used as radiotracers for srs pet imaging because they exhibit better tumor - to - background ratio . however , because nets are mostly well - differentiated tumors , they have a low metabolic activity and can not be visualized efficiently with f - fdg , which is more useful in , less differentiated nets without expressing ( sstr ) . our results also demonstrate similar findings as described with gallium labeled octreotide ( dota - noc ) showing the presence of a well - differentiated net primary in the gastric wall while the less differentiated secondaries in the liver were accurately shown on f - fdg imaging . it therefore represents a unique example of combining two functional imaging modalities , which are complementary to each other for accurately functional mapping of tumor lesions and predicting response to therapy . furthermore , the positivity of liver lesions on f - fdg imaging meant that this patient had a poor prognosis despite the primary showing increased uptake on ga - dota - noc imaging . in conclusion , our observations suggest that these functional imaging findings are characteristic of neuroendocrine cellular differentiation and are very useful in the pre - therapy localization / staging of tumors , predicting , and monitoring response to therapy and follow - up of the patient for future recurrences / relapse and also for prognostication .	because of the increasing clinical importance of gastric carcinoids and the difficulty in diagnosing them , the need for non - invasive diagnostic methods is growing . currently , the only reliable method is upper gastrointestinal endoscopy with biopsy . we report the case of a 32-year - old male where a combination of functional imaging studies ( 18f - fluorodeoxyglucose - positron emission tomography / computed tomography [ pet / ct ] and 68ga - dota - noc pet / ct ) not only helped in the correct staging , but also highlighted certain important biological aspects of these tumors , which are important from the management point of view and can prognosticate the patients .
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Proceed to summarize the following text: limited rest time , high intensity , and competition rate of strenuous sports emphasize on the necessity of following the most suitable approach to manage the functional overload of professional athletes . a proper nutritional strategy is helpful in order to achieve proper recovery , with multiple competing periods and several times per day . the content and time of nutrient consumption impact the resynthesis of fuel supply , reduction of muscle injury , and optimizing the competition performance . liquids are more tolerable in suppressed appetite of instant post - exercise period and they help in cell rehydration and substituting lost electrolytes due to sweating . beverage micro and macronutrient content and their utilization , during or post - exercise period , are effective in fuel restoration . studies reflect that the consumption of carbohydrate - protein beverages during post - exercise recovery periods can facilitate glycogen restore and muscle turnover speed . chocolate milk composition is similar to common sports drinks , and it can enhance blood sugar level , speed of muscle glycogen repletion , and protein turnover . its branched chain amino acids , carbohydrates , electrolytes , and easily absorbed casein and whey protein help athletes muscle stores . dough or persian salty yogurt drink with a consistency similar to milk which contains high amount of whey protein and critical electrolytes such as sodium and calcium can affect athletes performance . moreover , non - alcoholic beer as a source of carbohydrate , minerals , and vitamins is a popular and available supplement fluid . choosing the most proper recovery beverage can be useful for nursing and medical members of sports medicine to guide athletes . in the present study , we compared the effects of dough , non - alcoholic beer , and carbohydrate replacement drink on lactate dehydrogenase enzyme level , f2-isoprostane , lipid , and glucose blood level . the professionals were asked to stop their exercises for 24 hours before initiation of the intervention program . they were also asked to note their food intake in one - day food recall questionnaires . five milliliter venous blood was collected , and after 10-min warming up exercises , athletes followed standard protocol of running - based anaerobic sprint test ( rast ) . blood lactate was tested after running the protocol and 1-h post - rast by a calibrated lactometer ( scout company , germany ) . athletes at a 4 day interval , received 500 cc isocaleric beverages as dough , non - alcoholic beer , and chocolate milk . after taking the first beverage then , the participants venous blood was taken ; the other two beverages were consumed following the same guidelines . in other words , 21 participants who enrolled in one experimental study group consumed all three beverages . indirect vo2 max was determined by harvard step test and their 24-h recalls were assessed using a nutritionist iv software ( version 7.0 ; n - squared computing , salam , or , usa ) . serum triglyceride , total cholesterol level , and blood sugar assessed with enzymatic kits ( pars azmoon ins , tehran , iran ) . data were compared between different times using a simple repeated - measures analysis , and post - hoc comparisons were also performed too . all the analyses were done using the statistical package for the social sciences software ( spss inc , chicago , il , usa ) ( version 20 ) . paired t - test and analysis of variance ( anova ) test were also performed . ethics committee of iums approved the study process , and informed consent was gained from all of the subjects . ethics committee of iums approved the study process , and informed consent was gained from all of the subjects . the taekwondo players mean age was 23 2.7 years . levels of lipid profile and blood sugar before and after drinking beverages during pre and post - recovery periods are shown in table 1 . total cholesterol levels decreased after the three intervention periods , however , this reduction was not significant . comparison of total cholesterol change after intervention did not reflect a significant difference ( p > 0.05 ) . plasma concentrations of profile lipids and blood sugar before and after ingestion of beverage plasma triglyceride was lower after dough and carbohydrate replacement drink intake . the mentioned decrease was marginally significant in taekwondo players after dough consumption ( p = 0.076 ) , whereas there was a non - significant difference after carbohydrate drinking periods . in addition , non - alcoholic beer intake non - significantly increased triglyceride level . between groups was marginally significant after consuming non - alcoholic beer ( p = 0.083 ) , however , the mean change of plasma glucose did not show a significant increase for the three beverages . moreover , lactate dehydrogenase level reduced after all the intervention cycles . mean change of this plasma enzyme level was statistically significant after non - alcoholic beer consumption ( p = 0.048 ) . in addition , no significant increase was observed between mean pre and post - recovery f2-isoprostane values and between groups comparison did not show any statistically significant difference ( p > 0.05 ) . participants did not complaint about any side effects . mean and standard error of oxidative stress and muscle damage biomarkers before and after drinking beverages are shown in table 2 . in this study , we compared the effects of various types of beverages including dough , non - alcoholic beer , and carbohydrate - rich beverages on blood sugar , lipid profile , lactate dehydrogenase , and f2-isoprostane levels of elite taekwondo players . findings show that all three beverages at pre- and post - recovery periods enhanced blood sugar and f2-isoprostane levels , whereas these fluids supplements intake decreased plasma total cholesterol and lactate dehydrogenase levels . non - alcoholic beer increases the triglyceride level , and the other liquids lowered plasma triglyceride level . in a study by bishop et al . , plasma glucose levels reduced in the placebo group in comparison to the group that received carbohydrate - rich beverages , both at fatigue and at 1 hour after exercise . it seems that stress hormone release was accompanied with post - exercise blood glucose reduction . moreover , it can balance the fatigue perception , lowering quality of athletes performance . nutrient profile of milk and its products as carbohydrate , whey , electrolytes , and water can be effective on glucose level in athletes recovery periods . the observed effect of fluid drink on triglyceride and total cholesterol levels are comparable with previous findings . in a 6-week intervention trial with fermented milk product , ageraek et al . observed a significant reduction in cholesterol level of 58 healthy participants , whereas plasma triglyceride showed no significant change . cholesterol and triglyceride levels reduction shows an approximately similar trend , however , its strength is affected by sample size of the studied participants . the effect of yogurt , as a milk product , on lowering serum total cholesterol can be explained by its lactobacillus acidophilus content . bioactive compounds , calcium , conjugated linoleic acid fermentation bacteria , and probiotic components can play critical roles in reducing plasma cholesterol and triglyceride levels . exercise leads to a higher lactate dehydrogenase concentration as a converting enzyme with fuel supplying roles and also its level reflected the increased free radical concentration which is caused by stress of sport . in addition , its level affects lactate concentration of athletes muscle and their performance ability . karp et al . on comparing the effects of chocolate milk , fluid replacement drink , and carbohydrate replacement drink consumption in highly - trained cyclists observed increased post - exercise lactate level ; exhaustion time and glycogen - depleting exercise of participants can be managed by milk chocolate beverages . however , the mentioned effects were non - significant in a comparison of within - subject difference in the thomas trial on male trained cyclists . the non - significant within and between comparison of our supplement beverages on f2-isoprostane as a muscle injury and free radical arachidonic acids peroxidation indices can be explained using steensberg findings . they observed that plasma f2-isoprostane level decreases significantly in response to sport stress , however , this reduction is compensated in 1 hour after the recovery period . the trevor trial involving 127 men and women in the age group 30 - 65 years showed that low fat diet containing a daily 3-rich fish meal can reduce cell lipid peroxidation rate and lower urinary f2-isoprostane excretion . this reduction was higher in participants following aerobic exercises in addition to the mentioned diet . lack of reported studies on the effects of fluid supplements on f2-isoprostane level makes more comparison impossible . these findings can help nursing and medical team members of sports medicine to guide elites and professional athletes in rapid and most proper recovery periods . the limitations of our study were small sample size of participants and a before - after study design . moreover , measuring detailed nutrient and electrolyte content of beverages can be effective in our assessment . in regards to the strong points , the present study assessed the effects of isocaloric volume of dough intake in comparison to other available fluid supplements on lipid profile , blood sugar , muscle damage , and oxidative stress markers in professional athletes for the first time . in conclusion , we observed that dough , non - alcoholic beer , and carbohydrate replacement drink consumption at pre- and post - recovery periods can decrease plasma total cholesterol and lactate dehydrogenase level . non - alcoholic beer increases triglyceride level , and the consumption of other liquids was accompanied with lower plasma triglyceride in elite taekwondo players .	background : athletes recovery is important in improving their performance . nutritional strategies can be effective in enhancing recovery rate . choosing the best food items in appropriate intervals can play effective roles in resynthesis of fuels and recovery of muscle injury . beverage micro and macronutrient content are helpful in fuel restoration . in this study , we assess the effects of various kinds of beverages on oxidative stress , muscle injury , and metabolic risk factors in taekwondo players.materials and methods : this quasi - experimental study was performed on 21 taekwondo players of isfahan . after collecting fasting blood , they performed runningbased anaerobic sprint test ( rast ) . blood lactate was tested again and participants were divided into 3 intervention groups , that is , receiving 500 cc dough , non - alcoholic beer , and chocolate milk at 4 day intervals . after a 2-h recovery period , blood sampling was repeated . elites consumed other beverages in later phases . dietary intake and fasting triglyceride , cholesterol , blood sugar , lactate dehydrogenase , and f2-isoprostane concentrations were determined . data were analyzed with a simple repeated - measures test and post - hoc tests using the statistical package for the social sciences software.results:data showed that cholesterol levels non - significantly decreased after intervention . triglyceride level was lower after taking dough and carbohydrate replacement drink . blood glucose concentration increased after intervention periods , however , this increase was significant only after non - alcoholic beverage consumption . lactate dehydrogenase levels reduced after all cycles , however , f2-isoprostane level showed no significant change . there was not significant change in lactate dehydrogenase and f2-isoprostane levels.conclusions:non-alcoholic beer consumption can reduce lactate dehydrogenase concentration ; however , it leads to blood sugar increase . moreover , dough consumption significantly reduced triglyceride level in taekwondo players .
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Proceed to summarize the following text: since antibiotics were first discovered in 1928 , they have been widely used in human and animal medicine . however , most antibiotics are excreted and enter the soil or water environments through wastewater and fertilization with manure ( kmmerer , 2003 ) . antibiotic contamination of the natural environment has been documented in many countries , including those of europe , north america , and east asia , and areas of antibiotic contamination are widespread throughout the region ( segura et al . , 2009 ) . the pathways whereby residual antibiotics enter the environment are varied and include wastewater effluent discharge and runoff and leaching from land fertilized with agricultural or human waste . an increasing number of reports show a rise in the occurrence and distribution of antibiotics in surface waters in europe ( hirsch et al . , 1999 ; , 2001 ; kolpin et al . , 2002 ; kim and carlson , 2007 ) , and canada ( metcalfe et al . , 2004 ) . cases of antibiotic contamination of wastewater are also well - known ( gbel et al . , 2005 ; and others ) . some antibiotics can be found in ground water ( baquero et al . , 2008 ) . in china , antibiotics have been found in animal wastewater , pond water , animal farm effluents , and rivers . the most frequently detected antibiotics in china include sulfamethazine ( 75% ) , oxytetracycline ( 64% ) , tetracycline ( 60% ) , sulfadiazine ( 55% ) , and sulfamethoxazole ( 51% ) , which were found at maximum concentrations of 211 , 72.9 , 10.3 , 17.0 , and 63.6 ppb , respectively ( wei et al . , 2011 ) . in korea , environmental contamination with tetracyclines and sulfonamides was reported at the ppb level ( ok et al . , 2011 ) . recent excellent reviews by kmmerer ( 2009a , b ) summarize contamination of the environment with antibiotics and the spread of antibiotic resistance . this manuscript will focus on indochina environment having unique situation as following . unlike the socioeconomic - unified continents of europe and north america , asia is more geographically and culturally disjunct , and can not be readily grouped as a single geopolitical entity . it therefore remains difficult to obtain a generalized theory of antibiotic and antibiotic resistance contamination across all asian countries . this manuscript focuses specifically on the region of indochina and the major antibiotics in this area , including quinolones , sulfonamides , and tetracyclines . countries of indochina include vietnam , thailand , cambodia , lao pdr , and myanmar . of these , vietnam and thailand have particularly become a center of rapid industrial development and economic growth ; however this growth has far outpaced investment in public infrastructure , leading chemical pollution and sanitary issues . although indochina is globally one of the most active regions of industrial growth and development , there remains few studies on antibiotic contamination relative to their western and east asian counterparts ( le and munekage , 2004 ; nhiem et al . , 2008 ; zhang et al . , 2009 ; hoa et al . , 2011 ; takasu et al . , contamination levels are highly variable , ranging from scales of ppm to ppt ( le and munekage , 2004 ; managaki et al . , 2007 ; , 2008 ; hoa et al . , 2011 ; takasu et al . , 2011 ) . table 1 summarizes the various antibiotic contaminants that have been detected in indochina aquatic environments . until now for antibiotic resistance in this area , major reports consisted of studies on salmonella isolates ( ogasawara et al . , 2008 ; sirichote et al . , 2010 ; and others ) . , not quantifiable due to overlapped interfering peak . large amounts of antibiotics are used for human medicine , livestock farming , and aquaculture in southeast asia . in addition , we observed that antibiotics are used as additives in ice to prevent decay of harvested fish in fish markets . it is believed that high concentrations of antibiotics applied in this manner will remain in the fish , and will then be taken in by people upon consumption . such widespread antibiotic contamination of the environment and food supply likely promotes the development of antibiotic - resistant bacteria , both in the environment and within the human intestine . prior to the early 2000s , few studies addressed antibiotic contamination in indochina ( le and munekage , 2004 ) , although the occurrence of antibiotic - resistant bacteria in the region was reported ( kim et al . , 2004 ; le et al . , 2005 ; zhang et al . , 2009 however , recent studies on drug contamination , combined with detection of antibiotic - resistant bacteria and/or resistance genes , have provided a clearer picture of the problem in this area of asia ( hoa et al . , 2011 ; takasu et al . , aquaculture farming system known as vac ( vegetation , aquaculture , and cage ) is common . the vac system is a recycling farm , typically consisting of a vegetable field , an aquaculture pond , and caged animals , and has been practiced since the 1980s . in the vac system , livestock manure ( usually from pigs , chickens , and ducks ) is directly transported to fish ponds and to vegetable and rice fields . this untreated sewage and wastewater from the livestock operations is used for fish culture and for fertilization of the vegetable fields . the animal manure contributes to the eutrophication of pond water , which enhances phytoplankton growth . the vac system is considered a very economical method of recycling farming ( hop , 2003 ) ; however , the heavy use of antibiotics in livestock increases the prevalence of enteric antibiotic - resistant bacteria and the potential for antibiotic - resistant pathogenic bacteria to arise . subsequent discharge of antibiotic residues in farm waste likely increases the number of antibiotic - resistant bacteria in the environment , which in turn promotes the further selection and transfer of antibiotic resistance genes within the microbial community in the surrounding environment ( petersen and dalsgaard , 2003 ; heuer and smalla , 2007 ) . ( 2011 ) reported that enteric bacteria of pigs in vac showed high resistance to nalidixic acid and enrofloxacin , which would be dispersed to water environment . that is , the abundance of antibiotic - resistant bacteria and genes initially increases in the intestinal tracts of livestock as a result of exposure to high concentrations of various antibiotics . next , horizontal gene transfer occurs among various bacteria in the environment surrounding the vac operation . finally , the antibiotic resistance genes move to other environments via water use and the food supply . the antibiotic resistance genes are brought together , exchanged , and spread in vac systems . if the antibiotic - resistant bacteria come into contact with human pathogens and commensal bacteria , the risk to human health may increase significantly . although there are many snapshot reports of antibiotic resistance ( reviewed in zhang et al . , 2009 ) , further dynamic study is needed to evaluate the risks posed by the presence of antibiotic resistance genes in the aquatic environments of developing countries in asia . fluoroquinolones ( fqs ) are fully synthetic antibiotics that are widely used in humans , animals , and fish ( grave et al . , 1999 ; le and munekage , 2004 ; samanidou et al . , 2005 ; the antibacterial mechanism of fqs is based on inhibition of bacterial dna gyrase or topoisomerase iv , which are enzymes essential for dna replication . first - generation fqs are piromidic acid , oxolinic acid , and nalidixic acid , which were widely used in aquaculture in the 1970s in japan . in tropical asian countries , oxolinic acid still appears to be one of the major drugs used ( grslund et al . second - generation ( ciprofloxacin , cip and norfloxacin , nor ) and third - generation ( levofloxacin , lev and its enantiomer ofloxacin , ofl ) compounds are used in hospitals and animal husbandry in indochina ( takasu et al . , the fqs are photo - degradable , with a half - life in pure water of 105 and 90 min for nor and cip , respectively ( burhenne et al . , 1997 ) . however , fqs in the environment are relatively stable in water and sediment ( kmmerer , 2004 ; le and munekage , 2004 ) , which might be due to sorption onto particulates ( nowara et al . , 1997 ) . lai and lin ( 2009 ) reported that oxolinic acid and flumequine could be retained in sediment for 9.515 and 3.66.4 days , respectively . such long half - lives in the environment pose a selective pressure for environmental bacteria . although the bioavailability of antibiotics is suspected to decrease upon adsorption on clay and humic substances , no supporting evidence has been reported . ( 2003 ) reported that 74% of thailand shrimp aquaculture farmers use antibiotics , primarily nor . le and munekage ( 2004 ) reported detecting nor at 0.53.5 ppm in the water column and 2001500 ppm in the sediment of intensive ponds and improved extensive ponds in vietnam . oxolinic acid could be detected in the water column at a concentration similar to that of nor , but not in the sediment . the water column concentration indicates present inflow , while the sediment concentration indicates the value integrated over time ( takasu et al . , 2011 ) . the presence of antibiotics in both samples suggests that the compounds being used at present and are retained in the sediment . ciprofloxacin is commonly used with other drugs , such as griseofulvin , rifampicin , and oxytetracycline for shrimp larvae in vietnam ( thuy et al . , 2011 ) . the most recent quantification of fqs in indochina aquatic environments showed that the average concentrations were higher in thailand ( ofl , 7400 ppb ; nor , 209 ppb ; cip , 328 ppb ; lom , 67.4 ppb ) than in vietnam ( ofl , 255 ppb ; nor , 41.1 ppb ; cip , 162 ppb ; lom , 25.3 ppb ; takasu et al . both ofl and nor were confirmed as major environmental contaminants in both countries ; however , contamination at aquaculture sites was lower than at vac farms and city canals in both countries . a recent decrease in drug application and/or dilution effects may explain the improved contamination situation in aquaculture settings . since fqs are not natural compounds , it is believed that bacteria do not possess fq resistance genes . several resistance mechanisms have been reported , including mutation of dna gyrase ( fukuda et al . , 1990 ) , impermeability to fqs due to loss of porins , and extrusion by overexpressed efflux pumps ( hirai et al . , 1986 , 1987 ) . a number of plasmid - mediated quinolone resistance ( pmqr ) genes ( qnra , qnrb , and qnrs ) have also been identified ( robicsek et al . , 2006 ) . these pmqr genes are transferred horizontally among bacteria and encode a protein of the pentapeptide repeat family that has been shown to block the actions of fqs on purified dna gyrase and topoisomerase iv ( robicsek et al . , 2006 ) . the origin of these genes is thought to be chromosomes in aquatic bacteria ( poirel et al . thus , the aquatic environment is hypothesized to be a natural reservoir of fq - resistant bacteria and resistance genes . as mentioned above , the environmental concentration of fqs was found to be much higher in thailand than in vietnam . despite the lower level of contamination , the occurrence rate of fq - resistant bacteria was found to be higher in vietnam than thailand ( takasu et al . , 2011 ) . ( 2011 ) demonstrated that there is no relationship between the concentration of fqs in the environment and the rate of bacterial resistance . kmmerer ( 2003 ) reported that exposure to a sub - inhibitory concentration induces the emergence of resistant bacteria in aquatic environments . it was reported that fqs at concentrations 10100 times lower than the mic induce the expression of a functional gene ( herold et al . , 2005 ) , mutations ( gillespie et al . , 2005 ) , and morphological changes ( loubeyre et al . , 1993 ) at the single - cell level . low concentrations are thus effective inducers of drug - resistant bacteria . the effect of disinfectants on bacteria is considerably reduced in the presence of organic matter ( kawamura - sato et al . , 2008 ) , suggesting that the actual active concentration in the environment might be lower than analysis suggests . the importance of low concentrations of fqs to the development of antibiotic resistance should not be ignored , and additional research involving both instrumental analyses and bioassays is needed to estimate the actual concentration of fqs acting upon environmental bacteria . a broad range of bacteria can acquire resistance to fqs , including enteric bacteria ( escherichia coli ) , pathogenic bacteria ( e.g. , acinetobacter ) , and aquatic bacteria ( e.g. , brevundimonus ) . proteobacteria and actinobacteria are the major taxa of fq - resistant bacteria ( takasu et al . , 2011 ) , indicating that fq - resistant bacteria are not limited to specific groups . it was hypothesized that bacteria from humans and animals as well as natural aquatic bacteria are potential reservoirs of fq resistance genes . a number of resistance genes have been identified in fq - resistant bacteria , including qnra , qnrb , qnrs , and aac(6)-ib - cr ( takasu et al . , 2011 ) . since qnra , qnrb , and qnrs are thought to have originated from the chromosomes of water - dwelling bacteria ( poirel et al . , 2005a , b ) , it is possible that these genes can be transferred from aquatic bacteria to human bacteria ( hernndez et al . , 2011 ) . to date , pmqr genes including aac ( 6)-ib - cr , which are linked with other drug resistance genes and are transferable , have only been discovered in gammaproteobacteria such as vibrio ( poirel et al . , 2005a ) , shewanella ( poirel et al . , 2005b ) , and aeromonas ( cattoir et al . , 2008 ; picao et al . , the number of fq - resistant salmonella enterica isolates found in thailand increased in the late 1990s , and the resistant isolates possessed the same gyra region , suggesting rapid spread of the resistant bacteria ( hakanen et al . , 2001 ) . the gyra mutation is located on the chromosome , but recent research indicates that gyra can also be transferred ( ferrandiz et al . spreading of both resistant bacteria and chromosomal resistance genes might be a cause of the observed widespread drug resistance in asia . however , precisely how fq resistance genes are spread among various environments is not known , although mutation - based resistance and transferable genes should be considered . the dynamics of fq resistance genes are of interest and importance from the viewpoints of human clinical and gene evolution studies . sulfonamides , which are also synthetic antibiotics , have been widely used to treat bacterial and protozoan infections in humans , domestic animals , and aquaculture species since their introduction to clinical practice in 1935 ( perreten and boerlin , 2003 ; le and munekage , 2004 ; blahna et al . , 2006 ) . sulfonamides inhibit folate biosynthesis by competing with the natural substrate p - amino - benzoic acid for binding to dihydropteroate synthase ( dhps ) , an enzyme in the folic acid synthesis pathway . while the use of sulfonamides in humans has decreased in developed countries , they are still frequently used in developing asian countries due to their low cost ( macrolides are 13 $ /tablet , whereas sulfonamides are 2 cent / tablet in vietnam ) . sulfamethoxazole ( smx ) , a commonly used sulfonamide analog , was detected at ppm levels in shrimp ponds in vietnam in the late 1990s ( le and munekage , 2004 ) . although such levels are extremely high , smx levels in rivers in japan and vietnam are typically around 100 ppt in recent years ( managaki et al . , 2007 ) . this was confirmed in various locations in vietnam ( hoa et al . , 2011 ) , where concentrations were in the 6004000 ppt range throughout the year in a city canal . trimethoprim was detected in the 201800 ppt range as well , probably because it is often used with smx . in livestock farm wastewater , the concentration of smx was lower , in the 68600 ppt range , whereas that of sulfamethazine was much higher ( 4006000 ppt ) . other sulfonamides were either not detected or found at trace levels , suggesting that smx and sulfamethazine are the major sulfonamides in use in vietnam at present . since sulfonamides inhibit the formation of dihydrofolic acid ( perreten and boerlin , 2003 ) , bacterial resistance to sulfonamides can occur through mutations in the chromosomal dhps gene ( folp ) or through acquisition of an alternative dhps gene ( sul ) whose product has a low affinity for sulfonamides ( perreten and boerlin , 2003 ) . acquisition of sulfonamide resistance through sul genes is the most prevalent mechanism ( enne et al . sulfonamide resistance is globally prevalent among human and animal pathogens ( kerrn et al . , 2002 ; perreten and boerlin , 2003 ; antunes et al . , 2005 ) ; however , the presence of sul genes is not equally distributed among bacterial populations ( kerrn et al . , 2002 ; antunes et al . , 2005 ; hammerum et al . , the sul2 gene , which is related to class i integron , is common in acinetobacter isolated from fish farms and chicken manure in thailand ( agers and petersen , 2007 ) . however , hoa et al . ( 2008 ) reported that sul1 was the major sul gene among smx - resistant bacteria isolated from vac farms , city canals , and aquaculture sites in vietnam . most of these reports were based on isolated bacteria , which may bias interpretation of results due to the processes of isolation ; therefore , we can not obtain an accurate assessment of the distribution of antibiotic resistance genes from culturable bacteria alone . a direct quantification approach indicated that sul1 is the major sulfonamide resistance gene in lagoon and river waters in the usa ( pruden et al . , 2006 ) . it is likely that such non - culture monitoring of antibiotic resistance genes will increase in the near future ( for example , pei et al . , 2006 ) . a culture - dependent study of smx - resistant bacteria revealed that acinetobacter is one of the major reservoirs of sul genes in asian aquatic environments ( agers and petersen , 2007 ; hoa et al . , 2011 ) . since acinetobacter , especially a. baumannii , is known to be an important opportunistic pathogen with multi - drug resistance ( vila and pachn , 2011 ) , the risk posed by environmental a. baumannii to human health should be examined further . low concentration of smx can affect on bacterial nitrate metabolism and select bacterial species ( underwood et al . , 2011 ) , suggesting importance of trace contamination by smx . tetracyclines are a family of broad - spectrum antibiotics that includes tetracycline , oxytetracycline ( otc ) , chlortetracycline , doxycycline , and minocycline ( chopra and roberts , 2001 ) . these antibiotics inhibit protein synthesis in gram - positive and gram - negative bacteria by preventing the binding of aminoacyl - trna molecules to the 30s ribosomal subunit ( geigenmuller and nierhaus , 1986 ; ross et al . , 1998 ) . owing to their broad - spectrum activity and low toxicity , tetracyclines are used in the treatment of a number of human skin and dental diseases . tetracyclines are also used in agriculture as growth promoters in farm animals and are used widely as prophylaxes in plant agriculture and aquaculture around the world ( chopra and roberts , 2001 ) , including southeast asia ( thuy et al . , 2011 ) . the first - generation tetracyclines include otc , chlorotetracycline , and 6-demethylchlorotetracycloine , which were developed in the 1940s . minocycline and doxycycline were launched as second - generation drugs in the 1960s ( thaker et al . , 2010 ) . a new glycylcycline derivative , tigecycline , which is active against most otc - resistant bacteria , acinetobacter , mrsa , and extended - spectrum -lactamase ( esbl ) producers , was licensed in 2005 ( livermore , 2005 ) . despite the variety of available analogs , otc is still commonly used in animal production and aquaculture ( holmstrm et al . , 2003 ; nonaka et al . , 2007 ; and others ) , although use as growth promoters has been banned in eu until 2006 ( castanon , 2007 ) . as tetracycline - resistant bacteria , high occurrence of tetracycline - resistant salmonella from human ( 50% ) and animals ( pig 34% and poultry 76% ) are reported in vietnam ( vo et al . , 2010 ) , as well as enterococcus and e. coli ( both 80% < ; dang et al . , 2011 ) . similar results are known in thailand , e.g. , chicken ( 42% ) and chicken meat ( 45% ) , and pork ( 11% ) and pork meat ( 20% ; ogasawara et al . , 2008 ) . tetracycline resistance genes , tet series , from the resistant bacteria have been well - studied ( roberts , 2005 ; and others ) . studies have indicated that some tet genes are persistent in aquaculture site without selective pressure ( tamminen et al . , 2011 ) , pristine environments and in animals ( gilliver et al . , 1999 ; rahman et al . tracking of tet genes in aquatic environments , including pristine areas , has been examined in the usa ( pruden et al . , 2006 ; storteboom et al . , 2010a , b ) , japan ( kim et al . , 2004 ; nonaka et al . , 2007 ) , and other countries ( zhang and zhang , 2011 ) ; however , similar research in indochina aquatic environments is limited ( kobayashi et al . , 2007a ; suzuki et al . , 2008 ) monitoring and phylogenetic analyses are needed if we are to obtain a thorough understanding of the origin and spread dynamics of tet genes . kobayashi et al . ( 2007a ) detected tet(m ) , tet(s ) , and tet(w ) genes throughout the mekong river delta . they found that tet(s ) and tet(w ) have only one genotype each , while tet(m ) has at least two genotypes . one tet(m ) genotype is identical to the gene encoded in various plasmids and transposons of gram - positive and gram - negative bacteria , and another is similar to a gene encoded in tn1545 of enterococcus faecalis ( 99% identity in pcr product , 170 bp/171 bp ) . since the two types could be found in the same sample , it was assumed that more than one source of the gene exists . this hypothesis was partly confirmed by microbial diversity analysis using denatured gradient gel electrophoresis analysis , which indicated a positive relationship between the shannon index ( h ) value and the tet gene detection- and otc - resistant bacteria occurrence - rates across a wide area , ranging from the mekong river and tonle sap lake , cambodia to the south china sea ( suzuki et al . , 2008 ) . gene exchange and horizontal transfer among various bacterial species in natural environments have been demonstrated ( smets and barkay , 2005 ) . ribosomal protection protein ( rpp ) genes , including tet(m ) , tet(s ) , and tet(w ) , are transferred among bacteria ( chopra and roberts , 2001 ) , and this phenomenon was experimentally confirmed using combinations of marine bacteria ( gene donors ) and e. coli ( neela et al . , 2009 ) . ( 2007b ) , which holds that the rpp genes were derived through duplication and divergence of an ancient gtpase before the divergence of the three domains . the rpps may have originally provided ribosomal protection against other chemical substances in the environment . it is known that exposing microbial assemblages to one toxicant can result in indirect selection for bacteria with resistance to multiple , chemically unrelated toxicants ( baker - austin et al . , 2006 ; baker et al . , 2006 ) . for example , exposure to cd , ni , ampicillin , and tetracycline significantly increases the frequency of bacterial resistance to multiple , chemically unrelated metals and antibiotics ( stepanauskas et al . , 2006 ) . metals and antibiotics are known not only for being associated to similar efflux pumps , but also for inducing co - resistance ( baker - austin et al . aquatic environments in indochina are highly contaminated with many kinds of persistent organic pollutants , metals ( suzuki and takada , 2009 ) , and antibiotics ( managaki et al . , environmental bacteria can acquire drug resistance naturally over time as well as through horizontal gene transfer . both transferable paralogous resistance genes and canonical resistance genes might be present in natural environments . from the viewpoint of both drug resistance and evolutionary biology , rpp genes are thus of considerable interest ( kobayashi et al . , 2007b ) . indochina aquatic environments are particularly vulnerable to the development of antibiotic - resistant bacteria due to pollution with antibiotics and other chemicals in the absence of adequate wastewater treatment systems . recent important findings concerning antibiotic resistance are as follows : ( 1 ) sub - lethal concentrations of antibiotics accelerate the development of antibiotic resistance ( yeh et al . , 2009 ; gullberg et al . , 2010 ) , and ( 2 ) chemicals other than antibiotics can promote development of antibiotic resistance ( alonso et al . both of these scenarios are suspected to occur in natural environments , especially in asian countries . correlation between the number of antibiotic drugs detected and the occurrence of multi - drug - resistant bacteria in vietnam was found ( hoa et al . , 2011 ) , indicating that multi - drug contamination is almost certain to result in an increase in the prevalence of multi - drug - resistant bacteria in aquatic environments . it is important to note that contamination with various antibiotics correlates strongly with induction of multi - drug resistance , even if the contaminant concentration is low . multi - drug resistance is a serious issue , particularly in asia , and is likely to become of even greater concern in the future . furthermore , contamination of antibiotics with trace concentrations can alter microbial ecosystem in terms of metabolism and diversity ( underwood et al . , 2011 ) , indicating the importance to consider unexpectedly effect than antimicrobial resistance . there is still a considerable gap in knowledge regarding the environmental chemistry and microbiology in the environment . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .	southeast asia has become the center of rapid industrial development and economic growth . however , this growth has far outpaced investment in public infrastructure , leading to the unregulated release of many pollutants , including wastewater - related contaminants such as antibiotics . antibiotics are of major concern because they can easily be released into the environment from numerous sources , and can subsequently induce development of antibiotic - resistant bacteria . recent studies have shown that for some categories of drugs this source - to - environment antibiotic resistance relationship is more complex . this review summarizes current understanding regarding the presence of quinolones , sulfonamides , and tetracyclines in aquatic environments of indochina and the prevalence of bacteria resistant to them . several noteworthy findings are discussed : ( 1 ) quinolone contamination and the occurrence of quinolone resistance are not correlated ; ( 2 ) occurrence of the sul sulfonamide resistance gene varies geographically ; and ( 3 ) microbial diversity might be related to the rate of oxytetracycline resistance .
You are an expert at summarizing long articles. Proceed to summarize the following text: the use of therapeutic hypothermia ( th ) in clinical medicine is no longer a rarity . a variety of methods have been implemented in an attempt to cool patients faster . in a recent issue of critical care , skulec and colleagues evaluated the efficacy of cold normal saline as compared with a colloid solution containing hydroxyethyl starch in the induction of th in survivors of cardiac arrest by using a porcine animal model of ventricular fibrillation ( vf ) . a 20-minute infusion of either 45 mg / ml of 1c cold normal saline or 45 mg / ml of 1c cold colloid solution ( voluven , 6% hydroxyethyl starch 130/0.4 in normal saline ; fresenius kabi ag , bad homburg , germany ) comparison of pulmonary blood temperature and cerebral blood temperature demonstrated a statistically significant decrease in temperature in animals given normal saline ( -2.1 0.3 versus -1.6 0.2c , and -1.7 0.4 versus -1.1 0.3c , p < 0.05 , respectively ) . moreover , initiation of th with normal saline resulted in accelerated cooling when compared with colloid solution ( -91 22 versus -68 23c / min , p = 0.046 ) . the authors also performed a mechanical substudy evaluating the specific heat capacities of each fluid and demonstrated that normal saline resulted in statistically more significant cooling than the studied colloid solution ( -7,155 647 versus -5,733 636c / min , p = 0.008 ) , and this is consistent with the above clinical in vivo findings . these results demonstrating the differential cooling of normal saline compared with a colloid solution containing hydroxyethyl starch both in vivo and experimentally by skulec and colleagues are both novel academically and consistent with the understanding of specific heat capacity of solutions following the addition of solutes from thermodynamics . briefly , the addition of a solute to a solution results in an increase in the number of particles that can absorb energy and release it as heat , in turn resulting in an increase in the temperature of the solute - solution combination relative to the solution alone when a constant amount of energy is applied . as the authors demonstrated , the thermodynamic effect of adding solute to a solution resulted in statistically significant differences clinically with respect to hypothermia induction and temperatures attained , as well as rapidity of cooling . interestingly , the intracranial pressure ( icp ) was increased in animals treated with cold colloid solution containing hydroxyethyl starch as compared with normal saline solution ( end infusion icps of 25 5 and 16 6 mm hg , respectively ) , which persisted for up to 90 minutes following infusion . the association of increased icp with hydroxyethyl starch was also demonstrated by khan and colleagues , who evaluated the combined incidence of delayed cerebral ischemia , hydrocephalous requiring cerebrospinal fluid shunting , and rebleeding in patients following subarachnoid hemorrhage , and demonstrated a statistically significant increase in both the combined endpoint ( odds ratio 3.1 , 95% confidence interval 1.30 to 7.36 , p = 0.01 ) and the incidence of hydrocephalous requiring cerebrospinal fluid shunting ( odds ratio 6.1 , 95% confidence interval 1.63 to 22.95 , p = 0.004 ) . the increases in both icp and hydrocephalous requiring cerebrospinal fluid shunting in these two studies are most likely due to changes in cerebrospinal fluid absorption by the arachnoid microvilli related to hydroxyethyl starch . the use of colloid solutions containing hydroxyethyl starch for volume resuscitation of critically ill patients has been controversial , and the literature suggests both increased mortality and increased need for renal replacement therapy when compared with normal saline , and subsequently the us food and drug administration issued a boxed recommendation to health - care professionals not to use hydroxyethyl starch - containing solutions in patients admitted to the intensive care unit or in patients with pre - existing renal dysfunction [ 5 - 7 ] . the icp increase seen by skulec and colleagues in animals treated with colloid solution may be , in part , the mechanism associated with increased mortality in patients treated with hydroxyethyl starch solutions . this study not only elucidates the decreased efficacy of colloid solutions containing hydroxyethyl starch compared with normal saline in the induction of th in vivo but also provides insight into complications associated with hydroxyethyl starch and re - emphasizes the importance of specific heat capacity of the solution with respect to rapidity of cooling during th induction .	the prevention of ischemic injury to preserve both end - organ function and improve neurological recovery by the implementation of therapeutic hypothermia has been well established in the literature . however , not only the means by which body temperature is cooled but also the rate by which target temperature is attained remains an area of continued interest and research . the induction of therapeutic hypothermia to begin the process of body temperature lowering through the infusion of a cold solution intravenously into the body may be one variable that influences not only rapidity of cooling but also subsequent clinical outcome . in a recent issue of critical care , skulec and colleagues compared the induction of therapeutic hypothermia by cold normal saline versus cold colloid solution containing hydroxyethyl starch in a porcine animal model of cardiac arrest , assessing both the rate of temperature change and target temperature achieved , in addition to changes in intracranial pressure .
You are an expert at summarizing long articles. Proceed to summarize the following text: emergency department ( ed ) use is common for both children and adults not only in a country without universal health insurance like the united states but also in countries like france where virtually all residents have health insurance . in 2006 the annual rate of ed visits for parisian children less than 2 years old ranged , according to district , from 46.9 to 91.3 per 100 children . many of these visits are not for true emergencies but for what ed physicians , insurers , policy makers , and researchers consider as inappropriate or nonurgent , that is , minor complaints that could have been dealt with in primary care offices on either that or the following day . durand and colleagues found that , in studies in a variety of countries around the world , the estimates of nonurgent visits by adults and children ranged from 4.8% to 90% , with a median of 32% . one reason for the variation was the inconsistency in defining nonurgent , as pointed out also by mistry and colleagues for pediatric ed visits . recent figures on pediatric nonurgent visits include 58% in the united states , 57.1% in italy , and 39.9% in belgium . in many cases , parents do not even try to contact a primary care physician [ 79 ] . furthermore , they go to eds even though they often have long waits ; their children are likely to undergo more testing and treatment than by their own doctors ; the ed physicians do not know their medical histories and usually do not provide follow - up ; and in many cases the costs to them will be higher . many researchers have , therefore , tried to understand why parents decide to bring their children to eds . they hope to find out how to reduce unnecessary ed visits and thereby how to improve continuity of care ; to lessen overcrowding in eds , distraction from true emergencies , and diversion of ambulances to less crowded but more distant eds [ 11 , 12 ] ; and to decrease health care expenses [ 6 , 11 , 13 , 14 ] . reasons given by parents for their decisions to go to pediatric eds ( peds ) , even without referral by their children 's primary care physicians , include that they seek a quick and convenient solution to a health issue [ 1 , 8 , 10 , 1518 ] ; that they view the ped as the best place to go because of its better physicians and/or medical equipment [ 1 , 5 , 7 , 8 , 10 , 17 , 19 ] ; that they are dissatisfied with their primary care physician 's office or the physician 's diagnosis and/or treatment [ 8 , 15 , 20 ] ; that they are very worried about their child 's health [ 8 , 15 , 17 , 21 ] ; that they are in financial straits [ 17 , 20 ] ; that they have a habit of going to the ed [ 1 , 7 , 8 ] ; and that they have no access to other care [ 1 , 17 , 19 ] . the methodologies of the above - mentioned studies were not based on a broad theory of human motivation and may , as a result , have neglected important motives . in this study , therefore , we examined parental motives by applying michael apter 's metamotivational theory ( mtt ) [ 23 , 24 ] , which encompasses and systematizes the great diversity of human motives . by including all theoretically possible categories of motives in the questionnaire , we could ensure that it was psychologically complete , that is , that no possible parental motive was missed . objectives of the study were ( a ) to inventory the motives for going to peds ; ( b ) to order these motives , or categories of motives , as a function of their importance ; and ( c ) to find out how demographic characteristics were associated with these motives . the study was approved by the ethics committee of the children 's hospital of toulouse . the participants were parents attending the ped of the children 's hospital , a tertiary - level pediatric hospital in toulouse , france . the research assistant invited the parents of every child to participate , informed them about the survey , and obtained oral consent from those who agreed to participate . the first questionnaire contained 69 items referring to motives for going to a ped ( table 1 ) . forty - eight items were created by the members of the research team , inspired by mmt ( see table 5 ) , by the medical literature ( cited above ) , and by their personal experience as physicians . items were purposely and systematically created to correspond to all of mmt 's categories of motives . this questionnaire was then presented to a focus group of parents that reformulated items judged as ambiguous and suggested additional items based on their personal experiences as parents . the new questionnaire was presented to another focus group , and the process was repeated until saturation occurred . the common wording of all items in the questionnaireone of the reasons i came to the ped today with my childwas chosen to acknowledge that several motives could be operating at the same time . the second questionnaire asked about demographics , about previous ed use , and about prior doctor visits during this illness . participants responded individually in the waiting room , before their children were seen . if both parents were present , only one filled out the questionnaires but that one could confer with the other . the research assistant was , in most cases , not present during the process in order not to influence them . they indicated how much they agreed with each statement on the first questionnaire by putting a mark on the 10-point response scale under each one . the treating physician subsequently indicated the severity of each child 's illness on a scale of 1 ( not severe ) , 2 ( mildly severe ) , or 3 ( severe)and whether the child was hospitalized . the character of this study was both exploratory and confirmatory . through exploratory factor analyses using two - thirds of the sample , exploratory factor analysis is a statistical technique that is used to reduce a number of variables into a smaller set of unobserved factors . the variables within each factor are empirically highly correlated with each other but not with the other variables . if the correlation between items and any factor was lower than 0.30 and their means were very low , the items were removed from the analysis . confirmatory factor analysis is a statistical technique used to test whether a set of factors that has been obtained through exploratory factor analysis of data from one sample fits the data observed in another sample . as the exploratory factor analysis has to be conducted on all the useable items , the confirmatory analysis should only be done on a representative and more manageable subset of items , namely , on the three items with the highest loadings ( the highest correlations with the factor ) within each of the factors found in the exploratory analysis . therefore , the exploratory factor analysis requires a larger sample size than the confirmatory analysis , calculated in this case to be 2 to 1 . the sample size of 500 was determined in accordance with the technical requirements of factor analysis for a maximum expected number of factors of 10 . our choice of 10 was based on our analysis of the previous studies of reasons to go to the ed . table 1 shows the mean ratings ( on a scale of 1 to 10 ) of each item in the exploratory factor analysis using the first subsample ( n = 332 ) . thirteen of the 69 items did not load on any factor ( correlation between item and any factor < .30 ) , and their means were very low . six interpretable and independent factors emerged , namely , ( 1 ) being considered by others as responsible parents ( which explained 13% of the variance ) ; ( 2 ) empathic concern for child 's suffering ( 8% of the variance ) ; ( 3 ) seeking quick diagnosis , treatment , and reassurance ( 12% ) ; ( 4 ) dissatisfaction with previous consultation ( 7% ) ; ( 5 ) ped as the best place ( 7% ) ; ( 6 ) and external factors ( 5% ) , such as the family physician 's absence , the impossibility of borrowing money , and a recommendation from the service that provides urgent home visits . the results of the confirmatory factor analysis , conducted on the three items with the highest loadings on each of the six factors using the second subsample ( n = 168 ) , are shown in table 3 . the mean scores for each factor in the final model on a 10-point scale range from 1.79 ( dissatisfaction with previous consultation ) to 6.82 ( seeking quick diagnosis , treatment , and reassurance ) . table 4 shows correlations between demographic characteristics , severity indices ( as subsequently judged by physicians ) , and motives . seeking quick diagnosis , treatment , and reassurance was significantly more strongly endorsed by parents of one child than by parents of more than one . considering the ped as the best place to go was given a higher rating by younger fathers than by older ones . empathic concern for the child 's suffering was given a higher rating by parents with only one child . being considered by others as responsible parents was a stronger motive for parents of boys than of girls , for younger mothers , for less educated parents , and for parents who were more likely to go to eds without first consulting their regular physician , and a stronger motive in cases judged by the treating physician to be of lesser severity . finally , external factors were more strongly endorsed as motives by parents of older children who had siblings and in cases in which symptoms had more recently appeared . the aim of our study was to make an inventory of the motives of parents in bringing their children to peds using a broad theoretical framework . we found that , as in other studies [ 8 , 15 , 17 , 21 ] , parents endorsed a wide variety of motives . finding factors meant the guarantee that parents ' responses were meaningful , that is , that their responses were sufficiently coherent such that statistical analysis could reveal a clear structure to them . the emergence of a clear factorial structure also enabled measurement of the strength of each motive . telic in mmt ) seemed to be highly goal - oriented , going to a ped to obtain care they saw as important . this is consistent with findings in other countries [ 8 , 15 , 17 , 26 ] . ] described guardians as worried that symptoms represented serious illness and that it was important to seek medical attention . similarly , durand and colleagues , in their study of french adult patients , characterized them as rational consumers ( in contrast to the abusive and irresponsible consumers portrayed by the ed health professionals they interviewed ) . these parents are unlikely , therefore , to agree with the charge that large numbers of visits to peds are unwarranted . the second most highly rated factor was ped as the best place to go ( which would be called a conformist motive by mtt ) . their respect for the ped may have been stimulated by television shows and by reports in the media about the technology and specialization available in hospitals ( as well as , in the united states , by advertising ) . they may not have appreciated that technology and specialized care are not always beneficial to patients . empathic concern for child 's suffering received a lower overall rating . those parents who strongly endorsed these motives ( labeled as sympathy in mtt ) were no longer able to put up with their children 's suffering . they tended to be parents of a single child and , therefore , likely to be less experienced in coping with sick children . it is striking , however , that distress at their child 's current suffering was much less important than the goal - oriented aim of obtaining the most effective treatment . the other three factors were rated quite low overall but were endorsed by some parents . being considered by others as responsible parents was rated highly by parents who seemed to be concerned about their own image as parents ( labeled as these parents tended to be younger ( especially the mothers ) and less educated than other parents and to go to eds for themselves more frequently . their child 's condition was not likely to be judged as bad by the ed physician . physicians and health services experts , especially in the us , often cite structural factors as important determinants of excessive ed utilization [ 6 , 11 , 13 , 15 , 2730 ] . those few parents who endorsed these motives strongly indicated thereby that going to peds was not a truly personal decision . they followed the instruction of the urgent care service or could not go to another medical facility owing to the absence of a physician or to lack of money . their child 's illness tended to be more acute ; that is , the child was more likely than others to have experienced pain and/or fever for less than 12 hours . the lowest average rating was for dissatisfaction with previous consultation ( a negativist motive in the terminology of mmt ) . only a few parents were very unhappy with the care provided by their family physicians , in accordance with findings in other countries [ 8 , 31 ] . it was conducted in a single site in france , and the motives of the 35% of parents who declined to participate are unknown . the study 's results must , therefore , be generalized with caution to other populations of children and parents and to other sites . furthermore , even if the questionnaire is based on a broad theory of motivation , it has not been validated and should , therefore , be used prospectively again to confirm these results . nonetheless , our findings can help to illuminate the current debate about the overuse of eds . access to care is a key issue for parents , as well as for adult patients , and , therefore , is a focus of reform efforts . the 2011 national health interview survey in the united states found that , among adults who went to eds and were not admitted to the hospital ( 68.9% of those visiting the ed ) , 78.9% had had at least one access issue for going to the ed ( defined as indicating not having another place to go , doctor 's office or clinic being not open , ed being the closest provider ' , and/or clinic being not open ) . it seems possible to reduce ed visits , therefore , by improving access to other sources of care ( as in the state of north carolina ) . without this , as the medicaid experience in the state of oregon showed , expanding insurance will not reduce ed use ; it will increase it . accordingly , many reformers have proposed encouraging pediatric practices to expand the availability of urgent visits [ 10 , 30 ] ; france has tried to set up primary care units at or near eds ; and italy is requiring its primary care offices to be open 24 hours a day 7 days a week . wang and colleagues tested in the united states a pilot ped diversion program for children with medicaid using extended office hours , multiple access locations , and care coordination that reduced ed visits by 8 visits per 1000 members per month ( compared with the control group ) . because their diversion program was so expensive , however , the overall cost of care was not reduced . our study demonstrates the limits or barriers , from the parents ' perspective , to the success of such efforts . we found that parents generally bring their children to peds with serious , goal - oriented motives . similarly , in the united states , the 2011 national health interview survey found that 65.0% of adults not admitted to the hospital cited at least one acuity issue ( defined as indicating only hospital could help , advised by health provider to go to ed , and/or problem too serious for doctor 's office or clinic ) ; kubicek and colleagues found that 63% of parents bringing their children for reasons assessed as nonurgent perceived the visit as very or extremely urgent ; and kalidindi and colleagues found that parents assessed 94% of their visits as urgent , even though 27% of these urgent visits were assessed as nonurgent by the physicians . thus , even if access issues were greatly reduced in the us and other countries , the acuity issues would persist . because there is no consensus between the population and physicians about what is urgent and what is not , staff members in the eds tend to define many visits as nonurgent , to label them as inappropriate , and to feel frustrated [ 17 , 31 ] . in general eds in the united states , the presenting complaints of adult patients judged subsequently as primary care treatable do not in fact differ from those judged as needing ed care . furthermore , as cresson ] states , to speak of false urgency ' in situations where there is urgency for the patient and not for the doctors is to act as if the latter were the only ones capable of defining urgency . if overworked ed physicians , as well as researchers and policy makers , focus on ultimate diagnoses and wasted resources , they forget that the parents do not have the medical knowledge to make these diagnoses , do not know what the final diagnoses will be , and do not know how the illnesses are going to evolve . as adams concludes , trying to discern low - acuity conditions and putting up barriers to receiving care or denying payment after receiving care will work no better in future generations than in the past [ 38 , p. 1174 one logical solution would be to educate parents better [ 5 , 10 , 17 ] and specifically those parents whose own characteristics for example , in a us study , single parents , those who were themselves brought to eds as children , and those with medicaid make them more likely to use the ed . these efforts could involve targeted messaging to high utilizers or more formal instruction , but so far success has been limited . in one study , 130 families were randomly assigned to receive , or not , an educational intervention in the ped that involved reviewing with a research assistant a booklet about how to manage minor illnesses and watching a 10-minute video . at 6 months there was no difference between groups in ped utilization , and most visits were still for minor illnesses . similarly , a 90-minute , interactive educational activity at primary care offices increased 32 parents ' knowledge of fever , colds , and minor trauma and increased their after - hours telephone use ; but it did not decrease their ed use . education may , of course , have greater effects on other groups of parents than those studied and if done more intensively and repeatedly . yet our findings point to one possible reason for the limited impact of greater knowledge : the importance for many parents of emotional motives of anxiety about and empathetic concern for the child ( as would be expected of parents ) as well as , for some , of a desire to be seen as a caring parent . the setting able immediately to soothe their anxiety and distress is the hospital and its emergency department . it is not surprising , therefore , that the rate of ed visits has been increasing even in countries , like france , that have extended basic insurance to all residents . when parents in france , as elsewhere , think their children are suffering and might be seriously ill , they are goal - oriented and , often , frightened . they bring their children to the places that are immediately available and that , they think , are most capable of helping their children , to the peds . thus , as hendry , beattie , and heaney in the united kingdom concluded , perhaps it is the service design , rather than patient behavior , that is inappropriate the solution is not only to provide anxious parents with expanded daytime sick visits , 24-hour telephone access , and even night - time home visits , as is currently possible in many places . the solution may also be to expand the peds , that is , to provide urgent visit capacity for minor illnesses and injuries , at lower cost than truly urgent visits , in the places of reassurance and security , the peds themselves .	parents frequently bring their children to general or pediatric emergency departments ( eds ) , even though many of these visits are judged by others to be nonurgent and inappropriate . this study examined the motives behind parents ' decisions to take their children to a pediatric emergency department ( ped ) . at a ped in toulouse , france , 497 parents rated their level of agreement with each of 69 possible motives representing all categories of human motivation for coming to the ped that day . exploratory and confirmatory factor analyses found evidence for six separable motives , called ( in order of importance ) ( a ) seeking quick diagnosis , treatment , and reassurance ; ( b ) ped as the best place to go ; ( c ) empathic concern for child 's suffering ; ( d ) being considered by others as responsible parents ; ( e ) external factors ; and ( f ) dissatisfaction with previous consultation . conclusions . parents ' motives in bringing their children to the ped are primarily serious and goal - oriented . they are also often emotion based , as would be expected in parents of ill children . the parents would be unlikely to agree that these visits were inappropriate .
You are an expert at summarizing long articles. Proceed to summarize the following text: serotonin toxicity ( or serotonin syndrome ) is the result of excessive serotonin activity caused by the administration of selective serotonin reuptake inhibitors ( ssris ) , often in combination with serotonin receptor agonists.1 serotonin toxicity is characterized by myoclonus , hyperreflexia , autonomic nervous symptoms , and changes in mental status.1 given that an altered mental status , which includes agitation and anxiety , is common in depression , patients treated with ssris should be carefully examined for the presence of hyperreflexia . two serotonin receptor subtypes are candidates for the underlying pathophysiology of serotonin toxicity.1 however , the different effects of the stimulation of the 5-hydroxytryptamine ( 5-ht)1a and 5-ht2a receptors on the clinical exacerbation of serotonin toxicity have not yet been elucidated . in the present report , we suggest the possible involvement of the 5-ht1a receptor in mild serotonin toxicity without hyperthermia . a 64-year - old woman who experienced auditory hallucinations and insomnia , was treated with hypnotic benzodiazepines and perospirone ( 8 mg / day ; figure 1a ) . she had developed chronic renal failure , which had continued for several years , with high serum levels of creatinine and blood urea nitrogen ( figure 1a ) . she also experienced depressed mood , diminished interest , and anhedonia ; therefore , she was treated with paroxetine ( 10 mg / day ) . eighteen hours after paroxetine was added to her ongoing perospirone treatment , she exhibited finger tremors , sweating , coarse shivering , hyperactive knee jerks , vomiting , diarrhea , tachycardia , and psychomotor agitation . an 81-year - old woman who experienced persecutory delusions with mild dementia , was treated with neuroleptics ( figure 1b ) . she was admitted to a psychiatric unit , owing to increased hostility and aggression related to her delusions . the results of brain imaging tests revealed mild brain atrophy in the frontotemporal and hippocampal regions with no cerebrovascular lesions . after successful treatment of her paranoid state , the patient developed depressive symptoms , which were treated with 10 mg / day of paroxetine . the final neuroleptic dose was 8 mg / day of perospirone that had been administered for 3 weeks . fourteen hours after the addition of paroxetine to the patient s ongoing perospirone treatment , she exhibited tachycardia , finger tremors , prominent anxiety and agitation , and hyperactive knee jerks . the symptoms continued for days ( ie , for as long as perospirone and paroxetine were coadministered ) , and they disappeared 3 days after the discontinuation of both drugs . a 64-year - old woman who experienced auditory hallucinations and insomnia , was treated with hypnotic benzodiazepines and perospirone ( 8 mg / day ; figure 1a ) . she had developed chronic renal failure , which had continued for several years , with high serum levels of creatinine and blood urea nitrogen ( figure 1a ) . she also experienced depressed mood , diminished interest , and anhedonia ; therefore , she was treated with paroxetine ( 10 mg / day ) . eighteen hours after paroxetine was added to her ongoing perospirone treatment , she exhibited finger tremors , sweating , coarse shivering , hyperactive knee jerks , vomiting , diarrhea , tachycardia , and psychomotor agitation . an 81-year - old woman who experienced persecutory delusions with mild dementia , was treated with neuroleptics ( figure 1b ) . she was admitted to a psychiatric unit , owing to increased hostility and aggression related to her delusions . the results of brain imaging tests revealed mild brain atrophy in the frontotemporal and hippocampal regions with no cerebrovascular lesions . after successful treatment of her paranoid state , the patient developed depressive symptoms , which were treated with 10 mg / day of paroxetine . the final neuroleptic dose was 8 mg / day of perospirone that had been administered for 3 weeks . fourteen hours after the addition of paroxetine to the patient s ongoing perospirone treatment , she exhibited tachycardia , finger tremors , prominent anxiety and agitation , and hyperactive knee jerks . the symptoms continued for days ( ie , for as long as perospirone and paroxetine were coadministered ) , and they disappeared 3 days after the discontinuation of both drugs . in the present report , both patients were diagnosed as having serotonin toxicity because of the existence of tremor and hyperreflexia,2 and they presented without symptoms of muscle rigidity or hyperthermia.1 the possibility of neuroleptic malignant syndrome was not taken into consideration as a differential diagnosis , because the symptoms appeared just after adding paroxetine ; moreover , the present cases did not exhibit severe muscle rigidity and hyperthermia.3 in addition , there was no elevation of serum creatine phosphokinase in case 1 . the autonomic symptoms , neuromuscular hyperactivity , and altered mental status occurred upon initiation of the serotonergic drugs , and they ceased promptly after the treatment s discontinuation ; this is characteristic of serotonin toxicity.4 paroxetine is metabolized by cytochrome p450 oxidase ( cyp)2d6 in the liver and excreted in the urine . patients with renal failure are considered to be at an increased risk of developing side effects associated with a high serum concentration of paroxetine.5 the psychiatric diagnosis of case 1 was psychotic disorder not otherwise specified , and there was no evidence of a relationship between her renal insufficiency and her psychiatric symptoms or her serotonin toxicity symptoms . case 2 was diagnosed as having dementia with psychotic symptoms , including persecutory delusions , anxiety , and depressive symptoms . in this case , the serotonin toxicity persisted for days because of the unfortunate oversight of a possible serotonin toxicity induced by the administered psychotropics . the increased anxiety and agitation were believed to be derived from the behavioral and psychological symptoms of dementia . tachycardia was misdiagnosed as a result of the increased motor activity associated with her anxiety and agitation . serum paroxetine concentrations were not evaluated in either case presented here , and the possibility remains that the paroxetine concentration was sufficiently elevated in both cases . we considered that vulnerability induced by perospirone pretreatment is most likely to have exaggerated the mild serotonin toxicity in both cases . perospirone is a serotonin dopamine antagonist with unique agonistic effects on the 5-ht1a receptors,6 and is metabolized by cyp3a4.7 it was the second serotonin the receptor binding profiles of perospirone ( excluding 5-ht1a receptor binding ) and its pharmacological properties targeting the positive and negative symptoms of schizophrenia resemble those of risperidone.7 the ki values of perospirone for the dopamine receptor d2 , as well as for the 5-ht2a and 5-ht1a receptors are 1.4 nm , 0.6 nm , and 2.9 nm , respectively,7 whereas those of risperidone are 3.3 nm , 0.16 nm , and 250 nm , respectively.8 data on the ki values measured from various tissue sources can be viewed in the national institute of mental health psychoactive drug screening program ki database ( http://pdsp.med.unc.edu/pdsp.php ) by choosing a psychotropic drug for the test ligand . the 5-ht1a agonist activity of neuroleptics is expected to improve mood9 and cognition10 in schizophrenia . perospirone has also shown efficacy in reducing aggressive behavior in dementia patients.11 to treat the symptoms of aggression and anxiety exhibited by case 2 , previously administered olanzapine was switched to perospirone 10 days before the addition of paroxetine . when experimental animals were administered a 5-ht1a agonist , they developed tremors , forepaw treading , head - weaving and twitches , flattened body posture , hind limb abduction , the straub tail reaction , hyperhidrosis , and defecation.12 the similarities noted between the alterations in animal behavior and the symptoms of human serotonin toxicity suggest that the 5-ht1a receptor may be involved in the pathogenesis of serotonin toxicity . in another experimental animal model of serotonin toxicity , the activation of neuronal 5-ht2a receptors was identified as the cause of life - threatening hyperthermia.13 furthermore , the serotonin system has been implicated in a biphasic mechanism that controls thermoregulation . studies in experimental animals have indicated that a slight elevation of serotonin levels decreases body temperature through neural transmission involving the 5-ht1a receptors , whereas the considerable elevation of serotonin levels induces high body temperature through 5-ht2a neural transmission.14,15 therefore , it could be hypothesized that while a slight excess of serotonin causes side effects by activating the 5-ht1a receptor , a significant increase in serotonin levels can induce lethal side effects with hyperthermia , which can cause severe brain damage16 through the activation of the 5-ht2a receptor . the early observation17 that the affinity of endogenous serotonin for the 5-ht1a receptors ( ki = 5.1 nm ) is much higher than its affinity for 5-ht2a receptors ( ki = 420 nm ) supports this hypothesis . vulnerabilities to serotonin toxicity are increased by a polymorphism in the serotonin transporter or cyp , a competitive substrate of the cyp enzyme,18 and by coadministered monoamine oxidase inhibitors or lithium.19 the present cases underscore the importance of preventing the development of serotonin toxicity after the combined use of ssris and 5-ht1a agonistic drugs , even when a small amount of ssri is administered . several case studies have reported the development of serotonin toxicity by the combined use of ssris and 5-ht1a agonist anxiolytics;20,21 however , high body temperatures were not observed in these studies . the exacerbation of serotonin toxicity in the present cases by adding small doses of paroxetine while administering perospirone acting both as a 5-ht1a receptor agonist and a 5-ht2a receptor antagonist clearly indicates the involvement of the 5-ht1a receptor in mild forms of serotonin toxicity . although the involvement of both 5-ht1a and 5-ht2a receptors in the pathogenesis of serotonin toxicity has often been indicated,22 we considered it worthwhile to pay attention to serotonin toxicity profiles under the situation of an antagonistic blockade of 5-ht2a receptors in human subjects . taken together , the coadministration of ssris and neuroleptics , like risperidone , with a potent 5-ht2a antagonistic function and a scarce 5-ht1a agonistic function would produce a lower incidence of serotonin toxicity . a recent clinical trial23 indicated the effectiveness of adding a 5-ht1a partial agonist for the treatment of patients with depression who are resistant to citalopram . mirtazapine , which acts as a 5-ht1a agonist in addition to its norepinephrine - releasing function , has been proposed to enhance the clinical effectiveness of ssris.24 furthermore , aripiprazole , which acts as a 5-ht1a agonist , is used to augment the clinical effectiveness of ssri treatment.25 considering that recently available ssris , including vilazodone26 and vortioxetine,27 have considerable 5-ht1a agonistic function , more caution is needed to detect serotonin toxicity in depression treatments . in conclusion , the possible development of serotonin toxicity should be considered when neuromuscular toxicities including tremor , myoclonus , and hyperreflexia in the extremities are observed during the combined use of ssris and psychotropics with 5-ht1a agonistic properties , or during the use of ssris with 5-ht1a agonistic properties .	we propose the possibility of 5-hydroxytryptamine ( 5-ht)1a receptor involvement in mild serotonin toxicity . a 64-year - old woman who experienced hallucinations was treated with perospirone ( 8 mg / day ) . she also complained of depressed mood and was prescribed paroxetine ( 10 mg / day ) . she exhibited finger tremors , sweating , coarse shivering , hyperactive knee jerks , vomiting , diarrhea , tachycardia , and psychomotor agitation . after the discontinuation of paroxetine and perospirone , the symptoms disappeared . another 81-year - old woman , who experienced delusions , was treated with perospirone ( 8 mg / day ) . depressive symptoms appeared and paroxetine ( 10 mg / day ) was added . she exhibited tachycardia , finger tremors , anxiety , agitation , and hyperactive knee jerks . the symptoms disappeared after the cessation of paroxetine and perospirone . recently , the effectiveness of coadministrating 5-ht1a agonistic psychotropics with selective serotonin reuptake inhibitors ( ssris ) has been reported , and ssris with 5-ht1a agonistic activity have been newly approved in the treatment of depression . perospirone is a serotonin dopamine antagonist and agonistic on the 5-ht1a receptors . animal studies have indicated that mild serotonin excess induces low body temperature through 5-ht1a , whereas severe serotonin excess induces high body temperature through 5-ht2a activation . therefore , it could be hypothesized that mild serotonin excess induces side effects through 5-ht1a , and severe serotonin excess induces lethal side effects with hyperthermia through 5-ht2a . serotonin toxicity via a low dose of paroxetine that is coadministered with perospirone , which acts agonistically on the 5-ht1a receptor and antagonistically on the 5-ht2a receptor , clearly indicated 5-ht1a receptor involvement in mild serotonin toxicity . careful measures should be adopted to avoid serotonin toxicity following the combined use of ssris and 5-ht1a agonists .
You are an expert at summarizing long articles. Proceed to summarize the following text: periodontal pockets if left unaddressed inadvertently lead to further loss of the periodontal architecture . though elimination of pockets has been a common goal of periodontal therapy , regeneration is a favored modality , where there is a reconstruction of lost or injured tissues in such a way that both the original structure and their function are completely restored.1 supracrestal defects illustrated as horizontal bone loss radiographically , represents the least predictable periodontal defect type in the regenerative approaches and remain an unsolved dilemma for clinicians . currently , used regenerative procedures are not routinely applicable to this type of lesion.2 furthermore , open flap debridement ( ofd ) the accepted treatment modality for treatment of deep suprabony pockets results in significant gingival recession,3 it has also been reported that post - surgical recession is greater in sites with deeper periodontal pockets.4 thus , implying that post - surgical recession is directly related to initial probing depth . the varied explanations , for post - surgical recession include inadequate bone support , thin gingival tissue , limited blood supply , and post - operative shrinkage of the flap . with sequelae like poor aesthetics and hypersensitivity , its prevention should be considered as a desirable end goal of periodontal therapy . subepithelial connective tissue grafts ( sectgs ) have been used to treat labial and buccal gingival recession for attaining root coverage5,6 and clinical attachment gain.7,8 furthermore , histological evidence of new attachment in previously exposed root surfaces has been widely published.6,9 - 11 clinical gain of attachment has also been reported to be similar while comparing guided tissue regeneration ( gtr ) membrane and sectg12,13 however , lesser post - operative recession has been observed with sectg.13,14 it is possible to hypothesize that the use of sectg in conjunction with ofd in treatment of supracrestal / horizontal osseous defects could lead to better clinical results ; i.e. greater clinical attachment level gain , reduced probing pocket depth ( ppd ) , and decreased post - surgical gingival recession . most of the reported studies which have examined the efficacy of sectg along with ofd for treatment of horizontal bony defects have examined it in anterior segment,8,15,16 and in posterior segments as a treatment modality for furcation involvement.17 thus , an attempt was made to assess the efficacy of sectg along with ofd in the management of supracrestal defects of the posterior segments . the study was designed as a randomized , split - mouth clinical trial , comparing the clinical treatment outcomes of the use of sectg in conjunction with ofd to ofd in the treatment of horizontal osseous defects of posterior teeth . the study recruited 20 patients ( 10 males and 10 females , aged 18 - 56 years , with a mean age of 37.79 10.58 years ) , with no dropout . all patients treated were medically stable , non - smokers with no contraindications to periodontal surgery , had not been subjected to any periodontal therapy for at least 6 months prior , and had no history of antibiotics use within 6 months prior to treatment . to be included in the study , patients had to have radiographic evidence of at least two identical bilateral suprabony pockets with horizontal bone loss pattern , patients having an adequate band of keratinized tissue for ease in surgical manipulation and suturing . following the initial therapy , the selected sites displayed probing pockets depth of 4 mm and clinical attachment loss of 4 mm and horizontal bone loss detected by radiographic examination ( alveolar crest level [ acl ] - cementoenamel junction [ cej ] distance 4 mm ) at three adjacent posterior teeth . based on inclusion criteria , an informed consent was obtained from patients selected for study . post 6 weeks of phase 1 periodontal therapy , re - evaluation of patients was done . all the selected patients underwent phase 1 therapy . at study baseline , 6 months and 12 months after treatment , the following ancillary parameters were evaluated : plaque index ( pi),18 papillary bleeding index ( pbi).19 alginate impressions were recorded and study casts for each patient were prepared . furthermore , customized acrylic occlusal stent with a groove ( guide plane ) was fabricated on all these study casts . the groove acted as a guide for the clinical measurements , which were done by using a straight periodontal probe ( university of north carolina , unc - 15 ) ( figure 1 ) . the ppd was measured using the gingival margin as a reference to the base of the periodontal pocket . the relative attachment level ( ral ) as the distance from the apical end of stent to the base of the periodontal pocket . gingival marginal level ( gml ) was measured from the apical most end of the stent to the crest of the gingival margin , with the help of periodontal probe.20 all the acrylic stents were secured on the prepared study casts for the entire duration of the study to minimize distortion . customized acrylic occlusal stent with a groove ( guide plane ) and clinical measurements by using a straight periodontal probe ( university of north carolina , unc -15 ) . when measuring radiographic bone level ( bl ) , the location of both acl , and cej was determined.21 the cej position was identified according to schei et al.22 the positions of acl and cej were marked by a pencil on the radiographs and the distance acl - cej ( bl ) was measured by a millimeter grid ( figure 2 ) . linear distances between the most coronal interproximal bl and the cej were obtained by counting the grids.23 measuring radiographic bone level , alveolar crest level and cementoenamel junction using millimeter grid . intracrevicular incisions were carried out at buccal and lingual aspects followed by full thickness mucoperiosteal flaps elevation ( figure 3 ) . after the reflection of the flap , a sub - gingival calculus , plaque , pocket epithelium , and diseased granulation tissue were severed and surgical area was completely debrided . the sectg was harvested from the palate ( figure 4).24 the dimension of the graft was adequate to cover the root surfaces up to the cej ; the donor site was then secured with horizontal suturing technique . the harvested sectg was trimmed to remove all visible epithelium , shaped , and placed under coronal part of buccal flap up to cej ( figure 5 ) . harvested subepithelial connective tissue graft placed under coronal part of buccal flap up - to cementoenamel junction . surgical flaps were repositioned and primary closure was attained by the direct loop suturing technique using 3 - 0 silk suture ( mersilk - ethicon , division of johnson and johnson ltd . ) the surgical and donor site were protected by placing a non - eugenol periodontal dressing ( coe - pack - gc america inc . surgical flaps repositioned to the pre - surgical level and sutured with 3 - 0 silk suture . a similar surgical protocol was followed in control sites where only ofd was done without the use of sectg . clinical examination of patients were done at 1-week post - surgery at the time of suture removal , all patients exhibited uneventful healing . ancillary clinical parameters were recorded at 3 months , 6 months , and 12 months post - surgery , recording of clinical parameters and radiographic reassessment was done at 6 months and 12 months post - surgery . all along the means and standard deviations ( mean standard deviation ) values were calculated for all clinical and radiographic parameters.25 student s paired t - test was used to compare data from baseline with those at 6 months and 12 months in test sites and control sites . all the selected patients underwent phase 1 therapy . at study baseline , 6 months and 12 months after treatment , the following ancillary parameters were evaluated : plaque index ( pi),18 papillary bleeding index ( pbi).19 alginate impressions were recorded and study casts for each patient were prepared . furthermore , customized acrylic occlusal stent with a groove ( guide plane ) was fabricated on all these study casts . the groove acted as a guide for the clinical measurements , which were done by using a straight periodontal probe ( university of north carolina , unc - 15 ) ( figure 1 ) . the ppd was measured using the gingival margin as a reference to the base of the periodontal pocket . the relative attachment level ( ral ) as the distance from the apical end of stent to the base of the periodontal pocket . gingival marginal level ( gml ) was measured from the apical most end of the stent to the crest of the gingival margin , with the help of periodontal probe.20 all the acrylic stents were secured on the prepared study casts for the entire duration of the study to minimize distortion . customized acrylic occlusal stent with a groove ( guide plane ) and clinical measurements by using a straight periodontal probe ( university of north carolina , unc -15 ) . when measuring radiographic bone level ( bl ) , the location of both acl , and cej was determined.21 the cej position was identified according to schei et al.22 the positions of acl and cej were marked by a pencil on the radiographs and the distance acl - cej ( bl ) was measured by a millimeter grid ( figure 2 ) . linear distances between the most coronal interproximal bl and the cej were obtained by counting the grids.23 measuring radiographic bone level , alveolar crest level and cementoenamel junction using millimeter grid . intracrevicular incisions were carried out at buccal and lingual aspects followed by full thickness mucoperiosteal flaps elevation ( figure 3 ) . after the reflection of the flap , a sub - gingival calculus , plaque , pocket epithelium , and diseased granulation tissue were severed and surgical area was completely debrided . the sectg was harvested from the palate ( figure 4).24 the dimension of the graft was adequate to cover the root surfaces up to the cej ; the donor site was then secured with horizontal suturing technique . the harvested sectg was trimmed to remove all visible epithelium , shaped , and placed under coronal part of buccal flap up to cej ( figure 5 ) . harvested subepithelial connective tissue graft placed under coronal part of buccal flap up - to cementoenamel junction . surgical flaps were repositioned and primary closure was attained by the direct loop suturing technique using 3 - 0 silk suture ( mersilk - ethicon , division of johnson and johnson ltd . ) the surgical and donor site were protected by placing a non - eugenol periodontal dressing ( coe - pack - gc america inc . surgical flaps repositioned to the pre - surgical level and sutured with 3 - 0 silk suture . a similar surgical protocol was followed in control sites where only ofd was done without the use of sectg . clinical examination of patients were done at 1-week post - surgery at the time of suture removal , all patients exhibited uneventful healing . ancillary clinical parameters were recorded at 3 months , 6 months , and 12 months post - surgery , recording of clinical parameters and radiographic reassessment was done at 6 months and 12 months post - surgery . all along the means and standard deviations ( mean standard deviation ) values were calculated for all clinical and radiographic parameters.25 student s paired t - test was used to compare data from baseline with those at 6 months and 12 months in test sites and control sites . the full mouth mean pi and pbi scores decreased from baseline to 12 months follow - up . pi baseline 0.97 0.37 to 12 months 0.32 0.18 and pbi scores baseline 0.82 0.67 to 12 months score 0.20 0.16 , when the scores were compared statistically using paired t - test and the difference was statistically significant ( p > 0.05 ) in both the pi and pbi scores as shown in table 1 . ancillary clinical parameters : mean plaque pi and pbi scores at baseline , at 3 months , at 6 months and at 12 months ( mvsd ) . at baseline mean , ppd was 7.37 0.23 mm in the test group and 6.94 0.18 . mean ral at baseline in the test group was 15.91 0.45 and in control sites were 15.37 0.39 . the mean gml at baseline in the test group was 8.47 0.13 and control sites was 8.04 0.75 as shown in table 2 . clinical parameters : ppd , ral and gml at baseline , at 6 months and at 12 months of both test sites ( sectg+ofd ) and control sites ( ofd ) ( mvsd in mm ) . in the test group , the mean ppd from baseline to 6 months was 3.89 0.23 and baseline to 12 months was 4.19 0.25 both values were statistically significant . in the test group , the mean ral from baseline to 6 months was 3.09 0.47 which was not significant but baseline to 12 months was 4.21 0.55 values were statistically significant . in the test group , the mean gml from baseline to 6 months was 0.44 0.15 and baseline to 12 months was 0.47 0.21 both values were statistically not significant . in control sites , ral and gml does not show any significant change from baseline to 12 months at test site whereas in control site gml showed negative values both at 6 and 12 months compared to baseline thus implying presence of post - operative recession as shown in table 3 . the mean bl values at test sites baseline to 6 months is 0.14 0.11 and baseline to 12 months is 0.16 0.11 which were not statistically significant . similarly at control sites , the baseline to 6 months is 0.39 0.11 and baseline to 12 months is 0.38 0.11 which were not statistically significant as shown in table 4 . radiographic assessment : bl = aclcej , acl and cej in mm at baseline , at 6 months and at 12 months of both test sites ( sectg+ofd ) and control sites ( ofd ) ( mvsd in mm ) . the full mouth mean pi and pbi scores decreased from baseline to 12 months follow - up . pi baseline 0.97 0.37 to 12 months 0.32 0.18 and pbi scores baseline 0.82 0.67 to 12 months score 0.20 0.16 , when the scores were compared statistically using paired t - test and the difference was statistically significant ( p > 0.05 ) in both the pi and pbi scores as shown in table 1 . ancillary clinical parameters : mean plaque pi and pbi scores at baseline , at 3 months , at 6 months and at 12 months ( mvsd ) . at baseline mean , ppd was 7.37 0.23 mm in the test group and 6.94 0.18 . mean ral at baseline in the test group was 15.91 0.45 and in control sites were 15.37 0.39 . the mean gml at baseline in the test group was 8.47 0.13 and control sites was 8.04 0.75 as shown in table 2 . clinical parameters : ppd , ral and gml at baseline , at 6 months and at 12 months of both test sites ( sectg+ofd ) and control sites ( ofd ) ( mvsd in mm ) . in the test group , the mean ppd from baseline to 6 months was 3.89 0.23 and baseline to 12 months was 4.19 0.25 both values were statistically significant . in the test group , the mean ral from baseline to 6 months was 3.09 0.47 which was not significant but baseline to 12 months was 4.21 0.55 values were statistically significant . in the test group , the mean gml from baseline to 6 months was 0.44 0.15 and baseline to 12 months was 0.47 0.21 both values were statistically not significant . in control sites , ral and gml does not show any significant change from baseline to 12 months at test site whereas in control site gml showed negative values both at 6 and 12 months compared to baseline thus implying presence of post - operative recession as shown in table 3 . comparative parameters from baseline to 6 months and baseline to 12 months . the mean bl values at test sites baseline to 6 months is 0.14 0.11 and baseline to 12 months is 0.16 0.11 which were not statistically significant . similarly at control sites , the baseline to 6 months is 0.39 0.11 and baseline to 12 months is 0.38 0.11 which were not statistically significant as shown in table 4 . radiographic assessment : bl = aclcej , acl and cej in mm at baseline , at 6 months and at 12 months of both test sites ( sectg+ofd ) and control sites ( ofd ) ( mvsd in mm ) . specific surgical approaches have been reported to obtain primary flap closure and to preserve interdental and marginal gingival tissue loss . this study was a longitudinal 12 months test conducted with a prospective controlled , randomized design . the intent of the study was to clinically evaluate that , if the use of sectg in conjunction with ofd versus the classically used approach of ofd in treatment of horizontal osseous defects , could lead to better clinical , and radiographic results ; i.e. greater ral gain , reduced ppd , decreased post - surgical gingival recession and supracrestal bone growth in the maxillary posterior , as well as the mandibular posterior region . at the baseline examination , all participants of the present study showed good oral hygiene levels and healthy gingival conditions throughout the entire study period of 12 months as indicated by ancillary clinical parameters of pi and pbi scores . there were no significant differences in pi and sulcular bleeding index observed between control and experimental sites at any point during the study , suggesting that the different amounts clinical attachment gain between control and experimental sites were a result of the different treatment modalities tested and therefore unrelated to the amount of gingival inflammation present during healing . results revealed that both surgical treatments ( ofd + sectg and ofd alone ) significantly improved the clinical parameters compared to the pre - surgical conditions ; however , the addition of sectg resulted in a significantly greater reduction in ppd and gain in ral and a significantly lower gingival recession or apical displacement of gml both at 6 months and 12 months post - surgically . the added benefit provided by sectg is suggested by the greater ral gain scores and the overall proportion of pockets undergoing complete closure ( significantly greater ) . autogenous gtr membrane in this case , possibly allowing periodontal ligament cells , osteoblasts , and cementoblasts to repopulate the apical part of the wound . the good results for ral improvement obtained by using a connective tissue graft could be explained by the fact that in addition to providing a space for regenerating cells , the connective tissue graft may have better supported and protected the wound of the surgical site during the healing period . a primary goal of periodontal therapy is to reduce ppd so as to limit the risk of local re - infection . shallow pockets display a highly negative predictive value for future disease amelioration , whereas deep pockets in treated areas are a risk indicator for periodontal disease progression.26 in the present clinical study with an initial pocket range of 4 - 7 mm . at 6 months sectg group showed ( 3.89 0.23 mm ) a greater mean ppd reduction compared with the ( 2.88 0.30 mm ) of ofd group . at 12 months period the mean ppd reductions observed in the present study for both groups are comparable to the results reported.8,27,28 most of the improvements in clinical parameters were achieved during first 6 months post - surgery , and to a lesser extent at 12 months from baseline . horizontal bone loss represents 92% of the total bone loss in periodontal patients thus far exceeding the intrabony defects,29,30 which however have ironically received much surgical and regenerative therapeutic interventions , which in part can be attributed to the their favorable morphologic characteristic resulting in resolution associated with these treatment modalities.31 in clinical practice , ofd represents the non - resective surgical treatment of choice for horizontal bone defects , however , numerous attempts have been made32 - 36 for resolution of the defects . the lack of significant supracrestal bone growth observed in this study is in agreement with the data from jentsch and purschwitz37 and yilmaz et al.38 this finding is anticipated if we take into consideration that , there is a paucity of available space under the gingival flap in supra - alveolar - type defects to allow new bone formation . the present results agree with those previously reported in literature which demonstrate the efficacy of both treatment approaches , though statistical differences have been found with respect to improvement of various parameters between both arms of the experimental groups , long - term studies need to be conducted to prove the added benefits using sectg in conjunction with ofd . within the confines of the present study , results revealed that the use of sectg along with ofd compared to ofd alone resulted in significantly higher ral gain and significantly lower post - surgical gingival recession . . therefore use of sectg along with ofd , followed with regular maintenance therapy , has clinically predictable results in the treatment of periodontal horizontal osseous defects . long - term clinical trials are warranted to further substantiate the efficacy of this technique .	background : an improvement in clinical parameters along with regeneration is the desired outcome of periodontal therapy . the aim of this study was to analyze and contrast the efficaciousness of combined open flap debridement ( ofd ) and subepithelial connective tissue graft ( sectg ) to ofd in the management of periodontal supracrestal defects.materials and methods : totally , 20 paired sites exhibiting supracrestal defects were subjected to surgical treatment adopting the split mouth design . the defects were divided randomly for treatment with ofd and sectg ( test ) or ofd alone ( control ) . the clinical effectiveness of the two arms of treatment was evaluated at 6 months and 12 months post - operatively by assessing clinical and radiographic parameters . the measurements carried out included probing pocket depth ( ppd ) , relative attachment level ( ral ) , gingival marginal level , radiographic bone level ( bl).results : the mean reduction in ppd at 0 - 12 months was 3.20 0.82 mm and ral gain of 3.10 1.51 mm was observed , the ofd and sectg ( test ) group ; corresponding observations for ofd ( control ) were 2.10 0.63 mm and 1.90 0.57 mm . however , bl changes did not follow the pattern of clinical improvement on the radiographic assessment of either treatment group . post - operative evaluation was made . improvement in different clinical parameters was statistically significant ( p < 0.01).conclusion : treatment of supracrestal defects with a combination of ofd and sectg led to significantly better clinical results compared to ofd alone .
You are an expert at summarizing long articles. Proceed to summarize the following text: in health or disease , it is difficult to evaluate the state and changes in vascular function in a non - invasive manner . one such way is to study the post - occlusive response to a brief brachial artery occlusion . another way is to study superficial nutritive capillaries in the dorsal skin of the arm by laser doppler methodology in combination with iontophoresis of acetylcholine ( ach ) or sodium nitroprusside ( snp ) . when ach is administered it stimulates via muscarinic receptors the formation of nitric oxide ( via nitric oxide synthase ) which in turn activates guanylate cyclase in the smooth muscle cells causing relaxation . snp is a nitric oxide donor that can bind directly with guanylate cyclase to cause relaxation . thus , the iontophoresis of these agents may provide information on the function of the endothelin and the vascular smooth muscle cells , respectively . here we use this methodology in order to obtain information on the function of the vascular system in refractory angina . enhanced external counterpulsation ( eecp ) is a non - invasive counter pulsation technique used for treatment of patients with chronic stable angina pectoris refractory as defined by the european society of cardiology joint study group . the patients do not respond to revascularization procedures such as percutaneous interventions ( pci ) and coronary artery bypass surgery ( cabg ) and have persistence of anginal symptoms despite aggressive medical treatment . the basic principle of eecp is the diastolic augmentation of arterial pressure and lowering of systolic arterial pressure along with increasing venous return . the hemodynamic effects of eecp are similar to intra - aortic balloon pumping but the treatment yields long - lasting increases in coronary blood flow . , eecp is used to treat cardiovascular disease in assisting cardiac pumping effort by augmentation of the blood pressure and blood flow during diastole , decreasing resistance to pulse pressure upon systole . this form of counterpulsation increases coronary perfusion after a series of eecp sessions . besides the acute hemodynamic effects , the long - term improvement in coronary artery disease ( cad ) symptoms have been characterised by reduced angina pectoris episodes and glyceryl trinitrate use , increased exercise tolerance , positive psychosocial effects and increased quality of life. however , the mechanisms responsible for the beneficial effects of eecp remain unknown . increased coronary blood flow with diastolic one study in closed - chest infarction model in dogs had revealed increased distribution of microvessels in the region of the heart by long - term eecp , thus supporting the hypothesis that eecp promotes collateral microvascular development . in addition , shear stress and neurohormonal changes may also play a role in the clinical improvements experienced by patients treated with eecp . enhanced blood flow across the endothelial cell lining of blood vessels increases endothelial shear stress , which enhances endothelial function by stimulating the release of the vasodilatory mediator nitric oxide and reducing the release of the vasocontractile peptide endothelin-1 . , , the improved perfusion by eecp might , in addition , be related to release of angiogenesis factors , such as vascular endothelial growth factors , basic fibroblast growth factor and hepatocyte growth factor . recently , it was shown that eecp improved brachial artery flow - mediated dilatation and increased nitric oxide turnover / production and 6-keto - prostaglandin f2 while decreasing endothelin-1 levels . the primary hypothesis of our study was to investigate first if the skin microvascular circulation is reduced in refractory angina pectoris patients , and secondly if the microvascular responses are modified by eecp treatment . a recent study on patients with canadian cardiovascular society ( ccs)-class iii showed improved responses of the brachial artery flow - mediated dilatation . in the current study , we aim to test effects in the peripheral microcirculation in patients exhibiting somewhat worse symptoms ( ccs - class iii / iv ) . the study was performed at two departments : enrollment and implementation of eecp therapy was done at the department of medicine at the central hospital in kristianstad , kristianstad , sweden ; and the blood flow measurements were made at the clinical research unit , eb block at lund university hospital , lund , sweden . the study involved 20 patients ( 17 male , 3 female , 4883 years of age ) diagnosed with stable refractory angina pectoris and ccs - class iii despite optimized medical ( pharmacological ) and invasive therapy . medical therapies include the maximal tolerated use of anti - angina medications ( long and short - acting nitrates , -adrenoceptor antagonists or calcium channel blockers ) . the patients were referred for eecp therapy after a specialist cardiologist round , due to presence of angina pectoris refractory to a combination of medical treatment and further revascularization procedures such as cabg or pci . all patients had angiographically stated coronary stenosis ( > 50 % ) involving at least one major coronary artery . subjects with unstable angina , acute myocardial infarction less than three months , systemic hypertension more than 180/100 mmhg , permanent pacemaker that would interfere with eecp triggering , clinically evident peripheral vascular disease , deep vein thrombosis , phlebitis or abdominal aortic aneurysm or skin ulcers were excluded from the study . the study was non - blinded on two arms : all patients with refractory angina pectoris waiting for eecp treatment were assigned to enter the eecp group or a second group which served as controls and then given eecp at the conclusion of the study . assignments were randomly made by a coordinator at the department of medicine at the central hospital in kristianstad . the investigators of the study obtained medical history , ccs angina level coronary disease status and medication from the patients when entering the study . laser doppler iontophoresis and blood flowmetry ( except blood samples and ccs classification ) were performed by the same investigator in a standardized manner with the patient relaxing in supine position in an examining room after a rest period of 10 min . data were collected and analyzed at the first visit for both groups ( eecp and no eecp ) . data were then collected after a full 35-hour course of treatment in the eecp group or after a 7-week period in the no eecp group . in the eecp group , treatment was carried out within one week after inclusion in the study . in the other group , the eecp treatment was performed as soon as the second measurement , 7 weeks from baseline , was implemented . if any of the patients in the no eecp group showed symptoms of increased degree of angina pectoris during the study period , they were excluded from the study and immediately treated with eecp . the data were compared to a group of age matched healthy controls ( n = 20 ) : female 12 , male 8 and 3977 years of age randomly drawn from the regional population register and invited to participate in the study . blood pressure was measured non - invasively in supine position from the upper left arm with the cuff inflated at heart level . the diastolic value was accepted as korotoff 's phase v. the blood pressure was measured by the same investigator three times during visit 1 and visit 2 . arterial blood pressure ( systolic and diastolic ) and heart rate were measured at baseline with follow - up after eecp treatment or after a 7 week period in the no eecp group . blood samples were taken and analyzed at the department of clinical chemistry , central hospital of kristianstad , kristianstad , sweden or for interleukins at the clinical immunology laboratory at lund university hospital , lund , sweden . the periflux system ( perimed , jrflla , sweden ) was used to measure cutaneous blood flow . with this non - invasive system , the light reflects moving blood cells in the superficial cutaneous microvessels and undergoes a shift in frequency that is proportional to the number and velocity of the moving blood cells . the doppler effect is then used by the system in order to estimate blood flow . the patient rested at least 10 minutes before commencing with ambient room temperature of 22c23c ( remained constant throughout the study ) . the laser - doppler probe and the drug delivery systems were attached to the forearm . the chamber was filled with ach ( 2% dissolved in milliq water ; sigma , st . skin temperature was noted and basal blood flow was registered for two minutes . following this , ach was injected by iontophoresis ( 0.2 ma at the anode for one minute ) . louise ; mo ) was applied to a new chamber ( 0.1 ma at the anode ) . the current was switched on for one minute to a total of four periods with one minute rest . a probe was heated to 44 c and the increase in blood flow was registered for 10 min , in order to determine the maximum vasodilation possible under these experimental conditions . , patients refrained from long lasting nitrates , caffeine and smoking for six hours before the blood flow measurements . tby , sweden ) is based on the concept of counterpulsation and consists of three pairs of inflatable cuffs applied to the lower extremities . the eecp therapist sets cuff inflation and deflation timing to the cardiac cycle escorted by the finger plethysmogram waveform . cuffs are timed to inflate sequentially ( applying 260 mmhg of external pressure ) just after the onset of diastole , and then deflate simultaneously prior to the onset of systole . the instan taneous and simultaneous deflation of cuffs during systole enhances systolic unloading and reduces cardiac workload by reducing peripheral vascular resistance . group data is presented as mean se and 95 % confidence intervals ( ci ) were calculated . fisher 's exact test or the chi(2 ) test was used when comparing baseline characteristics between eecp patients and no eecp patients . differences between clinical characteristics , blood samples and blood flow responses were evaluated and analyzed by unpaired t test for two group comparisons and one - way analysis of variance ( anova ) , dunn 's post hoc test was used when comparing more than two groups . the study involved 20 patients ( 17 male , 3 female , 4883 years of age ) diagnosed with stable refractory angina pectoris and ccs - class iii despite optimized medical ( pharmacological ) and invasive therapy . medical therapies include the maximal tolerated use of anti - angina medications ( long and short - acting nitrates , -adrenoceptor antagonists or calcium channel blockers ) . the patients were referred for eecp therapy after a specialist cardiologist round , due to presence of angina pectoris refractory to a combination of medical treatment and further revascularization procedures such as cabg or pci . all patients had angiographically stated coronary stenosis ( > 50 % ) involving at least one major coronary artery . subjects with unstable angina , acute myocardial infarction less than three months , systemic hypertension more than 180/100 mmhg , permanent pacemaker that would interfere with eecp triggering , clinically evident peripheral vascular disease , deep vein thrombosis , phlebitis or abdominal aortic aneurysm or skin ulcers were excluded from the study . the study was non - blinded on two arms : all patients with refractory angina pectoris waiting for eecp treatment were assigned to enter the eecp group or a second group which served as controls and then given eecp at the conclusion of the study . assignments were randomly made by a coordinator at the department of medicine at the central hospital in kristianstad . the investigators of the study obtained medical history , ccs angina level coronary disease status and medication from the patients when entering the study . laser doppler iontophoresis and blood flowmetry ( except blood samples and ccs classification ) were performed by the same investigator in a standardized manner with the patient relaxing in supine position in an examining room after a rest period of 10 min . data were collected and analyzed at the first visit for both groups ( eecp and no eecp ) . data were then collected after a full 35-hour course of treatment in the eecp group or after a 7-week period in the no eecp group . in the eecp group , treatment was carried out within one week after inclusion in the study . in the other group , the eecp treatment was performed as soon as the second measurement , 7 weeks from baseline , was implemented . if any of the patients in the no eecp group showed symptoms of increased degree of angina pectoris during the study period , they were excluded from the study and immediately treated with eecp . the data were compared to a group of age matched healthy controls ( n = 20 ) : female 12 , male 8 and 3977 years of age randomly drawn from the regional population register and invited to participate in the study . blood pressure was measured non - invasively in supine position from the upper left arm with the cuff inflated at heart level . the diastolic value was accepted as korotoff 's phase v. the blood pressure was measured by the same investigator three times during visit 1 and visit 2 . arterial blood pressure ( systolic and diastolic ) and heart rate were measured at baseline with follow - up after eecp treatment or after a 7 week period in the no eecp group . blood samples were taken and analyzed at the department of clinical chemistry , central hospital of kristianstad , kristianstad , sweden or for interleukins at the clinical immunology laboratory at lund university hospital , lund , sweden . the periflux system ( perimed , jrflla , sweden ) was used to measure cutaneous blood flow . with this non - invasive system , the light reflects moving blood cells in the superficial cutaneous microvessels and undergoes a shift in frequency that is proportional to the number and velocity of the moving blood cells . the doppler effect is then used by the system in order to estimate blood flow . the patient rested at least 10 minutes before commencing with ambient room temperature of 22c23c ( remained constant throughout the study ) . the laser - doppler probe and the drug delivery systems were attached to the forearm . the chamber was filled with ach ( 2% dissolved in milliq water ; sigma , st . skin temperature was noted and basal blood flow was registered for two minutes . following this , ach was injected by iontophoresis ( 0.2 ma at the anode for one minute ) . louise ; mo ) was applied to a new chamber ( 0.1 ma at the anode ) . the current was switched on for one minute to a total of four periods with one minute rest . a probe was heated to 44 c and the increase in blood flow was registered for 10 min , in order to determine the maximum vasodilation possible under these experimental conditions . , patients refrained from long lasting nitrates , caffeine and smoking for six hours before the blood flow measurements . tby , sweden ) is based on the concept of counterpulsation and consists of three pairs of inflatable cuffs applied to the lower extremities . the eecp therapist sets cuff inflation and deflation timing to the cardiac cycle escorted by the finger plethysmogram waveform . cuffs are timed to inflate sequentially ( applying 260 mmhg of external pressure ) just after the onset of diastole , and then deflate simultaneously prior to the onset of systole . the instan taneous and simultaneous deflation of cuffs during systole enhances systolic unloading and reduces cardiac workload by reducing peripheral vascular resistance . group data is presented as mean se and 95 % confidence intervals ( ci ) were calculated . fisher 's exact test or the chi(2 ) test was used when comparing baseline characteristics between eecp patients and no eecp patients . differences between clinical characteristics , blood samples and blood flow responses were evaluated and analyzed by unpaired t test for two group comparisons and one - way analysis of variance ( anova ) , dunn 's post hoc test was used when comparing more than two groups . all patients in the eecp group and in the no eecp group completed the study regime , and there were no adverse cardiovascular events . none of the patients in the group of refractory angina pectoris but no eecp were excluded due to worsened ccs - class . eecp : enhanced external counterpulsation ; cad : coronary artery disease ; raas - blockade : renin - angiotensin - aldosterone system blockade ; * p < 0.05 . eecp : enhanced external counterpulsation ; bmi : body mass index ( weight ( kg)/heigh ( m ) ) ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; bnp : brain natriuretic peptide ; crp : sensitive c reactive protein ; hba1c : blood glucose levels in glycated hemoglobin a1c ; tnt : troponin t ; hdl : high density lipoprotein ; ldl : low density lipoprotein ; apoa : apolipoprotein a1 ; apob : apolipoprotein b ; il-6 : interleukin 6 ; sil-2r : soluble interleukin 2 receptor . * p < 0.05 , eecp vs no eecp , p < 0.05 , before eecp vs after eecp . patients treated with eecp showed a significant reduction in ccs - class , compared to ccs - class at baseline ( p < 0.05 ) . this was not seen in the no eecp group ( p = ns , figure 1 ) . systolic blood pressure ( sbp ) did change in the studied refractory groups . in the eecp group the sbp decreased from 131 16 mmhg to 123 13 mmhg after treatment ( p < 0.05 ) . there was also a decrease in sbp in the no eecp group ( 138 17 to 130 20 mmhg , p < 0.05 ) . patients in the group not receiving eecp had a significantly higher level of blood interleukin-2 receptor ( sil-2r ) concentration at the second measurement compared to the eecp treated patients ( p < 0.05 ) . in the no eecp group , sil-2r was significantly increased from baseline 455 185 to 638 282 ku / l ( kilo units/ litre ) at the 7 weeks of follow - up ( p < 0.05 ) . in contrast to the no eecp group , sil-2r decreased after eecp treatment from 786 603 ku / l to 608 261 ku / l ( p < 0.05 ) . six patients treated with eecp corresponded positively in reducing the plasma level of sil-2 receptor at the second visit . one patient reduced one ccs - class but increased the concentration of sil-2r in the blood . another patient increased both the ccs - classification and the amount of sil-2r in the blood sample at the second measurement . finally , two patients had no relief in the angina pectoris frequency but decreased the level of blood sil-2r concentration at visit 2 . remaining blood samples did not differ between the two studied groups of patients ( table 2 ) . the laser doppler flow method is semi - quantitative and the data is presented as percentage change compared with the baseline perfusion value ( pu ) . there was no difference in resting blood flow between the two groups of refractory angina patients ( eecp and non eecp ) at baseline during visit 1 as well as after eecp treatment , respectively at 7 weeks of follow - up ( visit 2 ) , or compared to that of no eecp . administration of ach or snp resulted in a local increase in pu that amounted to 1150% and 725% in healthy controls , respectively . these values agree well with our previous experiences . in the two groups of refractory angina pectoris , these responses were significantly lower , 694% and 486% , respectively , as compared to healthy controls ( p < 0.05 for both ) . after this one group of patients got 35 treatments with eecp while the other group was only monitored . the eecp treatment did not alter the cutaneous blood flow responses to ach or snp in refractory angina pectoris . at the first examination of the microvascular blood flow , responses in the two angina pectoris groups were significantly lower compared to the response seen in the healthy controls ( p < 0.05 ) . the response to the local heating ( + 44c ) in the two groups of refractory angina patients was also lower compared to that of the healthy subjects ( p < 0.05 , figure 2 ) . patients treated with eecp showed a significant reduction in ccs - class , compared to ccs - class at baseline ( p < 0.05 ) . this was not seen in the no eecp group ( p = ns , figure 1 ) . systolic blood pressure ( sbp ) did change in the studied refractory groups . in the eecp group the sbp decreased from 131 16 mmhg to 123 13 mmhg after treatment ( p < 0.05 ) . there was also a decrease in sbp in the no eecp group ( 138 17 to 130 20 mmhg , p < 0.05 ) . patients in the group not receiving eecp had a significantly higher level of blood interleukin-2 receptor ( sil-2r ) concentration at the second measurement compared to the eecp treated patients ( p < 0.05 ) . in the no eecp group , sil-2r was significantly increased from baseline 455 185 to 638 282 ku / l ( kilo units/ litre ) at the 7 weeks of follow - up ( p < 0.05 ) . in contrast to the no eecp group , sil-2r decreased after eecp treatment from 786 603 ku / l to 608 261 ku / l ( p < 0.05 ) . six patients treated with eecp corresponded positively in reducing the plasma level of sil-2 receptor at the second visit . one patient reduced one ccs - class but increased the concentration of sil-2r in the blood . another patient increased both the ccs - classification and the amount of sil-2r in the blood sample at the second measurement . finally , two patients had no relief in the angina pectoris frequency but decreased the level of blood sil-2r concentration at visit 2 . remaining blood samples did not differ between the two studied groups of patients ( table 2 ) . the laser doppler flow method is semi - quantitative and the data is presented as percentage change compared with the baseline perfusion value ( pu ) . there was no difference in resting blood flow between the two groups of refractory angina patients ( eecp and non eecp ) at baseline during visit 1 as well as after eecp treatment , respectively at 7 weeks of follow - up ( visit 2 ) , or compared to that of no eecp . administration of ach or snp resulted in a local increase in pu that amounted to 1150% and 725% in healthy controls , respectively . these values agree well with our previous experiences . in the two groups of refractory angina pectoris , these responses were significantly lower , 694% and 486% , respectively , as compared to healthy controls ( p < 0.05 for both ) . after this one group of patients got 35 treatments with eecp while the other group was only monitored . the eecp treatment did not alter the cutaneous blood flow responses to ach or snp in refractory angina pectoris . at the first examination of the microvascular blood flow , responses in the two angina pectoris groups were significantly lower compared to the response seen in the healthy controls ( p < 0.05 ) . the response to the local heating ( + 44c ) in the two groups of refractory angina patients was also lower compared to that of the healthy subjects ( p < 0.05 , figure 2 ) . the present study is the first to show that the microvascular dilator responses to cutaneous ach , snp and local heating are all reduced in patients with refractory angina pectoris compared to age matched healthy controls . this suggests a general reduction in the vascular reactivity of the microvasculature in refractory angina pectoris . other studies of man have revealed reduction in cutaneous vessel responses in hypertension , in aging , and in congestive heart failure . thus , the present method is well suited to monitor alterations in the peripheral vascular activity in one type of peripheral vascular beds . interestingly , therapy resulting in better status of congestive heart failure patients was found to improve the vascular responses to ach and snp . here we found that although eecp results in significantly reduced ccs - class , there was no improvement in microvascular responses . in support , a previous study has examined the skin microcirculation of healthy subjects compared to older patients with cad before and after one hour of eecp . reportedly the peripheral circulation was improved with superficial skin blood flow , transdermal oxygen and carbon dioxide pressure . our results suggest that the vasodilator capacity in patients with refractory angina pectoris is associated with reduced responsiveness in the peripheral microcirculation but the better status was not within the relatively short time period of up to three months not associated with better skin blood flow reactivity . although the cad might be associated with a general reduction in the circulation recent data suggest that brachial artery flow - mediated dilatation is improved by eecp . this would suggest that eecp has effects primarily on the more central parts of the circulation . flow - mediated dilation in the brachial artery is an often used method for assessing endothelial function in humans . however , there is controversy to whether endothelial function in the brachial artery is a manifestation of the status of coronary microcirculation . , considering the differences in the microvascular architecture , blood flow pattern and metabolic regulation between peripheral and coronary circulation , it could be assumed that reduced ccs - class related to eecp treatment is not associated with an improvement in responsiveness of the cutaneous micro circulation but coronary endothelial function . further , we can not establish that the vasodilator response to ach , snp and heating is an index of coronary microcirculation . the results of this study confirm that eecp treatment improves the ccs - class in patients with refractory angina pectoris . this is in accordance with previous studies.,,, in addition , the current study compared treated patients with a parallel group of matched subjects with refractory angina pectoris waiting for eecp treatment . a significant reduction in angina pectoris was measured by ccs - class in the eecp group compared to the no eecp group . the possible mechanisms responsible for the improvement of the angina status include enhancement of the endothelial function , promotion of collateral circulation and improved ventricular function . the consequences of these mechanisms are a reduction in ischemia , which produce pain relief . this is thought to activate factors that modify endothelial functions , which may be potent activators of angiogenesis , such as endothelial growth factors . however , we can not confirm that the endothelial function was improved , at least not in the cutaneous circulation . eecp did not improve the peripheral cutaneous reactivity to the endothelial vasodilator ach or the smooth muscle cell specific dilator , snp . in this study , both these mechanisms were shown to be reduced in refractory angina pectoris patients as compared to age matched healthy controls and patients not modified by eecp . hilz et al . reported enhanced skin oxygenation on carbon dioxide clearance during eecp treatment , as an acute effect following just one brief session of eecp for five minutes . a typical course of eecp therapy treatment ( as in our case ) , consists of 35 hours in total . , hashemi et al . investigated the long - term effect of eecp on endothelium - dependent vasorelaxation in patients with refractory angina pectoris by noninvasive , high - resolution ultrasound scans . their data suggest improvement in endothelial function , as a mechanism underlying the clinical benefit associated with eecp . this study did not measure symptomatic response to eecp and could not explain long - term clinical benefits of eecp due to improvement in endothelial function . this pharmacological treatment has shown cardio protective and symptom relieving effects , it was not ethical to discontinue their medical therapy in order to exclude possible interactions with the specific therapy , eecp . the medical therapy might somehow explain the reduced cutaneous blood flow response to ach or snp in refractory angina pectoris patients undergoing eecp treatment . furthermore , the presence of a matched group of patients not receiving eecp treatment supports our findings . the study by braith and colleagues showed that eecp improved responses to some vasodilators and reduced vasoconstrictor responses such as to that of endothelin-1 . we did not study these but the parameters that were comparable are in agreement ( table 2 ) . on the other hand , there was in the present study a reduction in sil-2 receptor level after eecp compared to its matched no eecp group after 3 months of only symptomatic treatment where the sil-2 receptor level was significantly increased . this result is in accordance with earlier studies investigating the effects of eecp on circulating levels of inflammatory biomarkers in patients with angina pectoris . experimental evidence suggests that shear stress in high volume along the blood vessel wall affect positively on proinflammatory cytokines . a study by kasapis and thompson showed that regular exercise yielded a long - term anti - inflammatory effect by improved endothelial function and reduced inflammatory markers . in a manner similar to regular physical exercise , eecp is able to increase cardiac output and blood flow . shephard et al . suggested that moderate training reduce the exercise - induced suppression of il-2 production . this study demonstrates that eecp is a treatment corresponding positively in reducing the plasma level of sil-2 receptor . this is in agreement to a study on congestive heart failure with inflammation and b12 deficiency . treatment with b12 supple mentation normalized homocysteine , reduced inflam matory markers and improved the condition of the vascular reactivity . the assignments were made by a coordinator which was not involved in the further contact with the included patients . thus , the control group was from a statistical point of view not different from the eecp group ( p > 0.05 ) . however the results indicate a general reduction in the vascular reactivity of the microvasculature in both groups of patients with refractory angina pectoris . thus , the lower reactivity may partly be caused by gender factors , however previous data would suggest that this is not the case . the results demonstrate that eecp is more effective in decreasing angina measured by ccs - class as compared with refractory patients only receiving pharmacological treatment . a direct comparison in a double - blinded the study was conducted on patients with severe angina pectoris and they were all considered not suitable for any more invasive investigations or surgery . the laser doppler , a non - invasive system in combination with iontophoresis of ach or snp was used in order to investigate the cutaneous microcirculation . the decreased angina measured by ccs - class in patients with refractory angina pectoris undergoing eecp was not accompanied by changes in the cutaneous microvasculature . the improved response of eecp in anginal status may therefore be limited to the coronary arteries . in year 1992 , stsch et al . demonstrated that the abnormal response of the microvasculature dilator stimulus in patients with microvascular angina was limited to the coronary arteries ( coronary sinus blood flow was measured by the coronary sinus thermodilulion technique ) and did not include the skin vessels . on the other hand , sax et al . used plethysmographic assessments of the forearm and showed that peripheral vascular responses correlated with those in the coronary circulation in patients with microvascular angina . further more , ijzerman et al . reported that increased coronary heart disease score was associated with impaired skin microvascular responses , both endothelium - dependent and endothelium - independent . which of the mechanism that is responsible for the improved anginal status measured by ccs - class in patients with refractory angina undergoing eecp can not be answered by the present study . taken together , refractory angina pectoris is associated with reduced microvascular responsiveness to endothelial and smooth muscle stimulants , e.g. , to ach and snp , as well as to local heating . however , eecp did not improve these responses compared to a control group of patients . together with other studies it is suggested that treatment with eecp may initially mainly improve the coronary circulation . the assignments were made by a coordinator which was not involved in the further contact with the included patients . thus , the control group was from a statistical point of view not different from the eecp group ( p > 0.05 ) . however the results indicate a general reduction in the vascular reactivity of the microvasculature in both groups of patients with refractory angina pectoris . thus , the lower reactivity may partly be caused by gender factors , however previous data would suggest that this is not the case . the results demonstrate that eecp is more effective in decreasing angina measured by ccs - class as compared with refractory patients only receiving pharmacological treatment . a direct comparison in a double - blinded , randomized manner is , however , required to verify this suggestion . the study was conducted on patients with severe angina pectoris and they were all considered not suitable for any more invasive investigations or surgery . the laser doppler , a non - invasive system in combination with iontophoresis of ach or snp was used in order to investigate the cutaneous microcirculation . the decreased angina measured by ccs - class in patients with refractory angina pectoris undergoing eecp the improved response of eecp in anginal status may therefore be limited to the coronary arteries . in year 1992 , stsch et al . demonstrated that the abnormal response of the microvasculature dilator stimulus in patients with microvascular angina was limited to the coronary arteries ( coronary sinus blood flow was measured by the coronary sinus thermodilulion technique ) and did not include the skin vessels . on the other hand , sax et al . used plethysmographic assessments of the forearm and showed that peripheral vascular responses correlated with those in the coronary circulation in patients with microvascular angina . further more , ijzerman et al . reported that increased coronary heart disease score was associated with impaired skin microvascular responses , both endothelium - dependent and endothelium - independent . which of the mechanism that is responsible for the improved anginal status measured by ccs - class in patients with refractory angina undergoing eecp can not be answered by the present study . taken together , refractory angina pectoris is associated with reduced microvascular responsiveness to endothelial and smooth muscle stimulants , e.g. , to ach and snp , as well as to local heating . however , eecp did not improve these responses compared to a control group of patients . together with other studies it is suggested that treatment with eecp may initially mainly improve the coronary circulation .	aimsto examine if the skin microvascular bed is altered and can be modified by enhanced external counterpulsation ( eecp ) in patients with chronic refractory angina.methodstwenty patients diagnosed with refractory angina were divided into eecp ( n = 10 ) or no eecp ( n = 10 ) groups . the data were compared to matched healthy subjects ( n = 20 ) . the cutaneous forearm microvascular blood flow was measured by laser - doppler flowmetry . the vascular responsiveness to iontophoretic administration of acetylcholine ( ach ) , sodium nitroprusside ( snp ) and local skin warming were studied . measurements of canadian cardiovascular society ( ccs)-class , blood pressure and plasma samples were registered.resultseecp patients showed reduced ccs - class compared to no eecp ( p < 0.05 ) . both eecp and no eecp ( p < 0.05 ) groups had decreased systolic blood pressure ( sbp ) as compared to sbp at baseline ( p < 0.05 ) . there was no difference in resting blood flow between the two refractory groups at baseline as well as after eecp and seven weeks of follow - up . responses to heating , the responses to ach and snp in the cutaneous microcirculation were lower in both groups of refractory angina patients as compared to healthy subjects ( p < 0.05 ) . eecp patients corresponded positively to the treatment shown by reduced plasma level of soluble interleukin-2 receptor and ccs-class.conclusionsrefractory angina patients have reduced responsiveness in their cutaneous microcirculation to ach , snp and heat compared to healthy subjects . although eecp reduced the ccs - class , this effect was not associated with improvements in responsiveness of the cutaneous microcirculation .
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Proceed to summarize the following text: the cerebellar ataxias are a highly heterogeneous group of neurological disorders that impinge on the cerebellum and its afferent and efferent connections . patients affected by cerebellar ataxia suffer from poor coordination of movements , imbalance , and a wide - based unsteady gait . moreover , patients often present with additional extra - cerebellar signs including marked cognitive dysfunction . ataxias are rare diseases with a varying prevalence between 1 and 40:100,000 [ 1 , 2 ] . the ataxias are broadly classified into hereditary ataxias , nonhereditary degenerative ataxias , and acquired ataxias [ 3 , 4 ] . the hereditary ataxias comprise a growing list of genetically diverse disorders with over 43 genetic loci known to cause the autosomal dominant ataxias and altogether more than 100 genes that primarily cause ataxia when mutated [ 13 ] . however , there are still several clinically distinct forms of hereditary ataxias for which the disease - causing mutations have not been identified , and additional genes underlying cerebellar ataxia remain to be discovered . the vast number of genes implicated in ataxia suggests that multiple pathogenic pathways can induce cerebellar dysfunction and atrophy . indeed , the pathogenesis of cerebellar ataxia appears to be highly heterogeneous , and a multitude of potential disease mechanisms have been proposed . many of these are not ataxia - specific but common disease features of neurodegenerative disorders in general , such as oxidative stress , protein aggregation , and transcriptional dysregulation [ 3 , 5 ] . the challenge remains to better understand the specific disease - causing mechanisms underlying the complex cerebellar ataxias and to identify common pathological pathways that could be targeted therapeutically . animal models provide invaluable insights into the normal function of the cerebellum and into the pathogenic mechanisms contributing to cerebellar ataxia . a large number of mouse mutants exhibiting a cerebellar ataxia phenotype have been reported ( reviewed in [ 6 , 7 ] ) . historically , the first ataxic mouse models that were intensively investigated were naturally occurring mutants , including the lurcher ( gain - of - function mutation in grid2 ) , staggerer ( rora deletion ) , and weaver mouse ( gain - of - function mutation in girk2 ) . for some of these spontaneous mutants , a direct clinical relevance was demonstrated only years after their initial discovery . for example , the mutation in the staggerer mouse was first observed at the jackson laboratory in 1955 and later identified as a null allele of the retinoid acid - related orphan nuclear receptor - alpha ( ror ) . more than 40 years after the initial description of the staggerer mouse , expression profiling studies suggested a mechanistic link between ror and spinocerebellar ataxia ( sca ) type 1 ( sca1 ) [ 9 , 10 ] . subsequently , ror was shown to physically interact in a regulatory transcriptional complex with atxn1 , the mutated protein in sca1 . similarly , it took 59 years after the initial discovery of the lurcher mutant , until the identification of the first human mutations in grid2 in patients with cerebellar ataxia [ 12 , 13 ] . these findings underscore the often tedious search for human genes implicated in the rare cerebellar ataxias , but also the power of ataxic mouse mutants in dissecting the disease - relevant molecular mechanisms of cerebellar dysfunction . in addition to the spontaneous mutants , many transgenic , knockout , and knockin mouse lines have been engineered for the human ataxias with varying degrees of success . some of these mutants , particularly the generated mouse models of the trinucleotide - repeat scas , phenocopy the human condition reasonably well and have been valuable tools in analyzing the underlying molecular pathogenesis and discovering potential new therapeutics for cerebellar ataxia . genome - wide random mouse mutagenesis represents a powerful complementary approach to gene knockouts and transgenics for the discovery of novel genes and allelic gene series with functional implications for the central nervous system [ 1517 ] . this phenotype - driven approach has been particularly successful in identifying cerebellar mutants , partly due to the relatively easy detection of motor phenotypes in the mouse . as part of a large n - ethyl - n - nitrosurea ( enu ) mutagenesis screen to identify new models of human cerebellar ataxia , we identified the dominantly inherited moonwalker ( mwk ) mouse . here , i review the genetic and functional characterization of the mwk mouse , which has provided new insights into the role of the transient receptor potential ( trp ) channel trpc3 in cerebellar development and disease [ 18 , 19 ] . i also examine the relationship of the mwk mutant to other mouse models of cerebellar ataxia and its relevance for the human disorder . mwk mice harbor a single non - synonymous point mutation in exon 7 of the trpc3 gene located on chromosome 3 . this gene encodes the trpc3 channel , a member of the transient receptor potential ( trp ) family of ion channels . the trp superfamily constitutes one of the largest ion channel families and is involved in numerous physiological functions [ 20 , 21 ] . the mammalian trp channel proteins form six - transmembrane cation - permeable channels that are grouped into six subfamilies including the canonical trpc family ( trpc 17 ) . trpc channels are widely expressed in different mouse tissues including the nervous system , where they function as non - selective calcium entry channels with distinct modes of activation [ 22 , 23 ] . microarray analysis of more than 80 different mouse cell lines and tissues shows that trpc3 is highly expressed in the nervous system , particularly in the cerebellum ( biogps gene portal ) ( http://biogps.org ) . notably , it is the alternatively spliced short isoform trpc3c that is preferentially expressed in the cerebellum . 1b ) , which is known to modulate trpc channel activity via competitive binding of the inositol triphosphate ( ip3 ) receptor ( ip3r ) and calmodulin ( cam ) , resulting in a channel with enhanced efficacy due to increased channel - opening frequency . within the cerebellum , trpc3 is most highly expressed on the soma as well as the dendrites of purkinje cells [ 25 , 26 ] ( fig . in fact , trpc3 was shown to be the most abundant trp channel in purkinje cells . in addition to purkinje cells , trpc3 was recently found to be selectively expressed in type ii unipolar brush cells ( ubcs ) , which are excitatory glutamatergic interneurons in the cerebellum ( fig . have demonstrated that trpc3 mediates metabotropic glutamate receptor subtype 1 ( mglur1)-dependent synaptic transmission in purkinje cells . moreover , trpc3 was recently suggested to be essential for the induction of long - term depression ( ltd ) in cerebellar purkinje cells . however , how loss of synaptic function in the trpc3 mouse mutants affects ltd remains to be determined.fig . in addition , intense labeling of unipolar brush cells ( ubcs ) in lobules ix and x is apparent . in situ hybridization image trpc3 contains four ankyrin - like repeats at the cytoplasmic n terminus , an n - terminal coiled coil region and a hydrophobic region , which does not span the plasma membrane ( pm ) , six transmembrane regions , a pore region , a characteristic trp domain in the cytoplasmic c - terminal region , followed by a coiled coil and truncated calmodulin and ip3r1-binding domain ( cirb ) . the localization of the mwk mutation ( t635a ) in the cytoplasmic s4s5 linker region is indicated . multiple sequence alignment between mouse mtrpc3 , human htrpc3 , and the related mouse mtrpc6 and mtrpc7 s4s5 linker shows a high degree of sequence conservation a trpc3 mrna expression in the adult cerebellum . in addition , intense labeling of unipolar brush cells ( ubcs ) in lobules ix and x is apparent . in situ hybridization image trpc3 contains four ankyrin - like repeats at the cytoplasmic n terminus , an n - terminal coiled coil region and a hydrophobic region , which does not span the plasma membrane ( pm ) , six transmembrane regions , a pore region , a characteristic trp domain in the cytoplasmic c - terminal region , followed by a coiled coil and truncated calmodulin and ip3r1-binding domain ( cirb ) . the localization of the mwk mutation ( t635a ) in the cytoplasmic s4s5 linker region is indicated . multiple sequence alignment between mouse mtrpc3 , human htrpc3 , and the related mouse mtrpc6 and mtrpc7 s4s5 linker shows a high degree of sequence conservation the mwk mutation results in a threonine - to - alanine amino acid change ( t635a ; refseq # np_062383)1 in the highly conserved cytoplasmic s4s5 linker region of the trpc3 protein ( fig . 1b ) . the mwk mutation does not alter the normal expression pattern of trpc3 . however , electrophysiological recordings from mwk purkinje cells revealed that mutant trpc3 exhibits altered gating properties that promote channel opening under conditions of low mglur1 activation . consistently , mwk but not wild - type trpc3 promotes increased calcium signaling and cell death upon overexpression in neuronal cell lines ( unpublished observations ) . the molecular mechanisms of how the mwk mutation leads to abnormal trpc3 channel gating remain to be fully elucidated . one possible mechanism might be the loss of an inhibitory phosphorylation by protein kinase c ( pkc ) . pkc has been shown to inhibit trpc3 channel activity in overexpression experiments in heterologous cell lines [ 2831 ] . indeed , threonine 635 , which is mutated in the mwk mouse , has been shown to be phosphorylated by pkc in an in vitro kinase assay . however , this inhibitory phosphorylation event has not yet been confirmed in vivo . moreover , nelson and glitsch have argued that native trpc3-dependent currents elicited in cerebellar purkinje cells are unlikely to be targets of conventional pkc or pkg kinases in contrast to the findings observed in overexpression experiments . alternatively , the mwk mutation in the s4s5 linker might disturb intramolecular interactions within trpc3 that are important for the gating mechanism of the channel . this mechanism might be similar to the established electromechanical coupling in the structurally related voltage - gated potassium channels , in which the s4s5 linker is critical for gating . recently , the s4s5 linker in trpc4 and trpc5 channels was identified as critical constituent of trpc4/5 channel gating , suggesting a similar key function for the s4s5 linker in trpc3 . future structural studies on trpc3 and related trp channels will undoubtedly shed more light on the intramolecular interactions that are crucial for trpc3 channel activation and function . from early postnatal days , mwk mouse mutants display growth retardation and remain about 60 % the size of their wild - type littermates throughout life . heterozygous mwk mouse mutants display motor and coordination defects from about 3 weeks of age ( fig . 2 ) . specifically , mwk mice exhibit retropulsion and a wider and shuffling gait compared to wild - type littermates . mwk mice are also severely impaired in their ability to maintain their balance in the static rod test . following their initial behavioral characterization , mwk mice are being extensively characterized as part of the europhenome project . in this large - scale phenotyping effort , mwk mutants were found to display significant hyperactivity , tail elevation , and hind limb grasping behavior , indicative of extra - cerebellar signs . show trpc3 expression at p8 , p13 , p18 , and p56 . developing mouse sections are more laterally compared to the midline section shown for the adult . importantly , dendritic abnormalities , purkinje cell ( pc ) dysfunction , and overt ataxia in the mwk mouse appear prior to the onset of pc loss . most of the unipolar brush cells ( ubcs ) are lost by 1 month of age ; it remains to be determined when the loss of these neurons starts . m months , mwk moonwalker mouse , p postnatal day , pc purkinje cell , ubc unipolar brush cell , wt wild - type progression of the mwk phenotype from the early postnatal period until adulthood . show trpc3 expression at p8 , p13 , p18 , and p56 . developing mouse sections are more laterally compared to the midline section shown for the adult . importantly , dendritic abnormalities , purkinje cell ( pc ) dysfunction , and overt ataxia in the mwk mouse appear prior to the onset of pc loss . most of the unipolar brush cells ( ubcs ) are lost by 1 month of age ; it remains to be determined when the loss of these neurons starts . m months , mwk moonwalker mouse , p postnatal day , pc purkinje cell , ubc unipolar brush cell , wt wild - type notably , the trpc3 knockout mice also exhibit an ataxic phenotype , albeit much milder than the mwk mice . the fact that both loss and gain of trpc3 function lead to cerebellar ataxia has been previously attributed as conflicting . however , it is commonly observed in other ataxic mouse mutants as well as human ataxia patients that both decreased and increased ion channel activities can lead to purkinje cell dysfunction and consequently cerebellar ataxia [ 6 , 37 ] . this established concept that functionally different mutations in the same ion channel gene can cause the same disease phenotype highlights the importance of the associated biological mechanisms such as maintenance of calcium homeostasis for proper purkinje cell function . histopathological analysis of the mwk brain revealed a slow but progressive loss of purkinje cells starting at 4 months of age ( fig . 2 ) . purkinje cell degeneration is particularly pronounced in the lateral hemispheres of the cerebellum . moreover , no evidence of dna fragmentation of activation of pro - apoptotic signaling molecules was found , suggesting a cell death mechanism other than apoptosis . given the underlying gain - of - function mutation in trpc3 and observed increase in calcium signaling , cell death is likely to occur due to calcium overload . this mechanism might also explain the fact that type ii ubcs are lost much earlier compared to purkinje cells in the mwk cerebellum . purkinje cells have an extensive calcium - buffering capacity due to the presence of high concentrations of calcium - binding proteins including parvalbumin and calbindin . the latter has been estimated to comprise more than 15 % of total protein in purkinje cells . hence , the ability to handle an excessive influx of calcium is likely to be much more rapidly exceeded in ubcs compared to purkinje cells . it would be interesting to further dissect the relative contribution of ubc loss and purkinje cell dysfunction with regard to the ataxic mwk phenotype in a conditional mouse mutant . type ii ubcs are predominantly found in the vestibulocerebellum , suggesting that their loss in the mwk mouse might contribute to the profound balance impairment of the mutant . significant cerebellar trpc3 expression starts within the second postnatal week and peaks at 3 weeks postnatally ( see also cerebellar development transcriptome database ) ( http://www.cdtdb.neuroinf.jp ) ( fig . 2 ) , suggesting a role for this non - selective cation channel during purkinje cell development . interestingly , mwk mice exhibit ataxic behavior long before loss of purkinje cells is observed ( fig . 2 ) , indicating that the ataxic phenotype is caused by the dysfunction rather than the loss of purkinje cells . indeed , electrophysiological alterations are evident in the mwk purkinje cells as early as 3 weeks of age [ 18 , 19 ] . cell recordings showed that only a small fraction of mwk purkinje cells are spontaneously active and suggested that mwk purkinje cells are generally depolarized . consistent with these observations , mwk purkinje cells responded differently to stimulation of mglur1 , with either no inward current or an inward current with significantly smaller spikes at a higher frequency . together , these findings suggest that purkinje cells in the developing mwk cerebellum are profoundly impaired in their intrinsic and evoked electrophysiological properties . developmental abnormalities in the mwk cerebellum are also evident in the altered dendritic morphology of the mutant purkinje cells . the postnatal increase in trpc3 expression coincides with the most intensive phase of dendritic arborization of purkinje cells , suggesting a role for trpc3 in this process . consistent with this idea , mwk purkinje cell dendritic arbors were found to be significantly less elaborate compared to wild - type purkinje cells ( fig . 2 ) . this phenomenon was particularly apparent in organotypic slice cultures of the developing cerebellum , where mwk purkinje cells exhibit a profoundly reduced dendritic arbor . this dendritic phenotype in the mwk mice that have increased cerebellar calcium signaling is consistent with other studies showing reduced purkinje cell dendritic growth upon activation of mglur1 [ 39 , 40 ] and pkc [ 4143 ] . recently , voltage - gated calcium channels ( vgccs ) were implicated in the regulation of purkinje cell dendritic growth after chronic mglur1 or pkc activation . interestingly , genetic knockout of trpc3 or pharmacological inhibition of trpc3 did not alter purkinje cell dendritic arborization . together , these findings suggest that it is the excessive calcium influx during the critical period of dendritic development in mwk mice that causes inhibition of dendritic growth . the inhibition of purkinje cell dendritic arborization by increased calcium signaling through activation of mglur1 , trpc3 , and pkc might be a powerful negative feedback mechanism to limit the size of the dendritic arbor after the establishment of a sufficient number of granule cell parallel fiber contacts . this model is consistent with earlier findings that electric activity controls purkinje cell dendritogenesis . in further support of this , purkinje cells of the lurcher mouse with a gain - of - function mutation in the 2 glutamate receptor ( glud2 ) , which changes the receptor into a leaky membrane channel resulting in chronic depolarization , also exhibit profoundly impaired dendritic growth . an extensive literature supports a role for calcium signaling in the dendritic morphogenesis of neurons including the local regulation of dendritic branch dynamics as well as the global control of gene transcription ( reviewed in [ 47 , 48 ] ) . however , surprisingly , little is known about the specific calcium - activated signaling mechanisms that regulate purkinje cell dendritic arborization downstream of mglur1 , trpc3 , and vgccs . pkcs have emerged as one of the kinases that regulate purkinje cell dendritic arborization in response to increased calcium influx ( reviewed in [ 43 , 49 ] ) . it appears that both pkc isoforms , pkc and pkc , contribute to purkinje cell dendritic development , depending on the strength of the stimulus . furthermore , calmodulin - dependent kinases ( camks ) have been implicated in purkinje cell dendritic growth . pharmacological block of camks has been reported to reduce the number of purkinje cell primary dendrites in cerebellar cultures . this effect was only observed between 5 and 15 days in vitro but not later , suggesting that camks might be involved in the early phases of purkinje cell dendritogenesis and before a critical function of trpc3 in this process . the specific substrates of pkcs and camks that regulate purkinje cell dendritic development remain largely unknown . the only reported downstream effector of camkii signaling in purkinje cells controlling local dendritic branching is the microtubule destabilizer stathmin , which is phosphorylated by camkii upon activation of vgccs and mglur1 . thus , further studies are needed to dissect the molecular mechanisms that control the calcium - dependent purkinje cell dendritic growth during development . the mwk mouse provides an excellent model system to study the transcription - dependent and transcription - independent effector pathways that regulate the developmental dendritic arborization of purkinje cells . efforts are underway to identify the genes and pathways that mediate purkinje cell dendritic growth upon trpc3 activation . importantly , as purkinje cells constitute significantly less than 1 % of the total cerebellar cell population , purkinje cell - specific transcriptional and biochemical profiling requires enriched cell preparations obtained by cell sorting or laser microdissection [ 53 , 54 ] . mouse knockout studies have demonstrated a key role for trpc3 in mediating mglur1-dependent synaptic transmission in purkinje cells . in the molecular layer , glutamate released from the parallel fiber presynaptic terminals binds to ampa receptors and mglur1 at the parallel fiber - purkinje cell synapse ( fig . 3 ) . influx of sodium ions through ampa receptor channels leads to a rapid postsynaptic depolarization , whereas mglur1s mediate a local dendritic calcium signal and a prolonged depolarization known as slow excitatory postsynaptic current ( slow epsc ) ( reviewed in ) . glutamate binding to mglur1 activates phospholipase c ( plc ) via g protein gq to produce ip3 and diacylglycerol ( dag ) . ip3 subsequently activates calcium - permeable ip3r1 receptor channels on the endoplasmic reticulum ( er ) membrane , resulting in a characteristic local dendritic calcium response that is required for the induction of ltd . the exact gating of trpc3 following of mglur1 activation remains to be elucidated but might involve rho gtpase - dependent activation of phospholipase d1 ( pld1 ) as well as ip3r and calmodulin and others ( reviewed in [ 59 , 60 ] ) . trpc3 activity has been proposed to be negatively regulated through phosphorylation by pkc and perhaps other kinases [ 2831 , 59 ] , but the exact nature of this inhibitory phosphorylation remains controversial .fig . trpc3 is central to the glutamate - triggered pathway that is vital for purkinje cell function and , when disrupted , results in cerebellar ataxia ampar ampa receptor , glud2 2 glutamate receptor , ip 3 r1 inositol triphosphate receptor type 1 , mglur1 metabotropic glutamate receptor subtype 1 , pkc protein kinase c , plc phospholipase c , pld phospholipase d , trpc3 transient receptor potential channel c3 mglur1-trpc3 signaling at parallel fiber - purkinje cell synapses . trpc3 is central to the glutamate - triggered pathway that is vital for purkinje cell function and , when disrupted , results in cerebellar ataxia . ampar ampa receptor , glud2 2 glutamate receptor , ip 3 r1 inositol triphosphate receptor type 1 , mglur1 metabotropic glutamate receptor subtype 1 , pkc protein kinase c , plc phospholipase c , pld phospholipase d , trpc3 transient receptor potential channel c3 importantly , disruption of any component of the mglur1-triggered signaling cascade results in cerebellar dysfunction and disease . similar to the mwk mouse phenotype , knockout mice deficient in the genes encoding mglur1 [ 61 , 62 ] , gq , ip3r1 , trpc3 , and pkc all exhibit cerebellar ataxia . moreover , auto - antibodies against mglur1 are associated with neoplastic as well as subacute cerebellar ataxia in human patients [ 6668 ] . gain - of - function missense mutations in the prkcg gene encoding pkc cause sca14 . interestingly , sca14-associated pkc mutants were shown to fail to phosphorylate trpc3 , resulting in a sustained calcium influx that might be central to the sca14 pathogenesis . overexpression of sca14-associated pkc mutations in developing purkinje cells also disturbs their dendritic development , which is consistent with the mwk developmental phenotype . recently , activation of mglur1 was shown to also trigger gating of the postsynaptic glud2 receptor . in another study , glud2 was found to associate with a mglur1-trpc3-pkc signaling complex and to regulate mglur1-mediated synaptic transmission in cerebellar purkinje neurons . interestingly , in this study , genetic loss of glud2 was found to increase extra - synaptic mglur1 and trpc3 expression in the hotfoot mutant mice , resulting in abnormal synaptic transmission . both loss - of - function and gain - of - function mutations in the grid2 gene encoding glud2 result in defects in purkinje cell dendritic arborization and cerebellar ataxia . recently , the first mutations in the human grid2 gene have been reported in patients with cerebellar ataxia [ 12 , 13 ] . in addition to the direct links between disrupted glutamatergic signaling and cerebellar ataxia described above , increasing evidence points towards a role of perturbed trpc3 signaling in other genetic forms of cerebellar ataxia . for example , the expression of trpc3 and other genes involved in calcium homeostasis was shown to be downregulated in a mouse model of sca1 . importantly , these expression changes occurred early and before any observed pathological changes in sca1 , suggesting that they might be a causal factor in the pathogenesis of sca1 . consistent with these findings , mglur1-trpc3-mediated synaptic signaling is disrupted in homozygous staggerer mice , an extreme model of sca1 . both expression of mglur1 and trpc3 were found to be dramatically reduced in these mice , and consequently , slow epscs were absent . similarly , mglur1-mediated synaptic transmission is completely absent in a transgenic mouse model of sca3 . collectively , these studies underscore that trpc3 is a central player in a glutamate - triggered signaling cascade that is vital for purkinje cell function and , when disrupted , results in cerebellar disease in mice and men ( fig . 3 ) . moreover , these findings suggest that trpc3 itself might be a promising candidate gene for human cerebellar ataxia . supporting this , a methylation - regulating trpc3 promoter polymorphism was found to be enriched in patients with idiopathic cerebellar ataxia . so far , candidate screening of the trpc3 gene did not identify potential mutations in patients with sporadic late - onset cerebellar or episodic ataxia , suggesting that trpc3 mutations are unlikely to be a common cause of cerebellar ataxia . however , trpc3 mutations might still contribute to cerebellar ataxia in specific subtypes of the disease that have not been screened yet . the increasing use of next - generation sequencing approaches for the genetic diagnosis of cerebellar ataxia is poised to fully elucidate the role that trpc3 mutations may play in human cerebellar ataxia . the gain - of - function mwk mouse has provided novel insights into the function of trpc3 in the normally developing cerebellum as well as in cerebellar ataxia . in particular , the link between trpc3-triggered aberrant purkinje cell development and cerebellar ataxia could not have been predicted from the trpc3 knockout phenotype , underscoring the power of genome - wide random mouse mutagenesis in identifying important mechanisms underlying nervous system dysfunction . accumulating evidence from other cell- and animal - based models of cerebellar ataxia suggests that abnormal purkinje cell development and early changes in purkinje cell physiology commonly contribute to cerebellar ataxia , thus challenging on our view of cerebellar ataxia as a neurodegenerative disorder [ 11 , 71 , 80 , 81 ] . despite major progress in deciphering the genetic and molecular mechanisms of cerebellar ataxia , one of the biggest challenges of ataxia research is the identification of drug targets and the development of novel therapeutic approaches . the mwk mouse and other cerebellar mutants have highlighted the importance of synaptic transmission at the parallel fiber - purkinje cell synapse and thus mglur1-trpc3 signaling in cerebellar function and disease . indeed , abnormal mglur1-trpc3 signaling seems to be a unifying feature of many hereditary ataxias including the more common trinucleotide expansion disorders sca1 and sca3 . thus , pharmacological regulation of this pathway might be beneficial not only in one particular ataxia subtype but in a larger group of ataxias that share a common pathological mechanism . modulating mglur1-trpc3 signaling in cerebellar ataxia is particularly attractive , as it would target neuronal dysfunction early on during the course of the disease . given the importance of the trpc3 pathway in cerebellar ataxia , the mwk mouse promises to be a valuable model to test novel compounds that might be beneficial in human cerebellar ataxia . hopefully , more selective and potent pharmacological modulators of trpc3 will be developed in the near future that could hold great promise as therapeutic agents for cerebellar ataxia .	the moonwalker ( mwk ) mouse is a recent model of dominantly inherited cerebellar ataxia . the motor phenotype of the mwk mouse is due to a gain - of - function mutation in the gene encoding the cation - permeable transient receptor potential channel ( trpc3 ) . this mutation converts a threonine into an alanine in the highly conserved cytoplasmic s4s5 linker of the channel , affecting channel gating . trpc3 is highly expressed in cerebellar purkinje cells and type ii unipolar brush cells that both degenerate in the mwk mouse . studies of the mwk mouse have provided new insights into the role of trpc3 in cerebellar development and disease , which could not have been predicted from the trpc3 knockout phenotype . here , the genetic , behavioral , histological , and functional characterization of the mwk mouse is reviewed . moreover , the relationship of the mwk mutant to other cerebellar mouse models and its relevance as a model for cerebellar ataxia are discussed .
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Proceed to summarize the following text: the production of fermented corn paste by natural fermentation of grains soaked in water and ground is an artisanal transformation process of maize commonly used in africa . such fermented corn paste can take many denominations in different countries . in nigeria , for example , the fermented paste is called ogi while in south africa the term commonly used is mawe . in cameroon , particularly in north region . this kutukutu has an important place in the sociocultural and nutritional plan . in the sociocultural plan , kutukutu is taken regularly during fast periods and is frequently used as complementary foods for infants . in cameroon , moreover , it is a major source of proteins , carbohydrates , and calories in the diets of large number of population . however kutukutu contains many antinutritional factors such as phytic acid , polyphenols , and tannins which reduce bioavailability and digestibility of proteins and carbohydrates through formation of complex with minerals and inhibition of enzymes . the technological processes such as mechanical , thermal , chemical , and biological processes are used to reduce antinutritional factors content and to improve the bioavailability of nutriments . unlike thermal , chemical , and mechanical processes which can deteriorate quality of food , fermentation is one of the processes that decreases the level of antinutrients in food grains and increases the starch digestibility , protein digestibility , and nutritive value . among the microorganisms used in food fermentation , the lab represents the principal group found on various substrates . lab are a large group of closely related bacteria that have similar properties such as lactic acid production , which is an end product of the fermentation . lactic fermentation is a common way of preparing traditional fermented food in africa like maize porridge , alcoholic beverages , and dairy products . several studies reported that lab improve the nutritional quality of foods during fermentation by increasing the protein content , reducing sugar content , reducing the antinutritional factors ( phytates , tannins , and polyphenols ) , improving the bioavailability of minerals , and increasing the energy density by hydrolyzing starch into simpler compounds such as glucose and fructose . although natural fermentation improves nutritional value and organoleptic qualities of foods , it has a major problem of fluctuation in the quality of different foods obtained . indeed , the spontaneous fermentation process that is carried out by the development of epiphytic microflora can lead to undesirable products on the organoleptic , microbiological , or toxicological quality . that is why the natural fermentation is often the main cause of diarrhea and malnutrition in children . to solve this problem , there is a crucial need to isolate and identify lab with specific physiological and metabolic properties , which can be used as starters in view to improve general food quality and nutritional value as suggested by few authors [ 1317 ] . the aim of this study is to reduce antinutritional factors and to improve the nutritional properties of kutukutu during fermentation with l. brevis g11 , l. brevis g25 , l. buchneri m11 , l. cellobiosus m41 , l. fermentum n33 , lactobacillus fermentum n25 , and l. plantarum a6 . the kutukutu was obtained after individual fermentation with seven lab under laboratory conditions . the strains like l. brevis g11 , l. brevis g25 , l. buchneri m11 , l. cellobiosus m41 , l. fermentum n33 , and l. fermentum n25 were isolated from fermented corn and kutukutu sampled in northern cameroon ( maroua , garoua , and ngaoundere ) . l. plantarum a6 was kindly provided by the microbiology laboratory of cirad montpelier , france . these lactic starters stored at 4c on agar slants were cultured by streaks on mrs agar and incubated anaerobically at 30c for 72 h. the perfectly insulated colonies were inoculated in test tubes containing 10 ml of mrs broth and incubated at 30c for 16 h. the resulting preparation was centrifuged at 3000 rpm for 10 min and the resulting pellet was washed in 10 ml of physiological peptone water ( peptone 1 g in saline solution ( 0.85% nacl ) , ph 7.2 ) and centrifuged again . the concentration of viable cells was adjusted at 10 cfu / ml using mcfarland standard tube number 4 . in order to evaluate the influence of lab on the nutritional properties of the kutukutu during fermentation with starters , the kutukutu was produced under laboratory conditions following the traditional process with some modifications . dry corn purchased from a local market in ngaoundere ( adamaoua , cameroon ) was decontaminated in sterile distilled water containing benzoic acid 6% ( w / v ) ( e210 ) for 24 h at room temperature . then sterile corn was soaked in sterile distilled water for 48 h at room temperature . grinding was proceeded after the determination of the water content ( 39.6% ) using a metallic grinding mill . the paste obtained was mixed ( 1/3 w / v ) with sterile distilled water and sieved through a sieve of mesh 200 m . after decantation for 24 h at room temperature , the paste was collected ( water content 73% ) in a sterile container and kept for inoculation and fermentation . flasks containing 700 g of previously described paste were inoculated separately with 1 ml containing 10 cfu of l. brevis g11 , l. brevis g25 , l. buchneri m11 , l. cellobiosus m41 , l. fermentum n11 , l. fermentum n25 , and l. plantarum a6 . these flasks were covered and kept at 25c for 120 h. the preparations were then homogenized on daily basis to enhance the distribution of bacteria in the medium . aliquots were collected every 24 h , dried at 45c for 24 h , and analyzed . diagram of inoculation of kutukutu with lab in laboratory is reported in figure 1 . to assess the physicochemical parameters , the lactic acid content was determined by titration according to obadina et al . and was expressed in grams of lactic acid per 100 g of sample . reducing sugar was determined by the method described by fischer and stein and the optical densities were read at 540 nm . the total nitrogen content ( n 6.25 ) was determined after digestion of the samples according to the kjeldahl method described by afnor and the coloration was determined by the method of devani et al . . minerals like iron ( fe ) , potassium ( k ) , manganese ( mn ) , magnesium ( mg ) , zinc ( zn ) , copper ( cu ) , calcium ( ca ) , and sodium ( na ) were determined by atomic absorption spectroscopy ( benton et al . ) . the phytates content was determined by the colorimetric method described by vaintraub and lapteva , modified by gao et al . , and the optical densities were read at 500 nm using a spectrophotometer . the formula below was used to determine the tannin content : ( 1)tannin mg/100 dm = total polyphenols mg/100 dmnontannin polyphenols mg/100 dm . the results were analyzed using statgraphics 5.0 ( 1998 ) software for the analysis of variance ( anova ) , calculation of averages , and standard deviations . generally , the ph of kutukutu fermented with the different lab trains decreased with time compared to the control ( figure 2 ) . however , kutukutu fermented with l. brevis g25 had the lowest ph ( 2.7 ) after 120 h. the decrease of ph is due to hydrolysis of carbohydrates during the fermentation which was followed by the production of organic acids . studies made by ali and mustafa showed a similar reduction of ph from 4.3 to 3.4 in the sorghum dough fermented with the lactobacilli strains ( l. fermentum , l brevis , and lactobacillus amylovorus ) after 6 h at 37c . contrarily to ph , acidity of kutukutu increased significantly with time ( p < 0.05 ) compared to the control ( figure 3 ) . it was noted that l. brevis g25 had the highest acidity range ( from 0.3 to 1.2% ) during fermentation of kutukutu . the increase of the acidity reflects the metabolism of sugars by lab during fermentation . from the organoleptic point of view , the acidity of kutukutu makes it more appetizing for anorexic children and may also reduce bacterial contamination [ 31 , 32 ] . this result is in agreement with the study of wedad et al . who showed increase in acidity of sorghum cultivar and cultivar karamaka from 0.36 to 1.6% and from 0.36 to 1.8% , respectively , after 16 h of spontaneous fermentation at 28c . the work of hounhouigan et al . also showed similar increase in acidity ( 88% ) of corn flour after 72 h of fermentation . the quantity of reducing sugars increased from 0 to 48 h of fermentation and then decreased after 48 h ( table 1 ) . an increase of 130% in reducing sugars ( from 168.2 to 387.6 mg/100 g dm ) of kutukutu fermented with l. buchneri m11 after 48 h was observed . contrarily to other lab species , the reducing sugars were produced by l. brevis g25 over a long period ( 96 h ) . according to osman , the increase of sugars during fermentation could be explained by the hydrolysis of starch due to amylases produced by the lab . osman showed an increase of glucose in millet flour from 6.8 to 11.35 g/100 g after 20 h of fermentation at 30c . osman also portrayed an increase in fructose ranging from 1.17 to 1.20 g/100 g after 20 h of fermentation at 30c . reducing sugars can equally be used during the fermentation by lab for the synthesis of various organic acids . this justifies the decrease of sugars in kutukutu fermented with l. brevis g25 ( 304.9 to 131.5 mg/100 g dm ) after 96 h and l. brevis g11 ( 282.9 to 246.3 mg/100 g dm ) , l. plantarum a6 ( 263.5 to 247.6 mg/100 g dm ) , l. buchneri ( 387.5 to 250.7 mg/100 g dm ) , l. cellobiosus ( 314.1 to 210.0 mg/100 g dm ) , l. fermentum n25 ( 321.7 to 236.1 mg/100 g dm ) , and l. fermentum n33 ( 353.9 to 215.7 mg/100 g dm ) after 48 h. these results corroborate with those of osman who showed reduction of glucose and fructose from 11.35 to 7.3 g/100 g and 1.2 to 0.6/100 g , respectively , for fermented millet flour between 20 and 24 h. the majority of starchy compounds in the kutukutu decreased significantly ( p < 0.05 ) during fermentation as compared to the control ( figure 4 ) . after 120 h of fermentation , we observed reduction of starch ranging from 1213.9 to 325.1 mg/100 g dm ( 73.2% ) in the kutukutu fermented with l. fermentum n33 . the hydrolysis of starch by the lab during fermentation reduces swelling of the starch granules and viscosity of the flours during the preparation of porridge . the decrease of starch content in kutukutu during fermentation could be due to the hydrolysis of starch due to amylases produced by the lab into simple sugars . . showed that lab isolated from fermented maize could have a strong amylolytic activity . . showed a decrease of starch from 65.6 to 23.6 g/100 g ( 64.0% ) after 72 h of spontaneous fermentation of dgu . a slight increase of the crude proteins content was observed during the fermentation of kutukutu with all selected strains excepted for l. fermentum n33 ( figure 5 ) . after 120 h of fermentation , crude proteins content in kutukutu fermented with l. brevis g11 , l. brevis g25 , and l. cellobiosus m41 increased from 5.8 to 6.9 g/100 g dm ( 18.9% ) for each one . however , l. fermentum n33 has a different behavior from the other bacteria . initially , an increase in proteins content ranging from 5.8 to 6.3 g/100 g dm ( 8.6% ) was observed after 48 h of fermentation , followed by a drop from 6.3 to 5.0 g/100 g dm ( 20% ) after 120 h of fermentation . the increase of crude proteins content could be attributed to the use of carbohydrates by lab . these results are in agreement with those of awade et al . , who showed an increase in crude proteins content by 14.63% after 14 h of fermentation of corn flour . however the decrease in proteins content observed in l. fermentum n33 fermented kutukutu may be explained by the fact that the lab used these proteins for their metabolic activities during fermentation . osman observed a similar reduction of protein content by 4.5% after 20 h of fermentation of millet flour at 30c . during the fermentation of kutukutu , a significant increase ( p < 0.05 ) in minerals was observed ( table 2 ) , but the highest content of mg , fe , and na was registered in kutukutu fermented with l. brevis g25 varying between 25.9 and 39 mg/100 g dm ( 50.5% ) , 9.2 and 15.7 mg/100 g dm ( 70.6% ) , and 1.2 and 1.3 mg/100 g dm ( 8.3% ) , respectively . there was also an increase in the k and p from 82.6 to 118.8 mg/100 g dm ( 43.8% ) and from 95.1 to 138.1 mg/100 g dm ( 45.2% ) , respectively , in kutukutu fermented with l. brevis g11 . l. fermentum n33 and l. brevis g25 increased the zn content in kutukutu with values ranging from 1.1 to 1.3 mg/100 g dm ( 18.2% ) . l. brevis g25 , l. brevis g11 , and l. buchneri increased the cu content in kutukutu from 0.1 to 0.2 mg/100 g dm ( 100% ) , while only l. brevis g25 and l. brevis g11 increased the mn content in kutukutu from 0.2 to 0.4 mg/100 g dm ( 100% ) . the increment in minerals could be explained by the reduction of antinutritional substances such as phytates and phenolic compounds which form complexes with minerals [ 10 , 42 ] . observed an increase of fe and zn from 5.8 to 5.9 mg/100 g and from 2.9 to 3 mg/100 g , respectively , in fermented millet flour of garira variety after 24 h of spontaneous fermentation at 37c . they also noticed an increase in p , zn , and fe content from 183.4 to 205.3 mg/100 g , 2.9 to 3.1 mg/100 g , and 6.5 to 10.2 mg/100 g , respectively , in the fermented millet flour variety gadarif the evolution of total polyphenols content in kutukutu during fermentation is shown in figure 6 . after 120 h of fermentation , the total polyphenols content was reduced from 425.8 to 66.3 mg/100 g dm ( 84.5% ) and from 425.8 to 86.8 mg/100 g dm in the kutukutu fermented with l. fermentum n33 and l. plantarum a6 , respectively . the reduction in polyphenols content during fermentation many studies on the improvement of nutritional quality of fermented grains such as millet showed a significant reduction of the levels of polyphenols [ 44 , 45 ] . adam et al . observed a reduction in polyphenols content ranging from 120.4 to 111.08 mg/100 g and from 125.1 to 107.2 mg/100 g , respectively , in millet cultivar ugandi and dembi yellow after 14 h of fermentation at 37c . tannins content was reduced significantly ( p < 0.05 ) during the fermentation of kutukutu compared to the control ( figure 7 ) . l. plantarum a6 and l. fermentum n33 reduced the tannins content in kutukutu from 215.1 to 2.5 mg/100 g dm ( 98.8% ) and 215.1 to 4.6 mg/100 g dm ( 97.9% ) , respectively , after 120 h of fermentation . indeed , some lab such as l. plantarum , l. pentosus , and l. paraplantarum are able to degrade tannins through their acylhydrolase tannin activity . this ability is often associated with the vegetable products and confers an ecological advantage to the lab . antony and chandra showed 52% reduction of tannins in millet flour during fermentation . in the same way onyango et al . reported a significant ( p < 0.05 ) reduction of tannins content after 8 days of fermentation of red sorghum flour , white sorghum and millet at 25c . the entire selected lab reduced the phytates content after 120 h of fermentation ( figure 8) . phytates content in kutukutu fermented with l. buchneri m11 was reduced from 278.7 to 12.4 mg/100 g dm ( 95.5% ) . this observed reduction of phytates can be due to phytases and phosphatases produced by lab which hydrolyze phytates to inositol and orthophosphates . studies made by ejigui et al . also illustrated a reduction in phytates levels ranging from 9.87 to 3.8 mg/100 g in corn flour after 96 h of fermentation at 30c . similarly , onyango et al . reported a significant ( p < 0.05 ) reduction of phytates in red sorghum flour , white sorghum , and millet after 8 days of fermentation at room temperature . the variables used to evaluate the improvement of the nutritional quality of kutukutu with lab were attached to a principal component analysis ( figure 9 ) . that helped to visualize correlations and to select among the 07 bacteria studied , ones who give the best results . the axis f1 explains 47.29% of information and the second axis f2 explains 32.13% of information . the analysis of the correlations between the different variables and the principal axis shows that the variables such as minerals ( mg ( 0.95 ) , mn ( 0.89 ) , iron ( 0.63 ) , and cu ( 0.82 ) ) , proteins ( 0.73 ) , lactic acid ( 0.85 ) , and ph ( 0.94 ) contribute significantly to the formation of the f1 axis , while variables such as polyphenols ( 0.95 ) , phytates ( 0.91 ) , starch ( 0.80 ) , and some minerals like zn ( 0.81 ) contribute mainly to the formation of the f2 axis . the supplementary variables ( reducing sugars ( 0.60 ) ) that are classified on the axis f1 also show a significant contribution on this axis . when lab are also represented in the axis system f1 f2 ( figure 10 ) , axis f1 corresponds to the variables induced by l. brevis g25 , l. fermentum n25 , while the f2 axis variables are induced by l. plantarum a6 , l. buchneri m11 , l. fermentum n33 , and l. cellobiosus m41 . however , l. brevis g25 ( 55.3% ) and l. fermentum n33 g25 ( 33% ) are those lab that contribute most to the formation of this axis system ( f1 f2 ) . this arrangement of variables and observations on f1 and f2 axis shows that l. fermentum n33 helps to reduce antinutrients factors such as phytates and polyphenols , while l. brevis g25 contributes to increased bioavailability of minerals ( mg , mn , cu , and fe ) , lactic acid , and protein contents . the fermentation of the kutukutu by selected lab induced many changes in nutritional properties as well as antinutritional factors . l. brevis g25 increased ( 80.7% ) reducing sugars content and increased the proteins content to 18.9% . it also increases availability of mg and fe , respectively , to 50.5% and 70.6% . l. plantarum a6 reduced the tannins content to 98.8% in kutukutu and l. buchneri m11 reduced the phytates content ( 95.5% ) in the kutukutu , while , for a best reduction of phytates and polyphenols , kutukutu must be fermented by l. brevis g25 . to improve protein content and minerals ( mg , mn , cu , and fe ) , kutukutu must be fermented by l. fermentum n33 . both of these bacteria can be used for improving the nutritional quality of kutukutu during fermentation .	the aim of this study is to reduce antinutritional factors and to improve the nutritional properties of kutukutu during fermentation with lactic acid bacteria ( lab ) . for that , kutukutu ( 700 g ) was prepared in the laboratory and inoculated with pure cultures of lab ( 109 cfu / ml ) . then , preparation was incubated for 120 h. every 24 h , kutukutu were collected , dried at 45c for 24 h , and analyzed . the results showed that lactobacillus brevis g25 increased reducing sugars content to 80.7% in kutukutu after 96 h of fermentation . lactobacillus fermentum n33 reduced the starch content to 73.2% , while lactobacillus brevis g11 , l. brevis g25 , and lactobacillus cellobiosus m41 rather increased the protein content to 18.9% . the bioavailability of mg and fe increased , respectively , to 50.5% and 70.6% in the kutukutu fermented with l. brevis g25 . l. plantarum a6 reduced the tannin content to 98.8% and l. buchneri m11 reduced the phytate content to 95.5% . the principal component analysis ( pca ) shows that , for a best reduction of antinutrients factors and improvement of protein content and minerals , kutukutu must be fermented by l. brevis g25 and l. fermentum n33 , respectively . these starter cultures could be used to ameliorate nutritional proprieties of kutukutu during the fermentation .
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Proceed to summarize the following text: patients attending the diabetes clinics at austin health routinely undergo regular measurements of renal function and 24-h urine collections for assessment of aer . patients with impaired renal function ( egfr < 60 ml / min/1.73 m using modification of diet in renal disease formula ) and aer within the normoalbuminuric ( < 20 g / min in two out of three consecutive 24-h urine collections ) or microalbuminuric range ( < 200 g / min in two out of three consecutive 24-h urine collections ) were invited to participate in the study . between 2005 and 2010 , research renal biopsies were performed in patients with normoalbuminuria and microalbuminuria . in the same period , clinically indicated biopsies were performed in macroalbuminuric patients with egfr < 60 ml / min/1.73 m. the study was approved by the human research ethics committee at austin health . in patients with normo- or microalbuminuria , renal biopsies were performed according to a research protocol , and egfr was supplemented with isotopic tc - diethylene triamine penta acetic acid ( dtpa ) measurement of gfr . patients with normoalbuminuria or microalbuminuria receiving a renin - angiotensin system ( ras ) inhibitor had this therapy withheld for 46 weeks prebiopsy . these medications were withheld prebiopsy in order to exclude the presence of normoalbuminuria due to ras blockade , so called pseudonormoalbuminuria . during this period , patients were reviewed at two weekly intervals . if blood pressure increased to > 140/80 mmhg , alternative , non - ras antihypertensive agents were used to control blood pressure . patients fasted from midnight the night before the renal biopsy and were admitted to the day treatment centre , austin health . a 14-gauge trucut biopsy needle was inserted into the kidney under real - time ultrasound guidance and one or two 12 cm cores of tissue were withdrawn . tissue for light microscopy ( lm ) was prepared using standard techniques ( formalin fixation , processing to paraffin wax , sections cut at 1-m thickness , stained with hematoxylin and eosin , periodic acid sections for either immunofluorescence ( using frozen tissue ) or immunoperoxidase ( using paraffin - embedded tissue ) were stained with antibodies to immunoglobulins and complement . tissue for electron microscopy ( em ) was prepared by fixation in 2.5% glutaraldehyde and resin embedded , and ultrathin sections were examined on a jeol transmission electron microscope . basic renal morphology was assessed at lm , including semiquantitative assessment of global glomerulosclerosis ( gs ) ( gs score 0 = < 10% , 1 = 1025% , 2 = 2650% , and 3 = > 50% ) , glomerular mesangial expansion ( score 0 = none or mild and focal , 1 = mild and diffuse , 2 = obvious and diffuse , and 3 = nodular ) , tubular atrophy ( ta ) , interstitial fibrosis ( if ) ( ta / if score 0 = < 5% biopsy area , 1 = 525% , 2 = 2650% , and 3 = > 50% ) , arteriosclerosis ( score 0 = none , 1 = vascular narrowing of < 25% luminal area in the most affected vessel , 2 = 2650% narrowing , and 3 = > 50% narrowing ) , and arteriolar hyalinosis ( score 0 = none , 1 = mild to moderate in at least one arteriole , 2 = moderate to severe in > 1 arteriole , and 3 = severe in many arterioles ) . evidence of glomerulonephritis and other nondiabetic causes for renal impairment was sought . all biopsies contained sufficient glomeruli for diagnostic evaluation . em assessment included quantitation of glomerular basement membrane ( gbm ) thickness and mesangial area ( by computer - assisted measurement using digimizer ( version 4.2.2 ) image analysis software ( medcalc software , ostend , belgium ) . mesangial area was obtained using an on - screen drawing tool to define the boundaries of each mesangial area . the normal range of mesangial area was calculated from examination of nondiabetic control patients from our institution in whom biopsies were performed for clinical reasons and showed qualitatively normal mesangium and in some cases thin basement membrane disease . lm was assessed by two experienced renal pathologists , and by consensus , each biopsy was classified according to the three patterns of renal injury described for microalbuminuric patients with type 2 diabetes , i.e. , normal or near - normal renal structure ( c1 ) , typical dn ( c2 ) , and atypical patterns of renal injury ( c3 ) , with absent or mild diabetic glomerular changes associated with disproportionately severe tubulointerstitial lesions , arteriolar hyalinosis , and/or global gs ( 11 ) . one - way anova was used to test for differences among micro- , normo- , and macroalbuminuric groups for continuous variables , which are reported as mean sd . patients attending the diabetes clinics at austin health routinely undergo regular measurements of renal function and 24-h urine collections for assessment of aer . patients with impaired renal function ( egfr < 60 ml / min/1.73 m using modification of diet in renal disease formula ) and aer within the normoalbuminuric ( < 20 g / min in two out of three consecutive 24-h urine collections ) or microalbuminuric range ( < 200 g / min in two out of three consecutive 24-h urine collections ) were invited to participate in the study . between 2005 and 2010 , research renal biopsies were performed in patients with normoalbuminuria and microalbuminuria . in the same period , clinically indicated biopsies were performed in macroalbuminuric patients with egfr < 60 ml / min/1.73 m. the study was approved by the human research ethics committee at austin health . in patients with normo- or microalbuminuria , renal biopsies were performed according to a research protocol , and egfr was supplemented with isotopic tc - diethylene triamine penta acetic acid ( dtpa ) measurement of gfr . patients with normoalbuminuria or microalbuminuria receiving a renin - angiotensin system ( ras ) inhibitor had this therapy withheld for 46 weeks prebiopsy . these medications were withheld prebiopsy in order to exclude the presence of normoalbuminuria due to ras blockade , so called pseudonormoalbuminuria . during this period , patients were reviewed at two weekly intervals . if blood pressure increased to > 140/80 mmhg , alternative , non - ras antihypertensive agents were used to control blood pressure . patients fasted from midnight the night before the renal biopsy and were admitted to the day treatment centre , austin health . a 14-gauge trucut biopsy needle was inserted into the kidney under real - time ultrasound guidance and one or two 12 cm cores of tissue were withdrawn . tissue for light microscopy ( lm ) was prepared using standard techniques ( formalin fixation , processing to paraffin wax , sections cut at 1-m thickness , stained with hematoxylin and eosin , periodic acid sections for either immunofluorescence ( using frozen tissue ) or immunoperoxidase ( using paraffin - embedded tissue ) were stained with antibodies to immunoglobulins and complement . tissue for electron microscopy ( em ) was prepared by fixation in 2.5% glutaraldehyde and resin embedded , and ultrathin sections were examined on a jeol transmission electron microscope . basic renal morphology was assessed at lm , including semiquantitative assessment of global glomerulosclerosis ( gs ) ( gs score 0 = < 10% , 1 = 1025% , 2 = 2650% , and 3 = > 50% ) , glomerular mesangial expansion ( score 0 = none or mild and focal , 1 = mild and diffuse , 2 = obvious and diffuse , and 3 = nodular ) , tubular atrophy ( ta ) , interstitial fibrosis ( if ) ( ta / if score 0 = < 5% biopsy area , 1 = 525% , 2 = 2650% , and 3 = > 50% ) , arteriosclerosis ( score 0 = none , 1 = vascular narrowing of < 25% luminal area in the most affected vessel , 2 = 2650% narrowing , and 3 = > 50% narrowing ) , and arteriolar hyalinosis ( score 0 = none , 1 = mild to moderate in at least one arteriole , 2 = moderate to severe in > 1 arteriole , and 3 = severe in many arterioles ) . em assessment included quantitation of glomerular basement membrane ( gbm ) thickness and mesangial area ( by computer - assisted measurement using digimizer ( version 4.2.2 ) image analysis software ( medcalc software , ostend , belgium ) . mesangial area was obtained using an on - screen drawing tool to define the boundaries of each mesangial area . the normal range of mesangial area was calculated from examination of nondiabetic control patients from our institution in whom biopsies were performed for clinical reasons and showed qualitatively normal mesangium and in some cases thin basement membrane disease . lm was assessed by two experienced renal pathologists , and by consensus , each biopsy was classified according to the three patterns of renal injury described for microalbuminuric patients with type 2 diabetes , i.e. , normal or near - normal renal structure ( c1 ) , typical dn ( c2 ) , and atypical patterns of renal injury ( c3 ) , with absent or mild diabetic glomerular changes associated with disproportionately severe tubulointerstitial lesions , arteriolar hyalinosis , and/or global gs ( 11 ) . one - way anova was used to test for differences among micro- , normo- , and macroalbuminuric groups for continuous variables , which are reported as mean sd . fourteen patients had research renal biopsies , and 17 patients with macroalbuminuria had renal biopsies for clinical reasons ( fig . 1 and table 1 ) . as part of the study protocol , after ceasing ras inhibitor therapy for 46 weeks prebiopsy , 2 out of 10 patients with normoalbuminuria developed microalbuminuria ( pseudonormoalbuminuria ) , making the total number of patients with normoalbuminuria eight and microalbuminuria six ( fig . fourteen patients had research renal biopsies , and 17 patients with macroalbuminuria had renal biopsies for clinical reasons . as part of the study protocol , after ceasing ras inhibitor therapy for 46 weeks prebiopsy , 2 out of 10 patients with normoalbuminuria developed microalbuminuria ( pseudonormoalbuminuria ) , making the total number of patients with normoalbuminuria eight and with microalbuminuria six . baseline characteristics of the study participants the baseline characteristics of the study participants are shown in table 1 . there was a trend for a higher proportion of women in the normoalbuminuria group ( p = 0.05 ) . renal structural patterns in the 31 patients in the current study are shown in table 2 . we identified typical glomerular changes of dn ( c2 ) in 22 of 23 patients : 5 of 6 patients with microalbuminuria ( mean egfr 48 ml / min/1.73 m ) and 17 of 17 patients with macroalbuminuria ( mean egfr 31 ml / min/1.73 m ) , compared with 3 of 8 patients with normoalbuminuria ( mean egfr 31 ml / min/1.73 m , p = 0.0002 , fisher exact test ) . in the study group as a whole with a mean egfr of 35 ml / min/1.73 m , a predominantly glomerular pattern ( c2 ) was seen in 25 of 31 patients . renal structure patterns in patients with type 2 diabetes considering subjects with normoalbuminuria , three out of eight had changes consistent with typical dn ( c2 ) . early diabetic changes on lm or em ( mild diffuse mesangial expansion and/or gbm thickening ) were seen in two out of eight and one had advanced nodular diabetic gs ( c2 ) . predominantly interstitial or vascular changes ( c3 ) were seen in 3 of 8 normoalbuminuric patients , and only 1 of 23 patients with micro- or macroalbuminuria ( p = 0.08 , fisher exact test ) . the remaining two out of eight patients with normoalbuminuria had nonspecific changes ( c1 ) . varying degrees of arteriosclerosis , although not necessarily the predominant pattern , were seen in seven out of eight patients with normoalbuminuria ( including patient number 8 with mild glomerular diabetic changes and disproportionate vascular and tubulointerstitial damage ) ( table 3 ) . a : patient 1 , from table 3 , demonstrating normal glomerulus and arteries , fioretto c1 . b : patient 3 , from table 3 , demonstrating advanced diabetic gs and arteriosclerosis ( inset ) , fioretto c2 . c : patient 8 , from table 3 , demonstrating minimal glomerular mesangial expansion and severe arteriosclerosis ( inset ) , fioretto c3 ( all images periodic acid schiff stain , original magnification 200 ) . biopsy findings in participants with normoalbuminuria of the 17 patients with macroalbuminuria , most ( n = 11 ) had a rapid renal function decline or increase in albuminuria and had biopsies performed for clinical reasons . some ( n = 5 ) had clinical features suggestive of a glomerulonephritis ( such as hematuria , positive anti - nuclear antibodies serology , or cryoglobulinemia ) . we found typical advanced dn with prominent glomerular changes ( diffuse or nodular gs ) , balanced with tubulointerstitial injury , arteriosclerosis , and arteriolar hyalinosis , in all 17 patients with macroalbuminuria . mesangial area was measured in a subgroup with em images ( normoalbuminuria [ n = 6 ] , microalbuminuria [ n = 3 ] , and macroalbuminuria [ n = 8 ] ) and compared with nondiabetic control patients ( n = 6 ) . the mean mesangial area in a glomerulus per subject was calculated ( mean of 12 measurements of mesangial area per subject ) . mesangial area was significantly different across the four groups ( one - way anova , p = 0.02 ) . mesangial area increased progressively from normal controls to patients with type 2 diabetes and normo- , micro- , and macroalbuminuria ( fig . 3 ) . mean mesangial area ( micron ) across normal controls ( normal c ) , patients with type 2 diabetes and normoalbuminuria ( normo ) , patients with type 2 diabetes and microalbuminuria ( micro ) , and patients with type 2 diabetes and macroalbuminuria ( macro ) . p = 0.02 , one - way anova for mean mesangial area across the four groups . we report that in patients with type 2 diabetes and renal insufficiency ( egfr < 60 ml / min/1.73 m ) , the typical glomerular changes of dn are less common in normoalbuminuric patients than in patients with micro- or macroalbuminuria . this suggests a multifactorial pathogenesis for the renal disease in these patients , with possible contributions from aging , hypertension , and vascular disease in addition to the specific contribution from diabetes . in nearly all ( 22 of 23 ) patients with micro- or macroalbuminuria , typical glomerular changes ( c2 ) of dn were observed . this was a significantly higher proportion than in a previous study in patients with type 2 diabetes , microalbuminuria , and a mean gfr of 101 27 ml / min/1.73 m , of whom 10 of 34 patients showed a predominantly glomerular pattern of renal structural changes ( table 2 , p < 0.0001 , ) ( 11 ) . this is likely due to the selective recruitment of patients with egfr < 60 ml / min/1.73 m in the current study . since it has been demonstrated that in patients with type 2 diabetes and increased aer , the reduction in gfr is correlated with the degree of mesangial expansion and gbm thickening ( 12 ) , it is not surprising that the vast majority of patients in the current study with reduced gfr and micro- or macroalbuminuria had classic diabetic glomerulopathy . furthermore , we found that the mesangial area increased progressively from normal control subjects to patients with type 2 diabetes and normo- , micro- , and macroalbuminuria ( fig . this is consistent with the concept that mesangial expansion is related to decline in gfr in diabetes ( 10 ) . although aer remains an important marker of dkd risk , the absence of an elevated aer does not identify patients with type 2 diabetes who are protected from a progressive decline in gfr ( 5 ) . compared with patients with similarly decreased gfr and macroalbuminuria , we have previously found that patients with normoalbuminuria were more likely to be older and female ( 5 ) . in the current study , although the mean age was similar in the three groups , 5 of 8 subjects with normoalbuminuria were females , compared with 1 of 6 in the microalbuminuria and 3 of 17 in the macroalbuminuria groups . there was a predominance of typical dn ( c2 ) in all micro- and macroalbuminuria patients . in subjects with normoalbuminuria , seven of eight also had varying degrees of renal arteriosclerosis , implying that aging , blood pressure , and intrarenal vascular disease may be playing pathogenic roles . previously , we have demonstrated a role for intrarenal vascular disease in renal insufficiency in diabetes . in that study ( 13 ) , patients with an egfr < 60 ml / min/1.73 m had a higher resistance index of the renal interlobar arteries compared with patients with an egfr 60 ml / min/1.73 m. intrarenal vascular disease was associated with increased age and bmi and decreased egfr and blood pressure . however , there was no relationship between aer status and intrarenal vascular resistance ( 13 ) . it is likely that aging and hypertension are the chief contributors to the atypical nephropathy ( c3 ) changes seen in patients with renal insufficiency and normoalbuminuria . studies in animal models , such as the cohen diabetic rat , may also provide further insights into normoalbuminuric dkd , because in this model , urinary protein excretion does not increase with the development of diabetes despite decreasing creatinine clearance and typical changes of dn on renal biopsy ( 14 ) . however , this does not mimic the atypical pattern of renal damage with no or mild glomerular changes seen in the c3 patients . earlier studies of renal structure in type 2 diabetes and overt nephropathy have shown conflicting results , with some suggesting that the incidence of nondiabetic kidney disease is uncommon ( 15,16 ) , similar to patients with type 1 diabetes , whereas others have reported a higher incidence of nondiabetic kidney disease in patients with type 2 diabetes ( 1719 ) . the characteristic histological lesions of dn , mesangial expansion and thickening of the gbm , correlate with rising proteinuria ( 20 ) , and the decline in gfr is at least partly explained by the loss of filtration surface that accompanies mesangial expansion ( 20 ) . by contrast , the structural basis of low gfr without an increase in albuminuria includes not only mesangial expansion but also tubulointerstitial and vascular changes . prominent interstitial pathology , including if , ta , and mononuclear cell infiltration , correlates with renal microvascular alterations in long - standing dn , and it has been suggested that this is due to chronic ischemia ( 16,21,22 ) . furthermore , if has been proposed as a codeterminant of decline in renal function in dn ( 23,24 ) . an earlier biopsy study demonstrated atubular glomeruli and atrophic tubules in patients with type 2 diabetes and low levels of proteinuria and suggested that both glomerular and interstitial damage may lead to the development of atubular glomeruli in patients with type 2 diabetes ( 20 ) . however , in the current study , the existence of atubular glomeruli was not assessed . although this is the first biopsy study exploring the association between normoalbuminuria and impaired renal function in patients with type 2 diabetes , this was a single - center study including only six patients with microalbuminuria . there was slow recruitment of participants in the current study due to the reluctance of both clinicians and patients to perform a renal biopsy as this is an invasive procedure with risk ( 25 ) . two of the patient renal biopsies were complicated by bleeding episodes requiring blood transfusions . in the current study , 17 of 17 patients with macroalbuminuria and type 2 diabetes undergoing a clinically indicated renal biopsy had typical changes of diabetes , suggesting that nondiabetic disease is relatively rare in these patients . this finding is in concordance with a previous study designed to evaluate the prevalence of nondiabetic renal disease in type 2 diabetes , in which 29 of 33 patients with type 2 diabetes and proteinuria had typical diabetic lesions ( 16 ) . in our study , diagnostic categories were assigned based on lm appearance ( according to fioretto ) , supported in most cases by em findings . assignment to fioretto c1 did not exclude the possibility of early mild dn , which requires quantitative em assessment for diagnosis . we used a classification system developed by fioretto et al . ( 11 ) , which differs from a recent classification system proposed by the research committee of the renal pathology society ( rps ) , histopathological classification based purely on glomerular lesions ( 26 ) . under this classification , our normoalbuminuric cases would be assigned as unclassified ( four cases ) , class i ( one case ) , class iia ( two cases ) , and class iii ( one case ) ( see table 3 ) . the more severe lesions correspond to our fioretto c2 cases , stratified by severity of diabetic glomerular changes . two of the unclassified cases were assigned to fioretto c3 , which illustrates a shortcoming of the classification system proposed by the research committee of the rps in that it underemphasizes the contribution of vascular and tubulointerstitial injury in diabetes . given the heterogeneity of the renal lesions underlying dn and the complex natural history of the disease , the rps scheme is not yet ready for clinical application but should be considered an important first step toward the development of a clinically useful classification system ( 27 ) . a study of 50 patients compared clinical features with the rps classification of dn and found a decrease in mean egfr and decreased 5-year renal survival as glomerular lesions increased from class i to iv ( 28 ) . in summary , in subjects with type 2 diabetes and reduced renal function , normoalbuminuria is associated with heterogeneous changes in renal structure . furthermore , in patients with type 2 diabetes and reduced gfr , microalbuminuria and macroalbuminuria are associated with a predominantly glomerular pattern of renal damage , in contrast to the mixed pattern previously shown in microalbuminuric patients with type 2 diabetes and normal gfr ( 11 ) . in type 2 diabetes , renal structural changes , both glomerular and nonglomerular , are more heterogeneous in normoalbuminuric than in albuminuric renal insufficiency . this suggests that aging , perturbations in blood pressure , and the development of intrarenal vascular disease may contribute to decreases in renal function independently of changes in albuminuria .	objectivethe structural basis of normoalbuminuric renal insufficiency in patients with type 2 diabetes remains to be elucidated . we compared renal biopsy findings in patients with type 2 diabetes and estimated glomerular filtration rate ( egfr ) and measured gfr of < 60 ml / min/1.73 m2 , associated with either normo- , micro- , or macroalbuminuria.research design and methodsin patients with normo- ( n = 8) or microalbuminuria ( n = 6 ) , renal biopsies were performed according to a research protocol . in patients with macroalbuminuria ( n = 17 ) , biopsies were performed according to clinical indication . findings were categorized according to the fioretto classification : category 1 ( c1 ) , normal / near normal ; category 2 ( c2 ) , typical diabetic nephropathy ( dn ) with predominantly glomerular changes ; and category 3 ( c3 ) , atypical with disproportionately severe interstitial / tubular / vascular damage and with no / mild diabetic glomerular changes.resultsin our study population ( mean egfr 35 ml / min/1.73 m2 ) , typical glomerular changes ( c2 ) of dn were observed in 22 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 subjects with normoalbuminuria ( p = 0.002 ) . by contrast , predominantly interstitial or vascular changes ( c3 ) were seen in only 1 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 normoalbuminuric subjects ( p = 0.08 ) . mesangial area increased progressively from normal controls to patients with type 2 diabetes and normo- , micro- , and macroalbuminuria . varying degrees of arteriosclerosis , although not necessarily the predominant pattern , were seen in seven of eight subjects with normoalbuminuria.conclusionstypical renal structural changes of dn were observed in patients with type 2 diabetes and elevated albuminuria . by contrast , in normoalbuminuric renal insufficiency , these changes were seen less frequently , likely reflecting greater contributions from aging , hypertension , and arteriosclerosis .
You are an expert at summarizing long articles. Proceed to summarize the following text: the prevalence of asthma ranges from 9.3% in children younger than 18 years to 7.3% among adults , with an incremental cost due to asthma of $ 56 billion in the united states in 2007.1 asthma medication compliance or adherence is often poor and is a significant contributor to poor health outcomes . many factors can contribute to medication nonadherence in asthma , including economics , social and cultural barriers , attitude , and physician behavior.2 when pharmacy records are compared to patient interview data in older patients on medication use , kappa statistics range from 0.37 to 0.86 for chronic agreement between history and drug use records.3 history is not completely reliable as the sole means of measuring medication adherence . in reviewing the impact of medication regimens on adherence to chronic treatments , ingersoll and cohen specifically noted the importance of dose frequency and regimen complexity as important factors.4 coleman et al , in a meta - analysis evaluating medication use in chronic disease , reported that once - daily dosing had the best adherence while twice - a - day dosing reduced adherence by 6.7%.5 because of the importance of dosing frequency in asthma treatment , this paper reviews the potential use of a new inhaled cortical steroid ( ics)/long - acting beta2-agonist ( laba ) ( fluticasone furoate [ ff]/vilanterol [ vi ] ) combination dry - powder inhaler in the treatment of asthma . ics continue to be the preferred initial controller agent in persistent asthma and play an important role in the treatment of both chronic obstructive pulmonary disease ( copd ) and asthma copd overlap syndrome ( acos).6,7 the national asthma education and prevention program expert panel 3 asthma guidelines8,9 and various reviews have focused on the importance of ics therapy in the maintenance treatment of patients with persistent asthma and acos.7,10 the anti - inflammatory actions of glucocorticoids in asthma are believed to be a result of direct inhibition of multiple cell types involved in airway inflammation ( eg , mast cells , eosinophils , basophils , and lymphocytes ) and of inflammatory mediators produced by these cells ( eg , histamine , leukotriene , and cytokines ) as part of the asthmatic response.11 the mechanism of action is thought to involve binding of corticosteroids to the glucocorticoid receptor , inducing a conformational change that allows the activated glucocorticoid receptor to bind to deoxyribonucleic acid ( dna ) . this binding occurs at the glucocorticoid - responsive dna sequence that , subsequently , promotes synthesis of anti - inflammatory proteins and inhibition of transcription of many proinflammatory cytokines.12 currently available ics products include dry powder , liquid for nebulization , or metered - dose inhaler delivery systems . assessing individual response to ics products can be difficult because significant response variability has been reported with ics use in persistent asthma and acos patients.7,13 the predicting response to inhaled corticosteroid efficacy ( price ) study demonstrated that short - term response to ics predicts long - term asthma efficacy , but that as many as 40% of the patients were ics nonresponders.14 a systematic review of current ics - alone products failed to show an economic benefit from one product compared to other similar products.15 ff is currently available as an inhaled dry powder with once - daily dosing . in a randomized , double - blind , crossover design study of 190 patients aged 12 years with persistent moderate asthma , patients were given either dry - powder inhalations of ff 200 g or fluticasone propionate ( fp ) 200 g once daily , ff 100 g or fp 100 g twice daily , or a matching placebo , for 28 days . patients treated with the once - a - day or twice - a - day fp or ff demonstrated superior lung function compared to those on placebo.16 the absolute mean differences in forced expiratory volume in 1 second ( fev1 ) from placebo were 108 ml ( 95% confidence interval [ ci ] : 64153 ; p0.001 ) for ff 200 g daily ; 98 ml ( 95% ci : 54142 ; p0.001 ) for ff 100 g twice daily ; 87 ml ( 95% ci : 14161 ; p=0.020 ) for fp 200 g daily ; and 132 ml ( 95% ci : 59205 ; p 0.001 ) for fp 100 g daily . the least effective treatment was fp 200 g once daily , supporting its currently approved twice - daily dosing interval . the use of ff 200 g once daily was not inferior to ff 100 g twice daily.16 an 8-week multicenter , double - blind , randomized study of 627 patients with persistent moderate - to - severe asthma evaluated placebo and ff 200 , 400 , 600 , or 800 g dry - powder inhalation once daily or fp 500 g twice daily.17 there was no dose response relationship seen in peak expiratory flow ( pef ) in the ff doses studied , and all the doses of ff or fp were more effective ( p<0.001 ) than placebo.17 improvement in pef was significantly greater ( p<0.001 ) compared to placebo in all active groups , with a 5.1 l / minute decrease seen with placebo and a 16.3 l / minute increase with ff 800 g ; a 14.5 l / minute increase with ff 400 g ; and from 11.1 to 11.9 l / minute increases for the other ff and fp groups . the predefined 200 ml fev1 ( p<0.001 ) improvement over placebo was confirmed for each dose of ff and fp at week 8.17 in another study , 575 patients with persistent asthma were randomized to inhaled dry - powder ff 100 g daily ( morning ) , ff 100 g daily ( evening ) , ff 250 g daily ( evening ) , or placebo in a double - blind , double - dummy , placebo - controlled , parallel - group study.18 the greatest improvement in pef was with the ff 250 g dose , but this was a small difference compared to either of the two daily ff 100 g doses ( evening or morning ) . the change from baseline in trough pef compared to placebo was an increase of 19.2 l / minute ( 95% ci : 8.230.2 ; p<0.001 ) for ff 100 g ( morning dose ) ; an increase of 15.9 l / minute ( 95% ci : 4.926.9 ; p=0.005 ) for ff 100 g ( evening dose ) ; and an increase of 24.64 l / minute ( 95% ci : 13.635.7 ; p<0.001 ) for ff 250 g ( evening dose ) . all of the ff doses demonstrated significantly better pef than placebo.18 taken together , the above data support the use of inhaled ff in the treatment of persistent moderate - to - severe asthma . inhaled labas play an important role in providing bronchodilation in many patients suffering from asthma , copd , or acos . controversy exists about the use of inhaled labas when prescribed as monotherapy in persistent asthma . chronic high - dose exposure to 2-adrenoceptor agonists demonstrates proinflammatory effects.19 in vitro , they enhance the type 2 helper t - cell ( th2 ) inflammatory pathway by inhibiting interleukin-12 and interferon gamma.19,20 the us food and drug administration ( fda ) , in 2010 , performed its own meta - analysis examining inhaled laba therapy use in asthma without simultaneous use of ics compared to non - laba therapy.21 according to this meta - analysis , the fda estimated that 3.63 per 1,000 more clinical trial patients treated with the inhaled laba had significant asthma - related events , including death , the need for intubation , or the need for hospitalization.21 when the inhaled laba was paired with an ics , the asthma - related event difference was a nonsignificant increase of 0.25 per 1,000 clinical trial patients . as a result , the fda placed a black box warning to discontinue the use of labas in asthma as monotherapy and to remove laba use in stable asthmatics , but they did not advise against continued use of laba therapy with an ics in persistent symptomatic asthma patients.21 the risks of laba therapy in asthma patients was felt to be counterbalanced by the meaningful clinical symptomatic improvements seen when labas were added to ics therapy . inhaled labas remained the preferred add - on drug to ics in the 2007 national asthma education and prevention program expert panel report when ics alone are ineffective in achieving asthma control.9 table 1 summarizes currently available combined ics + laba preparations . available 2-agonist bronchodilators act on 2 airway receptors to cause relaxation of airway smooth muscles and improve airflow , thereby decreasing lung hyperinflation . the presumed mechanism of cellular action is through the stimulated receptors ability to modulate intracellular adenylyl cyclase resulting in the generation of cyclic adenosine monophosphate ( camp ) . the camp then results in the activation of effector protein kinases and guanine nucleotide exchange functions.22 the mechanism for the longer duration of action of labas is not known , but may be due to their greater lipophilicity , agonist efficacy , and microkinetic behaviors.2224 vi is a new potent , selective laba with longer duration and greater intrinsic efficacy than salmeterol ( sal ) and a greater potency than indacaterol and salbutamol.25 in addition , vi has been shown , using human recombinant 1/2/3-adrenoreceptor camp assays , to have significantly greater 2-adrenoceptor selectivity ( versus 1 or 3 receptors ) than formoterol , indacaterol , and salbutamol.26 in studies in patients with persistent asthma , inhaled vi in daily doses of 25100 g dry powder was shown to have significant bronchodilation effects and to be well tolerated.27 in a double - blind , placebo - controlled 28-day trial of inhaled vi ( 3 , 6.25 , 12.5 , 25 , and 50 g ) daily in persistent asthma patients , statistically significant ( p0.016 ) increases in morning fev1 were reported for vi doses of 12.550 g.25 systematic reviews have shown that adding a laba to low - dose ics in poorly controlled asthma patients is more effective in reducing the risk of asthma exacerbations than using higher doses of ics.28 asthma patients controlled with low - dose ics therapy who are then switched to either a laba or placebo alone were shown to have more treatment failures and asthma exacerbations than those left on low - dose ics . both active treatments were superior to placebo.29 the use of combined ics / laba products was evaluated in a systematic and economic review.15 cost savings were shown when using the combined products compared to the use of individual laba and ics inhalers . the review was unable to show a difference in asthma patients between combined fp + sal versus budesonide + formoterol inhalers . adherence with prescribed ics therapy has been studied , with compliance rates ranging from only 30% to 70%.3033 low adherence is one of the major barriers to achieving asthma control and may also be associated with increased adverse outcomes . many factors contribute to poor patient medication adherence , including cost , language , socioeconomic status , cultural , and insurance barriers ; physician attitudes and behaviors ; and patient health literacy and disease / treatment understanding.2 a systematic review of 51 studies of medication adherence in various chronic diseases found that the prescribed daily frequency of dosing was a major predictor of adherence , with a 6.7% , 13.5% , and 19.2% reduction in medication adherence when once - a - day dosing was compared to twice - daily , three - times daily , and four - times daily dosing , respectively.5 another review of medication adherence in chronic disease found that dose - frequency medication regimen complexities are important factors.4 using electronic ics actuation counters , onyirimba et al32 reported that control subjects used their ics medications only 51% of the time and that the frequency of use decreased during a 10-week study . the direct clinician - to - patient feedback group demonstrated adherence rates greater than 70% during the entire study . a recent review of nonadherence in patients whose asthma was difficult to control confirmed a nonadherence range between 30% and 70% , with associated poor health outcomes.34 no single approach can correct nonadherence to chronic asthma medication use , but reducing the frequency of dosing is likely to improve adherence . by simplifying the dosing regimen , inhaled ff in combination with the laba vi , administered once daily , may increase patient adherence , leading to favorable results . for this reason , there is much interest in this combination treatment for persistent asthma . one of the mainstay pharmacotherapies for the treatment of copd consists of ics therapy in combination with labas , which , until recently , required twice - daily dosing . the novel once - daily dosing of ff 100 g / vi 25 g combination dry - powder inhaler is fda - approved for use as a maintenance therapy for copd . in a 4-week randomized , double - blind , placebo - controlled study , patients with moderate - to - severe copd were treated with either placebo or ff / vi 400 g/25 g once a day . results for 60 patients ( mean age of 64 years ; ff / vi n=40 ; placebo n=20 ) showed the ff / vi group had statistically greater improvements compared to placebo in trough fev1 ( mean difference of 183 ml [ 95% ci : 87179 ] ) and in the 0- to 4-hour post - dose weighted mean fev1 ( mean difference of 236 ml [ 95% ci : 154319]).35 two other multicenter , randomized , double - blind , placebo - controlled studies compared variable doses of inhaled corticosteroid use and aimed to assess the efficacy and safety over 24 weeks in copd patients.36,37 doses for inhaled ff ranged from as high as 200 g to as low as 50 g and were combined with a set dose of vi at 25 g . subjects ( n=1,224 and n=1,030 ) with moderate - to - severe copd experienced significant ( p<0.001 ) fev1 improvements ( range of increase 103209 ml ) for all ff doses combined with vi 25 g when compared to placebo , but no difference was found when comparing inhaled ff / vi 200 g/25 g versus ff / vi 100 g/25 g treatments.36,37 as a result of this and similar data , inhaled ff / vi is fda - approved for once - daily maintenance treatment and prevention of exacerbations of copd using a combination multi - dose , dry - powder inhalation device containing ff 100 g and vi 25 g . evidence from these studies as well as others38,39 suggests that the combination ff / vi once - a - day inhaler offers an effective alternative to other available twice - daily dosing ics / laba combination inhalers ( table 1 ) for the treatment of copd . although the initial studies performed for inhaled ff / vi were in copd patients , it is obvious from the individual data on ff and vi in asthma that this combination of ics / laba therapy could also be applied to the treatment of asthma . combination inhaled ics / laba therapy continues to be common in the treatment of persistent asthma , due to improved adherence as well as convenience.8,9 a recent randomized , double - blind study consisting of 806 patients compared the efficacy of inhaled ff / vi 100 g/25 g administered once daily with fp / sal 250 g/50 g administered twice daily over 24 weeks in patients aged greater than 12 years with persistent asthma that was poorly controlled on medium doses of ics alone.40 the efficacy of once - daily ff / vi was similar to that of twice - daily fp / sal in improving lung function in these patients . over the 24-week trial , the observed 0- to 24-hour weighted mean fev1 improvement over baseline was 341 ml for ff / vi and 377 ml for fp / sal ( adjusted treatment differences 37 ml ; 95% ci : 88 to 15 ml ; p=0.162).40 there were no differences in asthma exacerbation between the groups ( 3% fp / sal versus 2% ff / vi).41 no differences were found in baseline asthma quality of life + 12 questionnaire , asthma control test , and european quality of life-5 dimensions asthma health outcomes assessments between the two treatments . both the ff / vi and the fp / sal groups showed improvement from baseline in the asthma quality of life + 12 questionnaire , asthma control test , and the european quality of life-5 dimensions asthma health assessments , with no difference seen between treatment groups.40 another randomized , multicenter , double - blind study conducted between june 2010 and october 2011 at 63 centers in six countries ( germany , japan , poland , romania , russia , and the usa ) compared the efficacy and safety of once - daily inhaled ff / vi 200 g/25 g to once - daily inhaled ff 200 g or twice - daily inhaled fp 500 g in 586 patients older than 12 years of age with moderate - to - severe persistent asthma , in whom a significantly greater improvement in lung function was observed with ff / vi versus either ff alone or fp alone.42 trough fev1 at week 24 was improved with all therapies compared to baseline , with ff / vi patients showing a 394 ml improvement , ff patients a 201 ml improvement , and fp patients a 183 ml improvement in fev1 . when the improvement in fev1 seen with daily ff / vi was compared to daily ff alone , a 193 ml ( 95% ci : 108277 ; p<0.001 ) difference was seen . when daily ff / vi improvement fev1 was compared to twice - daily fp , a 210 ml ( 95% ci : 127294 ; p<0.001 ) difference was seen . improvement in rescue - free 24-hour periods was seen with ff / vi compared to ff alone . all treatments were generally well tolerated with no safety signals observed.42 this study demonstrated the benefit of once - daily inhaled combined ff / vi compared to once - daily inhaled ff or twice - daily inhaled fp alone in patients with moderate - to - severe asthma . in a crossover study of 52 subjects with mild asthma , inhaled ff / vi 100 g/25 g and inhaled ff 100 g alone were dosed once daily in the evening for 28 days and compared to placebo to evaluate their capacity to provide bronchoprotection against early asthmatic response ( ear ) and airway hyperresponsiveness ( ahr ) stimulated by an inhaled allergen challenge.41 bronchoprotection against ear was assessed by change from post - saline baseline weighted mean fev1 for the first 2 hours post - allergen challenge on day 29 ( 22- to 23-hour post - final dose on day 28 ) . the ear was also assessed using maximum percent decrease from post - saline baseline and minimum absolute fev1 . both ff / vi and ff significantly suppressed the ear to an allergen challenge relative to placebo . allergen challenge caused reductions of fev1 between 0 and 2 hours of 1.091 l ( 95% ci : 1.3440.837 ) for placebo , 0.826 l ( 95% ci : 1.0700.581 ) for ff , 0.955 l ( 95% ci : 1.2090.702 ) for vi , and 0.614 l ( 95% ci : 0.8580.370 ) for combined ff / vi . measurements of ahr 24 hours after allergen challenge were significantly better with both ff / vi and ff compared to placebo . the use of ff / vi was superior to both ff and vi.41 the ahr was significantly reduced with ff / vi and ff compared to placebo . inhaled ff / vi was superior to both ff and placebo.41 a randomized , double - blind variable - duration ( 2478 weeks ) trial comprising 2,019 patients aged 12 years who had had one or more asthma exacerbations in the previous year compared the effects of inhaled ff 100 g / vi 25 g once daily to inhaled ff 100 g once daily on the frequency of asthma exacerbations.43 the use of ff / vi demonstrated a rate reduction of 25% ( 95% ci : 5%40% ) , compared to ff alone . once - daily inhaled ff / vi reduced the risk of severe asthma exacerbations ( hazard ratio 0.75 ; 95% ci : 0.6420.985 ) and improved lung function compared to inhaled ff alone , with excellent tolerability.43 in another study , both morning and evening once - daily dosing of inhaled ff 100 g / vi 25 g produced comparable improvements in lung function in subjects ( n=26 ) with persistent asthma after 14 days.44 the difference in weighted mean fev1 from placebo was 377 ml ( 95% ci : 293507 ) for morning ff / vi and 422 ml ( 95% ci : 337507 ) for evening ff / vi dosing . no significant difference was seen in weighted mean fev1 between morning and evening dosing of ff / vi ( 44 ml , 95% ci : 125 to + 36 ) . consistent with other reports , the fixed combination of ff / vi once daily appears to be more effective than a similar once - daily inhaled dose of ff alone42 , but clinical trials to date do not demonstrate a clear overall efficacy difference among ics / laba combinations approved for asthma therapy.45 data support that inhaled ff / vi is just as efficacious as previous asthma regimens in terms of fev1 and symptomatic relief in asthma , with the added benefit of it being a once - daily dosed inhaler . to date , this is the only combination ics / laba therapy that is dosed once daily with the potential to improve adherence in persistent asthma . the safety and adverse side effects of inhaled ff / vi can initially be evaluated by examining the effects of the individual components . inhaled corticosteroids continue to be the anti - inflammatory therapy of choice in adult asthma due to their remarkable efficacy and apparent safety.9 the most commonly recognized aes of ics therapy are oropharyngeal candidiasis , dysphonia , and pneumonia.46 other potential systemic effects of inhaled corticosteroids include adrenal suppression , bone loss , increased cataract formation , skin thinning , increased bruising , glucose / metabolic changes , and behavioral abnormalities.46 some studies have reported mild aes , including mouth ulcerations thrush and dysphonia , with ics.38,39,49 other frequent aes with ics include headache and upper respiratory infection.41 in one study , the frequency of adverse events , including upper respiratory infections , pneumonia , and urinary tract infections , was similar when inhaled ff / vi was compared to inhaled twice - daily fp / sal.40 another study of asthma patients44 found oral candidiasis / oropharyngeal candidiasis was more common with ff / vi ( 6%7% ) than with fp ( 3% ) alone . significant cortisol suppression was seen with fp compared to both ff / vi groups ( 100g/25g and 200g/25g ) at weeks 12 and 28 , but no suppression or differences were seen at week 52 for ff / vi 100 g/25 g and for ff / vi 200 g/25 g.44 a study of copd patients showed no significant differences in end - of - treatment period 0- to 24-hour weighted mean serum cortisol levels with inhaled ff / vi 50 g/25 g , 100 g/25 g , or 200 g/25 g doses compared with placebo.38 the use of ff / vi dry - powder inhalers at doses of 100 or 200 g ff and 25 g vi was noninferior to placebo on hypothalamic pituitary adrenal axis function.47 a meta - analysis of eight studies concluded that hypothalamic pituitary adrenal alteration resulting in suppression of cortisol levels would routinely require ff levels several times higher than average clinically used doses.48 no significant changes were reported in non - fasting glucose , potassium , qt interval corrected using fridericia s formula , or ophthalmic assessments.38,40,49 in a large , double - replicate , multicenter , randomized , double - blind , parallel - group copd study comparing three strengths of ff / vi with one strength of vi , nasopharyngitis was the most frequently reported ae for both groups.39 pneumonia and fractures were also reported more frequently with ff / vi than with vi alone . similar to other ics studies50,51 in copd , a small increase in incidence of deaths from pneumonia was also noted in the inhaled ff / vi group when compared with the inhaled vi group alone.39 more pneumonia - related deaths occurred in the inhaled ff / vi 200 g/25 g - treated group , which raises concern that a dose - related adverse event function exists.39 similar consistent data in asthma studies demonstrating an increased risk for pneumonia are lacking . the second component in the combination inhaler is a laba , and commonly reported aes for inhaled labas include arrhythmias , palpitations , tremor , headache , and metabolic effects that may also be dose related . the increase in death for monotherapy labas in asthma patients was reported in the salmeterol multicenter asthma research trial ( smart)52 and in several systematic reviews.15,21 this resulted in an fda black box warning for all inhaled labas and the recommendation of discontinuation of laba therapy in well - controlled asthmatics . in asthma patients , no difference using 24-hour holter monitoring was found in ventricular ectopy over 52 weeks between inhaled daily ff / vi ( 100 g/25 g ) , daily ff / vi ( 200 g/25 g ) , and twice - daily fp ( 500 g).49 current data suggest that dry - powder inhaled ff / vi is well tolerated , with a favorable side effect profile , in the treatment of asthma . current guidelines for the treatment of persistent asthma offer management suggestions for the treatment of chronic asthma and stress the role of combined inhaled ics / laba in the treatment of persistent asthma.79 however , asthma remains poorly controlled in many patients , challenging physicians to reevaluate the current therapy , which should continue to be aimed at improving symptoms and in preventing asthma exacerbations . approaches to maximizing medication adherence with personalized pharmacotherapy regimens that reduce asthma - related activity impairment and the risk of exacerbation in patients with persistent asthma is a major goal . recent studies provide preliminary support for the use of inhaled ff / vi in the treatment of persistent asthma . it appears to be efficacious in patients with persistent asthma , with similar aes to previous twice - daily inhaled ics / laba treatment options and with the added benefit of being a once - daily dosed inhaler . the excellent patient tolerability of inhaled ff / vi along with a likely increase in medication adherence should improve asthma outcomes and help prevent acute severe exacerbations . there is limited current data on the long - term use of ff / vi in persistent asthma patients . to date , no data demonstrating the efficacy of ff / vi over other combined ics / laba products in persistent asthma patients exist . cost and safety data also remain limited in the asthma population , but the extensive data in copd patients is reassuring .	despite the 2007 national asthma education and prevention program expert panel 3 guidelines for the treatment of uncontrolled asthma , many patients with poorly controlled asthma still continue to tax the health care system . controlling asthma symptoms and preventing acute exacerbations have been the foundation of care . using long - term controller treatments such as inhaled corticosteroids ( ics ) and inhaled long - acting beta2-agonists ( labas ) is a common approach . while patient responses to recommended pharmacotherapy may vary , poor adherence to therapy also contributes to poor asthma control . a once - daily combination inhaler , such as fluticasone furoate , an ics , in combination with vilanterol , a laba , offers increased convenience and potential improved adherence , which should result in enhanced clinical outcomes and reduced exacerbations . the ics / laba combination inhaler of fluticasone furoate and vilanterol is currently approved in the united states for use in the maintenance of chronic obstructive pulmonary disease and to reduce exacerbations . this paper reviews the expanding literature on the efficacy of fluticasone furoate and vilanterol in treating asthma .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 50-year - old male reported to the ophthalmic outpatient department ( opd ) with complaints of inability to see with the left eye for the past three months and with the right eye for the past one month . he gave history of difficulty in seeing after sunset for the past few years . he gave history of chronic diarrhea , especially following meals , associated with marked weight loss for the past four years . he denied history of ocular trauma , fever , cough , hemoptysis , chest pain , breathlessness or any systemic medication . patient belonged to poor socioeconomic strata and his diet consisted of only rice with occasional curry . there was dry keratinization of the palpebral conjunctiva with a corrugated appearance akin to skin . the visual acuity was reduced in each eye to perception of light with accurate projection of rays . the cornea of the right eye was reduced to a thin opaque membrane seen in parts only and was covered with mucopurulent strands . the entire iridocorneal mass was protuberant with a whitish / pinkish - appearing patch in the center [ figure 3 ] the cornea of the left eye was shrunken to about 6 mm in diameter , opaque , thinned out and flattened with nasal vascularization . rest of the details of the anterior chamber could not be made out [ figure 4 ] . patient was provisionally diagnosed as keratomalacia right eye and atrophic bulbi with xerophthalmia left eye . he was evaluated to determine any contributory causes for loss of weight and chronic diarrhea . blood sugar , serum electrolytes , stool examination , renal and liver function tests and chest x - ray were normal . hbsag and c - reactive protein were negative , electrocardiogram showed sinus bradycardia and abdominal ultrasound showed splenomegaly . he was given intramuscular injection of vitamin a 200,000 iu on the day of admission , the next day and then after one week . in addition he was treated with topical antibiotics and artificial tears . within a week of the treatment there was a reversal of all changes of conjunctival xerosis and keratinization and the conjunctiva became pink and velvety [ figure 5 ] . however , there was no change in the corneal condition . the patient sought discharge against medical advice despite being advised about his condition . aids caused by hiv is characterized by the deficiency of t helper ( cd4 ) lymphocytes , leading to an inability to combat opportunistic infections . a provisional diagnosis of aids was made based on the world health organization criteria2 which , was subsequently confirmed in accordance with the national aids control organization guidelines.3 besides two major features , weight loss and chronic diarrhea , and one minor feature in the form of generalized lymphadenopathy , the patient was serologically positive too , established by positive elisa . the diagnosis of keratomalacia was based on the findings of a melted cornea in a relatively quiet eye associated with clinical features of xerophthalmia with reversal of the conjunctival signs on parenteral vitamin a therapy . the patient could not afford serum retinol levels and cd4 cell count or plasma viral load estimation . hypovitaminosis a is well documented in children , especially following severe measles where vitamin a stores are rapidly depleted due to increased metabolism.4 active corneal xerophthalmia is extremely rare in older children and adults , except in severe famines . they are a medical emergency and require urgent treatment by high - dose vitamin a supplementation.5 retinol deficiency is quite frequent in the population of hiv - infected individuals . serum retinol levels of less than 1.05 micromol / l determine a 3.5 to five times higher death risk.6 the recommended treatment for hiv - positive individuals is similar to their hiv - negative peers.5 liang et al . , have found that hypovitaminosis a , e , and b12 accelerated the development of aids , whereas their normalization retarded the development of immune dysfunction.7 in ethiopia , vitamin a deficiency is recognized as a serious public health problem among patients with chronic diarrhea , with or without hiv infection . micromol / l ) was observed in 52.7% and 45.5% of diarrheic patients with and without hiv co - infection , respectively . about 13% of healthy controls and 29.3% of asymptomatic hiv - infected blood donors were deficient in vitamin a.8 though hypovitaminosis a is well documented in patients with hiv infection and is aggravated by the associated diarrhea , keratomalacia as presenting ocular manifestation of aids was not found on a medline search of literature . this case is being reported on account of the unusual presenting ocular manifestation of aids .	a 50-year - old male who presented with bilateral keratomalacia and on subsequent evaluation was found to be human immunodeficiency virus ( hiv ) positive is being reported . a medline search of the literature did not reveal any report of keratomalacia as the initial presenting feature of hiv/ acquired immune deficiency syndrome .
You are an expert at summarizing long articles. Proceed to summarize the following text: cemento ossifying fibroma ( cof ) is a benign osseous tumor , which is very closely related to other lesions such as fibrous dysplasia , cementifying periapical dysplasia or cemento - osseous florid dysplasia . it has multipotential cells capable of forming cementum , lamellar bone and fibrous tissue . under pathological conditions more aggressive lesions usually involve the maxillary antrum where extensive growth is unimpeded by anatomic obstacles . tumor manifest as slow - growing , asymptomatic , intraosseous masses , most frequent in females between 35 and 40 . cofs of the mandible are common , but cofs of the maxillary sinus are rare and few have been reported in the literature . the present case report is about a 45-year - old female patient who reported to our department ; in with the chief complaint of huge swelling on the right side of the face , which was gradually increasing in size since last 6 year . he had a history of tobacco chewing since last 15 years . on examination swelling was 9.0 cm 10.0 cm in size at right maxillary region displacing the right alae of nose , columella and septum on the left side . superiorly the swelling displaced the lower eyelid compressing the eyeball giving the sun - set appearance of the eye . laterally the swelling has involved zygomatic region and up to the preauricular region [ figure 1 ] . superficial skin was normal and non - adherent to lesion , but stretched with prominent vascular margin . on palpation the swelling was non - tender , non - compressible and non - reducible . were not palpable . extra oral presentation of lesion intra orally swelling was extending from left central incisor to right second premolar with palatal extension up to mid palatal line obliterating the buccal sulcus . the swelling has displaced all teeth from right central incisor to second premolar and with normal appearing mucosa but stretched with multiple erythematic patches . histological examination of specimen revealed many delicate interlacing collagen fibers , seldom arranged in discrete bundles , interspersed by a large number of active proliferating fibroblasts with occasional presence of cementum like droplets . on the basis of these computed tomography scan revealed a 8.0 cm 8.5 cm 10 cm expansile lesion arising from the right half of the hard palate and maxillary alveolar arch . the lesion showed mildly enhancing high attenuation material with foci of calcification dispersed in it . the lesion showed well defined anterior margin and irregular and eroded posterior and lateral margin . medially the lesion was involving the right nasal fossa resulting in deviation of the nasal septum towards the left side . the soft - tissue overlying the lesion all over is thinned by the lesion [ figures 2 and 3 ] . orthopantomogram demonstrate extent of lesion computed tomography scan demonstrate extent of lesion as per histological findings patients was planned for excision of the lesion . the lesion was excised until healthy margin of bone were achieved , through extra oral weber - ferguson approach under general anesthesia . per operatively it is removed with minimal bleeding and minimal injury to vital structure [ figure 4 ] . excised specimen [ figure 5 ] was submitted for histopathological examination , which confirms the diagnosis of ossifying fibroma [ figure 6 ] . post - operative profile view histomicrograph of specimen post - operatively patient had mild post - operative hematoma of superficial skin on the right side of the face . the level of the right eye was restored to normal which was elevated pre - operatively due to massive size of the lesion . anatomic structure such as alae of nose , columella , septum and upper lip was also restored to normal posture . the above mentioned case is presented hereby because of its huge size causing facial asymmetry which is very rare . it was a case in which the lesion rarely presents in the maxilla with a weight of the lesional mass was 350 g. fibro - osseous lesions of craniofacial skeleton are rare and believed to be the result of replacement of normal bony architecture by fibrous tissue , which may , mineralize in various forms such as woven , lamellae bone , or cementum and include a broad spectrum of distinct entities with different clinical presentations and microscopic appearance . most of the calcified fragments are immature cementum ; these tumors are named central cementifying fibroma . in other cases , central cementifying fibroma and central ossifying fibroma arise from the same progenitor cell but produce variable amounts of bone and cementum within any one lesion . the hybrid term central cof may be used to designate the presence of both types of tissue within the same lesion because of the difficulty in being able to distinguish reliably immature bone from immature cementum and because of the presence of both of these substances in many of the lesions . thus , central cof is the most accurate term ; it can be interchanged with either central ossifying fibroma or central cementifying fibroma . there is no apparent clinical or radiologic difference between the central cementifying fibroma or central ossifying fibroma . the mean age of patients at the time of diagnosis is 34 years ( range , 16 - 62 years ) . the mandible is involved more often than the maxilla , especially the premolar and molar regions . predilection for the mandible has been demonstrated in various studies . in the 64 cases of cof reported by eversole et al . , the lesions were found most frequently in the molar region ( 52% ) , followed by the premolar ( 25% ) , incisor ( 12% ) and cuspid ( 11% ) regions . swelling displaced alae of nose , columella and septum towards the left side , lower eyelid displaced superiorly giving rise sun - set appearance due to aggressive growth which is consistent with features described in the literature . bone swelling or expansion at the buccal and/or lingual cortical plates is the most frequent clinical sign of cof ( 96% , ) . root displacement has also been demonstrated in 17% and 18% of cofs reported by eversole et al . and sciubba and younai respectively . fibrous dysplasia and cof are clinically and radiologically distinct disease entities that nevertheless are not always histologically distinguishable . cof with a completely radiolucent lesion may be misdiagnosed as early stage of cemento - osseous dysplasia , odontogenic cyst , periapical granuloma , traumatic bone cyst , ameloblastoma , or central giant cell granuloma . cof with mixed radiographic features might be given a nonspecific diagnosis of fibroosseous lesion , or misdiagnosed as a calcifying odontogenic cyst ( gorlin cyst ) or an adenomatoid odontogenic tumor . other differential diagnoses of cof with mixed radiographic features may include rarefying and condensing osteitis , intermediate stage of cemento - osseous dysplasia , fibrous dysplasia , calcifying epithelial odontogenic tumor ( pindborg tumor ) , or odontogenic fibroma . furthermore , cof with completely radio - opaque radiographic features may be misdiagnosed as retained root , odontoma , idiopathic osteosclerosis , condensing osteitis , late stage of cemento - osseous dysplasia , or osteoblastoma . early lesions may be radiolucent as they mature , they become a mixed radiolucent and radio - opaque lesion and finally become radio - opaque . microscopically , cofs showed trabeculae or spherules of mineralized materials in a cellular fibrous connective tissue stroma . the characteristic microscopic criteria for diagnosis of cof include presence of a mixture of woven and lamellar bones and cementum - like materials in a cellular fibrous connective tissue stroma in addition , osteoblastic rimming is usually found . the well - defined borders of the central cof differentiate it from the aggressive sarcomas and carcinomas . fibrous dysplasia has a characteristic ground glass appearance not seen in the central cof . the radiologic differentiation of central\cof from gorlin cysts and pindborg tumors is difficult ; the final diagnosis is based on histologic appearance . complete removal of the lesion at earliest possible treatment been suggested by majority of the authors . appropriate treatment for a benign fibro - osseous lesion , irrespective of its aggressive nature includes either curettage or enucleation of the lesion , until healthy margins are reached . lesions that did not produce marked deformity or obstruction at initial presentation , curettage or peripheral ostectomy alone appeared to be adequate management along with long - term clinical and radiographic follow - up . recurrence rates ranging from 30% to 58% and 0% to 28% have been described by mintz and velez and chang et al . respectively . since recurrence rate is variable therefore patients should be followed - up regularly .	ossifying fibroma of bone is a central neoplasm of bone and it is more common in young adult with marked predilection for mandible and also it is more common in female . lesion is generally asymptomatic until growth produces a noticeable swelling and mild deformity . it presents an extremely variable roentographic appearance depending upon the stage of development . this lesion is composed basically of many delicate interlacing collagen fibers , seldom arranged in discrete bundle , interspersed by large numbers of active , proliferating fibroblasts . the lesion should be excised conservatively . we present a case of huge ossifying fibroma arising from maxilla .
You are an expert at summarizing long articles. Proceed to summarize the following text: miller fisher syndrome ( mfs ) is characterized by the acute onset of external ophthalmoplegia , ataxia of cerebellar type , and the loss of tendon reflexes . it is considered a variant of guillain - barr syndrome ( gbs ) , because some patients who present with mfs progress to gbs . in contrast , patients who show drowsiness , brisk reflexes , extensor plantar responses and hemisensory disturbance are usually considered to have bickerstaff 's brainstem encephalitis ( bbe ) rather than mfs . the fact that bbe and mfs share a common autoantibody suggests that they are closely related . bickerstaff and cloake speculated that the etiology of bbe is similar to that of gbs because they found areflexia and cerebrospinal fluid albuminocytologic dissociation ; however , a case of overlapping mfs , gbs and bbe has not been fully reported . a 72-year - old woman developed diplopia and unsteady gait in the morning ( day 1 ) a week after an upper respiratory infection . she was admitted to our hospital due to weakness of both upper limbs on day 3 . on admission , the general physical examination was normal . on neurological examination , she had bilateral blepharoptosis . her eyeballs were fixed in the central position . she had weakness of both upper limbs . hematological investigation showed an elevated white blood cell count of 9,500/l ( segmented leukocytes , 86.5% ; lymphocytes , 10.5% ) , and her cerebrospinal fluid ( csf ) showed 2/l ( 100% mononuclear cells ) and 30 mg / dl protein . magnetic resonance imaging ( mri ) showed a normal brain . we diagnosed mfs on day 1 , and she developed gbs on day 3 . on day 5 , she became drowsy , with a worsened respiratory status and weakness in all four limbs . this condition was considered to be overlapping bbe . on day 12 , electroencephalography ( eeg ) showed background activity at 78 hz with no epileptiform discharges . on day 21 , a nerve conduction study of the right median nerve showed abnormal amplitude reductions in both the forearm and upper arm . motor nerve conduction velocity in the left median nerve was decreased ( 44.3 m / s ) . the sensory nerve action potential amplitude in both median nerves was reduced , and sensory nerve conduction velocity was normal . on day 51 , anti - gm1 igg , gd1a igg , gq1b igg , and gt1a igg antibodies were positive , and anti - gq1b igg antibody titer was the highest . she underwent intravenous immunoglobulin therapy from day 3 to 7 and seven sessions of immunoadsorption therapy from day 14 to 28 . on day 30 we added intravenous immunoglobulin therapy from day 34 to 38 . on day 40 , she began to open her eyelids and move both eyeballs . on day 54 , her consciousness became alert . on day 56 , she was taken off the respirator . on day 63 , she could stand up , but the ataxic gait remained . on day 90 , she changed hospital for rehabilitation ( fig . we report a case of overlapping mfs , gbs and bbe during the course of illness . neurologically , the responsible lesions were suggested as the pyramidal tract , nuclear or infranuclear of the ocular motor nerves , brainstem reticular formation , cerebellum and peripheral nerves of the limbs . we diagnosed her with mfs on day 1 , gbs on day 3 and overlapping bbe on day 5 . overlapping mfs / gbs and bbe / gbs syndromes have been reported [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ] ; however , only two case reports have described overlapping mfs / gbs / bbe syndrome ( table 1 ) . arai et al . described a patient with bbe on day 1 , mfs on day 2 and gbs on day 5 in the presence of anti - gq1b antibody . stevenson et al . described a patient with bbe on day 1 , gbs on day 9 , and mfs on day 10 in the presence of anti - gm1 antibody . although these two patients underwent plasma exchange and intravenous immunoglobulin therapy , respectively , our patient required mechanical ventilation and underwent immunoadsorption therapy and two courses of intravenous immunoglobulin therapy . anti - gm1 igg , gd1a igg , gq1b igg , and gt1a igg antibodies were positive in our case . the abundant and synaptic - specific binding of anti - gq1b , -gt1a , and -gd1b ganglioside antibodies and the rich capillary supply in the human extraocular muscles may partly explain the selective paralysis of these muscles in mfs . some large neurons of the dorsal root ganglia were immunostained with anti - gq1b monoclonal antibody . gq1b also exists in nerve endings near the skeletal muscle spindle and anti - gq1b igg antibody may thus be associated with ataxia as well as ophthalmoplegia . our case supports a previous proposal by odaka et al . that bbe , mfs , gbs and acute ophthalmoparesis are all part of a continuous clinical spectrum , which is an antibody - mediated process . anti - gq1b antibody is detected in gbs , fs and bbe , so it is called anti - gq1b igg antibody syndrome , and is useful for understanding the etiological relationships among those illnesses . because of the similarities in the clinical presentation and autoimmune etiology of mfs and bbe , a terminology fisher - bickerstaff syndrome may be helpful for nosology . although the clinical picture , neurophysiology , and csf findings are usually sufficient to indicate therapies , the presence of antiganglioside antibodies is a useful guide to diagnose this group of conditions .	we report a 72-year - old woman with overlapping miller fisher syndrome ( mfs ) , guillain - barr syndrome ( gbs ) and bickerstaff 's brainstem encephalitis ( bbe ) . she developed diplopia and unsteady gait a week after an upper respiratory infection on day 1 . she had weakness of both upper limbs on day 3 and became drowsy , and her respiratory status worsened on day 5 . neurologic examination revealed ophthalmoplegia , ataxia , symmetrical weakness , areflexia , and consciousness disturbance . we diagnosed her with mfs on day 1 , gbs on day 3 and overlapping bbe on day 5 . she underwent immunoadsorption therapy and two courses of intravenous immunoglobulin therapy . ten months after onset , her symptoms had fully recovered . anti - gm1 igg , gd1a igg , gq1b igg , and gt1a igg antibodies were positive . our case supports the notion that mfs , gbs , and bbe are all part of a continuous clinical spectrum , which is an antibody - mediated process .
You are an expert at summarizing long articles. Proceed to summarize the following text: in norway we find the most gender segregated labour market in europe and a vast market of part time jobs mostly occupied by women . according to esping - andersen it has been possible to combine high female participation with high birth rates only at the cost of extraordinarily gender segregated employment . the majority of contemporary scandinavian women opt for the dual - role model , intent on lifetime employment , but unwilling to sacrifice motherhood . in scandinavia the rising birth rates are closely related to public expansion of welfare , health , and education services . is it on this background , esping - andersen asks , possible to conceive a society with a labour market that optimises not only women s ability to work and have children , but one that also lessens gender segregation ? his conclusion is that the key issue of gender equality ( like any inequality ) lies in life course dynamics ; true gender equality will not come about unless , somehow , men can be made to enter a more feminine life course . to some degree men in scandinavia are doing this ; increasingly they take parent leave , contribute more at home , and they often refuse to travel in business . this has however so far had little influence on the gender segregation in the labour market and at the arena of education . there are still a majority of women in health and social studies , and a majority of men in engineering , and it ; a difference that is connected with value and recognition . teaching , supervising and doing research within the arena of health and social work has taught me how difficult it is for female professionals to get recognition for their work , often also by themselves . this holds at a personal level as well as at an institutional level . for four years i was elected member of the board of the university college at which i work . soon i became aware of how my department of health and social studies is the least recognised of all departments . the staff at this department is working more hours and having less time to do research than their colleagues at the more male staffed departments such as engineering , teaching , and it . in my view this is owed to the fact that the majority of the workforce in my department is female , and that the areas of care and social work are perpetually linked to women . the leaders of my department realise that they are acting as good girls , following all the rules and expectations . thus , in spite of a constant frustration , every one of us contributes to the cementation of this societal and academic hierarchy . as i write this article , nurses in norway are pictured with a beard demonstrating that the inequality in wage paid for the same level of competence is gender biased . maybe these bad girls of the next generation represent a hope for the change that good girls shall never experience . inspired by various ethical theories and own empirical research i suggest placing care in a broader phenomenological understanding . like tronto , i maintain that there is no starting point for care ; it is part of life itself and thus part of the responsibility of every human being whichever culture , gender , position , and age . in the following i will shortly resume the history of care ethics and present some challenges of today . secondly i will look into some ethical approaches addressing care as part of the basic ontology of human existence . together with my own empirical studies revealing how power and gender conflate our relations and interactions privately and publicly from birth to death care is an ontological given in the sense that to live is to care in the framework of interdependency . when care is defined as an existential condition of life , demanded from all of us , it opens up for a more gender equal and just society with lesser gender segregation in the labour market and at the arena of education . since gilligan the scholarship on care and care ethics has exploded . in ethics of care and feminist ethics diemut bubeck renders a thorough review of the publications , discussions , problems and perspectives of care in the last 15 years . in spite of many differences the commonality between them is that care so far has taken place under oppressive conditions . care is probably the most significant and important category of work that is accorded to women via the sexual division of labour , and a lot of it continues to be done unpaid or badly paid if the latter , then often by women from less privileged class and ethnic backgrounds.care is thus inextricably intertwined with oppressive gender divisions : care is done by women , it is associated with women and it expresses and symbolises femininity [ 2 , p. 26 ] . according to bubeck the enthusiasm for theorising care neglects the extent to which theory is corrupted by the oppressive conditions under which care is practised . as a result the only themes of distortion discussed by care ethicists are self - sacrifice and self - denial . on this background diemut bubeck suggests theories of care to take into account that practise of care reflects the conditions under which it takes place , otherwise it will paint a distorted picture of care . it is necessary to look into questions such as : the boundaries between carer and cared- for example the danger of solving the problems for the one in need of care rather than letting him / her find a solution in their own time and on their own condition . closely linked to this is the conflict of need between carer and cared - for ; the problem of the care person seeing the other person s needs when they are in conflict with his or her own . together these questions amount to the more general question of use and misuse of power in the best intention . more recently some authors have addressed these questions , however mostly within the frame of celebrating care and care ethics , while others , like joan tronto and selma sevenhuijsen have been concerned about the less fortunate conditions under which care often is practised which again influences recognition and payment . in this article , however , i shall focus on some ethical approaches placing the questions raised by bubeck in an ontological and phenomenological context , universal and relevant for all human beings . care is thus inextricably intertwined with oppressive gender divisions : care is done by women , it is associated with women and it expresses and symbolises femininity [ 2 , p. 26 lgstrup is well known in denmark and norway and lately his philosophy has caught increasing interest abroad . the title infinitely demanding reveals just how inspired simon critchley is by lgstrups philosophy of the ethical demand . lgstrup points to the ethical significance of phenomenology and has been presented as giving a key critique of modern rationality . being a danish citizen i studied theology , including the subject of ethics , at rhus university where lgstrup was a professor gathering listeners from within and outside the academia until his death in 1981 . the ethical theory of lgstrup is linked mostly to his early philosophy , especially his book the ethical demand , published in 1958 ; in english 1972 , 1989 . the ethical demand springs from this interdependency requiring each human to take care for the other without taking responsibility from him or her . whether this other is capable of formulating her want or not , the challenge of the ethical demand is that it requires that i i.e . any one of us use our knowledge , understanding and love to consider what is in the other s best interest . if lgstrup had addressed it , he would have been concerned about the powerlessness of the patient and the power of the institutional system [ 8 , 11 ] . and most probably lgstrup would have consented to bubeck arguing that there are no pockets of non - distorted caring and that selfloss is a risk , not only in the practise of care but as an existential danger to everyone living in a relation . the concept , even the word care , may not cover the intention of lgstrup . while traditionally care is recognised as something you can do or not do , care in the framework of interdependency is a necessary condition of life . within the framework of interdependency we , men and women , as human beings , are dependent and independent throughout life . this differs radically from the tradition that has taught us to develop from dependency into independent and autonomous adults . by taking ontological interdependency as a point of departure interdependency means that every one of us holds some of the life of the other in our hand . continuously confronted with each other s lives it is not possible not to be involved , to use another word than care . if i choose to do nothing , also my not doing or not saying will make a difference . we are interdependent in the sense that we influence each other with what we do and say and by what we do not say and do ; we are each other s authors , to use a metaphor of max frisch . the danger of transgressing boundaries is always there , because the ethical demand is including all of us always . in the relation between any two persons there is a tension between dependence and independence : between the other being totally in my hand and the other being responsible for herself and vice versa . this is maybe the most common ethical dilemma of everyday life , well known to all of us . what is in the best interest of the other person whether she is my child , spouse , client , or colleague ? according to lgstrup it does not suffice to do what the other person tells me to do . although i am not supposed to take responsibility away from the other person , i am supposed to take the ethical demand seriously . and i therefore may have to oppose her verbal or non - verbal wishes if i find it to be in her best interest in the long run . this way lgstrup maintains the interdependence from which the ethical demand springs while at the same time emphasizing the responsibility of each human person for him or her self and for the other . that the ethical demand in lgstrup s philosophy is unilateral and radical , underlining it is possible to read lgstrup as supporting the subject / object system in the sense that the other is an object left to my subjective evaluation of what is best for her . and it is possible to maintain that ethics for lgstrup fundamentally is about thinking in first persons terms . as pointed out by sren holm this leaves the health care areas with the challenge of establishing stable and predictable actions . a health care ethics suitable for modern health care need not only answer lgstrup s questions of how should i act , but also to the question of how should we act . the balance between i and we is relevant ; it is however , exactly what lgstrup s approach inherently criticizes . in my view , the critics neglect that radicalism of the demand has to be seen in the framework of interdependency . each of us is both subject and object , independent and dependent , throughout life . in other words , by turning the question of paternalism versus non - intervention into an ongoing responsibility of both parts , lgstrup in fact demolishes the subject - object system , consistently concerned about deconstructing the myth of the independent autonomous human being [ 3 , 4 ] . as soon as it turns into rules , how we should act , as relevant as it may be at a ward or a hospital , it is necessary to be en garde and observant . the ethical approach of lgstrup , his claim of interdependence , offers a productive framework in that it is open to different ways of responding to the ethical demand of the other . interdependence means that every one of us are i , s as well as others . not one approach to such requests is ethical or unethical per se , which is to say that it is possible to care in many ways . in my study of five social pedagogues in their first year of work they kept returning to the concept of nearness ; one of them even described that the only way she managed to work was by being when i asked them to define nearness , and why they found it important , they unanimously said that nearness was necessary to establish a good relation . when i probed what they meant by a good relation , one of them suddenly , and evidently shaken , burst out : because then the client does what i want her to do ! these informants had , since the last year of their bachelor study , been deeply concerned about how to transfer ethical theories into practise . it was shocking for them to realise how they came to exert the professional power they so determined to avoid . by way of nearness and a good relation they made the client how come that these otherwise fairly professional informants did not see their misuse of power in the best intention ? i was supervisor for a male student in his period of practise at a nursing institution for old people . he was asked to attend to a resident , also male , who was paralyzed . after some weeks of his practise , the student asked me if he was allowed to focus his report on the difference between men and women . encouraged by me the student presented a report on a very successful practise , concluding that it is important to get more men into the caring profession ! while the conclusion is trivial , his report is brilliant in documenting and analysing how his work came to be such a success . it appeared that the old man was an incarnated patriarch , resisting aggressively help from any of the female staff . the student could well understand why the old man s wife only came visiting now and then . the student was a mature man around 40 ; adding what he had learnt at school to his own life experience he managed both to encourage the old man and to train him to become much better in managing his handicap . acknowledging that he had done good professional work , the student insisted that he would never have had a chance if he had not been gendered male . when the student added that the only solution was to employ more men , i thought at first that he just made it easy for himself . from the perspective of the student his colleagues were very professional in their way of working ; due to their gender , however , they rarely got a chance to prove their competence as a professional one . while his work was seen and recognised as professional work , their work was reduced to care , as something that any one can do . without being able to theorise his experience it is , however , as he indicated , not gender per se that is the problem . it is the power , the factual and the symbolic power that is allocated to gender that is problematic , not least within the arenas of health and social work . how come that the interdependency works out differently for the sexes ? in order to understand what is at stake in the relation of interdependency in general and in the asymmetrical relations of professional care i had to look out for other ethical approaches than lgstrup s . although lgstrup had a phenomenological understanding of the body , seeing the body as something we are and not something we have , he did not address the question of the difference that the sexual embodiment makes . merleau - ponty in his work on the phenomenology of perception , also like lgstrup neglected the implications of sexual embodiments . he did , however , inspire simone de beauvoir who in a review of his book declared that his philosophy has made a new ethics possible by way of abolishing the opposition between subject and object and claiming that the human being is a body . in the nineteenforties and onwards , when lgstrup was developing his philosophy of the ethical demand , simone de beauvoir was developing her ethics of ambiguity . like lgstrup , beauvoir based her ethical understanding on the existential condition of interdependency as common to all of us . while unfolding her ethical understanding , beauvoir reluctantly had to realise that her intention of replacing the asymmetrical subject / object relation with a relation of symmetry , with inter - subjectivity , had to begin with the question : what is a woman , what is the meaning of the being a woman when she is not seen nor sees her self as a subject ? beauvoir s main point is that woman s specific ethos , no less than a man s , is constituted in a social context as well as in relation to others . like lgstrup , in fact i have often wondered what position she would have taken if she had considered that the main occupation of women then as well as today , is to care , due to a gender segregated society . in developing beauvoir s ethical understanding diprose shows how it inherently presents a challenge to how ethics of care has developed since gilligan . recognising that ethics of care has put the self - other relation on the ethical map diprose criticizes it for assuming that the self - other relation is already in place , and that the identity and difference of the parts involved is given , prior to care . when identity and difference are given , the central theme in an ethics of care is how the other s difference can be considered in one s resolution of moral dilemmas . , the focus on how to understand the difference of the other leads to a celebration of women s relatedness and women s apparently shared moral perspective , keeping women in their place . instead diprose suggests giving attention to the implication of two important claims : ( a ) that the self develops an embodied and sexed identity within and as an effect of a specific social context , and ( b ) that one s identity is constituted in the context of and through relations with others . my studies [ 15 , 16 ] confirm diprose s theory in how the work of my informants not only is influenced by , but also is inseparable from their bodies and their sexed identity . their identities as well as the identity of the client- are fixed and not fixed at the same time , depending on relation and situation , action and interaction . at the level of practise as well as at the level of knowledge production and politics , gender identity , or preferably sexual difference , is part of an ongoing negotiation and definition . there are many examples of how dangerous nearness is for women ; most often it implies a female monopolisation of responsibility , damaging for both parts in the professional caring interaction , leaving them with only little recognition and demolishing their professionalism . at the same time nearness is necessary in order to take the ethical demand from each human being seriously . also the male student had to come close enough to find how to help the old resident in the best possible way . unlike the female informants he does not use the word nearness as he is never conceived as being too near . while most men do not see , or are not raised and trained to see , themselves as gender , most women feel or become aware of their difference , striving to find a way of working that will be acknowledged professionally . often this results in an ambiguity : partly working the way the culture demands , partly working in her own way ( nearness ) , the female professional often works without getting recognition , not even from her own self . unable to relate or implement her practise to the theory she was taught at school , she does not see her practise as creating professional knowledge . consequently she enters a vicious circle of silence , of not contributing to knowledge production [ 15 , p. 204 ] . in a sad way the male student came to confirm my analysis , that care work is reflecting how power doing studies in various fields of health care made me understand the limitation of lgstrup s ethical demand . it is as important to identify gender and how it is conflated with power , as important it is underline that gender is not to be used as an excuse . not one ethical approach can cover the complexity of the challenging dilemmas of health and social arenas . it is , however , necessary that ethical theory and practise takes into account that all of us are embodied and gendered human beings , dependent and independent , constructing and being constructed by various factors in our personal and public lives . thereby reflecting the argumentation of walker that differently placed people have different knowledges , which again presupposes being able to see and reflect critically on one s own cultured social , moral and gendered construction . only such an ethics will be able to criticise traditional normative ethics , for having failed to implicate the social context for the constitution of identity , and thus for how we respond to the most challenging processes in our society today . the most challenging processes today are according to andersen the processes of othering and eldering . these processes begin when each of us enters the need of help . othering and eldering are two different processes . othering is the process that makes us the other , the different one , the one we strongly dislike to be , the one who costs too much . while eldering is something that happens to everyone , othering is something we do to someone . othering is a psychological and a political process in which we create the other with perceptions and attitudes , a process that is not restricted to old people in need of help . by not mentioning that othering and eldering are processes conflated with gendered power relations , andersen comes to underline ( with lgstrup ) that the ethical demand is posed to every one of us . at the same time it is important to see and identify how all our relations are inextricably gendered for good and for worse ( beauvoir , diprose ) the main character walt kowalski , played by clint eastwood , is a conservative grumpy old white man who after the death of his wife finds himself estranged by the people surrounding him , different as they all are in colour , culture and religion . he neither likes them nor their children , and this goes for his own children as well . in walt kowalski is not hit by a stroke , but of an attempt to steal his car , the gran torino . a menacing gang has forced the boy in the house next to kowalski s to do this as an initiation . his sister intercedes for her brother and kowalski reluctantly accepts that the brother makes amends by working in the service of kowalski . with this event the movie explodes into an example of how intersubjectivity and interdependency works out in daily life . both have inter as prefix , indicating a space between the subjects . in this space power is at stake . exposed to othering and eldering , and exposing others to the same , walt kowalski experiences that the self only exists in the complex web of its varied relations , and that it is possible to change by means of accepting the responsibility each of us has as human beings independent of sex , religion , age and culture . gender identity is part of an ongoing negotiation and definition at the level of practise as well as at the level of knowledge production and politics . to pave a way towards a less gender segregated society in the arena of the labour market and in the arena of education i suggest to conceive care as the existential condition of life , thus freeing care from the identification with women . within the framework of interdependency both men and women are , as human beings , dependent and independent throughout life . this differs radically from the tradition that has taught us to develop from dependent into independent and autonomous adults , which again makes a difference in the understanding of care . in this tradition care a necessary condition of life is a perspective solidly unfolded in the above mentioned ethical approaches , each in their way describing how every human being from the very beginning of life is inserted as subject , as a citizen , with a need to care and to be cared for . when care is defined as an existential condition of life , it opens up for various ways of answering the ethical demand of the other . from childhood to old age we live our private lives in a continuous challenge to balance difference and sameness , independence and dependence . one of the most challenging dilemmas of professional life is the same : to live this balance in relations and interactions like the ones between staff and clients , teachers and students . narratives like gran torino remind us that there is only one real citizen , a citizen who locates her / himself within the bonds of human interdependence . to neglect this we come to reduce as walt kowalski does in the beginning of the movie the world to objects , left to our analysis , and forget how differentiated the world , the language and the bodies in it are . to navigate in the landscape of care requires observing and analysing critically how deeply our personal and societal structures are influenced with ethics , power and gender .	the theory and practise of care is defined and enacted differently in different national as well as cultural contexts , illuminating how differently constructed the personal and societal structures in europe are . a common trait is however that care work paid or non - paid , private or public is identified with women . to navigate in the landscape of care and ethics requires taking into account the constitutive relation between one s identity , embodiment and position . the author suggests conceiving care as an existential condition of life demanded from all human beings . this will free care from the identification with women and pave a way towards a more gender equal and just society with less gender segregation in the labour market and at the arena of education .
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Proceed to summarize the following text: si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck . oceanapia sagittaria ( sollas , 1902 ) , order haplosclerida , family oceanapiidae , was collected by scuba diving in the gulf of thailand near koh samed ( 3 meter depth ) in april 2005 and frozen immediately at 20c prior to extraction . sumaitt putchakarn and the voucher registered as bims - i 2004 was deposited at bangsaen institute of marine science , burapha university , thailand . the sample ( 3.5 kg net weight ) was thawed , homogenized with etoh ( 7l ) , allowed to stand for 24 h in the dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2x7l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca . the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 13.1 g ) . the crude etoac extract ( 13.1 g ) was applied over a flash chromatography column of si gel 60 ( 150 g ) and eluted with a solvent gradient system of petrol - chcl3 , chcl3 , chcl3,-meoh , etoac and meoh , with 200 ml fractions being collected as follows : frs . 12 ( petrol - chcl3,1:4 ) , 37 ( chcl3 ) , 817 ( chcl3,-meoh , 49:1 ) , 1822 chcl3,-meoh , 19:1 ) , 2324 ( chcl3,-meoh , 9:1 ) , 2526 ( etoac ) , 2730 ( meoh ) . 34 were combined ( 2.6 g ) and recrystallized from a mixture of petrol and chcl3 to give white crystal of 24 -methylcholestanol ( 1 , 436 mg ) . 67 were combined ( 870 mg ) and chromatographed over si gel 60 ( 20 g ) and eluted with petrol - chcl3 , chcl3-me2co , and chcl3-meoh , 200 ml subfractions were collected as follows : sfrs . 17 ( petrol - chcl3 , 2:3 ) , 815 ( petrol - chcl3 , 1:4 ) , 1624 ( chcl3 ) , 2530 ( chcl3-me2co , 19:1 ) , 3134 ( chcl3-me2co , 9:1 ) , 3538 ( chcl3-me2co , 4:1 ) , 3943 ( chcl3-me2co , 3:2 ) , 4445 ( meoh ) . 36 were combined ( 156 mg ) and purified by tlc ( si gel , chcl3 ) to give 33.2 mg of a mixture of p - hydroxybenzoic acid ( 3 ) and phenylacetic acid ( 4 ) . 715 were combined and purified by tlc ( si gel , chcl3-me2co - hco2h , 18:1:0.1 ) to give kuanoniamine a ( 6 , 20 mg ) . 810 were combined ( 390 mg ) and purified by tlc ( si gel , chcl3 , me2co , hco2h , 95:5:0.1 ) to give p - hydroxybenzaldehyde ( 2 , 15.7 mg ) and 3-formylindole ( 5 , 18.6 mg ) . 1113 were combined ( 346 mg ) and purified by tlc ( si gel , chcl3 , meoh , hco2h , 98:2:0.1 ) to give kuanoniamine a ( 6 , 32 mg ) . 2022 were combined ( 355 mg ) and applied over a flash chromatography column of ods gel ( 10 g ) and eluted with a gradient system of meoh - h2o and meoh - chcl3-h2o , 100 ml fractions being eluted as follows : sfrs 12 ( meoh - h2o , 7:3 ) , 34 ( meoh - h2o , 4:1 ) , 56 ( meoh - h2o , 9:1 ) , 710 ( meoh ) , 1114 meoh - chcl3-h2o , 7:3:0.5 ) . 56 were combined ( 81 mg ) and recrystallized from a mixture of chcl3 and me2co to give yellow crystals of kuanonamine c ( 7 , 44 mg ) . fetal bovine serum ( fbs ) , l - glutamine , phosphate buffered saline ( pbs ) and trypsin were from gibco invitrogen co. ( scotland , uk ) . acetic acid , cyclosporin a , dimethyl sulfoxide ( dmso ) , doxorubicin , ethylenediaminetetraacetic acid ( edta ) , histopaque-1077 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , penicillin , phytohemagglutinin ( pha ) , propidium iodide ( pi ) , rnase , streptomycin , sulforhodamine b ( srb ) , triton x-100 , trypan blue were from sigmachemical co ( saint louis , usa ) . magnesium chloride , paraformaldehyde , tricloroacetic acid ( tca ) , tris , sodium citrate and sodium dodecyl sulphate ( sds ) were sourced from merck ( darmstadt , germany ) . vectashield , vectashield containing dapi ( 0.5 g / ml ) were from vector laboratories ( uk ) . facscalibur flow was from becton dickinson ( mountain view , ca ) , [ h]-thymidine from amersham ( illinois , usa ) and scintillation liquid from perkinelmer ( boston , usa ) . stocks solutions of kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) were prepared in dmso and kept at 20c . five human tumour cell lines , mcf-7 ( breast adenocarcinoma , estrogen - dependent er ( + ) ) , mda - mb-231 ( breast adenocarcinoma , estrogen - independent er ( ) ) , nci - h460 ( non - small cell lung cancer ) , sf-268 ( glioma ) and uacc-62 ( melanoma ) and one non - tumour cell line , mrc-5 ( diploid embryonic lung fibroblast ) were used . mcf-7 , mda - mb-231 and mrc-5 were obtained from the european collection of cell cultures ( ecacc , salisbury , uk ) while nci - h460 , sf-268 and uacc-62 cell lines were provided by the national cancer institute ( nci , bethesda , u.s.a . ) . cells , growing as monolayer , were routinely maintained in rpmi-1640 medium supplemented with 5% heat - inactivated fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) , at 37c in a humidified atmosphere containing 5% co2 . exponentially growing cells were obtained by plating 1.510cells / ml for mcf-7 , mda - mb-231 , sf-268 and mrc-5 , 1.0 10cells / ml for uacc-62 and 0.75 10cells / ml for nci - h460 , followed by 24 h incubation . the effect of the vehicle solvent ( dmso ) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration ( 0.5% ) of dmso used in each assay . the effects of the kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) on the growth of human tumour and non - tumour cell lines were evaluated according to the procedure adopted by the national cancer institute ( nci , usa ) in the in vitro anticancer drug discovery screen that uses the protein - binding dye sulforhodamine b to assess cell growth [ 18 , 19 ] . exponentially growing cells were then exposed for 48 h to five serial concentrations of kuanoniamine a ( 0.14 ; 0.69 ; 3.46 ; 17.30 and 86.51 m ) and kuanoniamine c ( 0.11 ; 0.53 ; 2.67 ; 13.37 and 66.84 m ) that were starting from a maximum of 25 g / ml ( 86.51 m and 66.84 m , respectively ) . following this exposure period adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with 0.4% srb solubilized in 1% acetic acid . the bound stain was solubilized in 10 mm tris and the absorbance was measured at 492 nm in a microplate reader ( biotek instruments inc . , powerwave xs , wincoski , usa ) . for each cell line a dose - response curve was obtained and the growth inhibition of 50% ( gi50 ) , corresponding to the concentration of kuanoniamine a and kuanoniamine c that inhibited 50% of the net cell growth was calculated as described elsewhere . the effect of kuanoniamine a and kuanoniamine c on the mitogenic response of human lymphocytes to pha ( 10 g / ml ) was evaluated using a modified version of the colorimetric mtt - assay . briefly , mononuclear cells , isolated from heparinized peripheral blood of healthy volunteers by histopaque-1077 density centrifugation , were adjusted to 23 x 10 cells / ml in rpmi-1640 supplemented with 10% fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) . they were then harvested in 96-well plates and exposed for 4 days to seven serial concentrations of kuanoniamine a ( 0.12 ; 0.36 ; 1.07 ; 3.20 ; 9.61 ; 28.84 and 86.51 m ) and of kuanoniamine c ( 0.09 ; 0.28 ; 0.83 ; 2.48 ; 7.43 ; 22.28 and 66.84 m ) at 37 c . following this incubation period , the mtt solution ( 1 mg / ml ) was added and plates were incubated for more 4 h. the water insoluble formazan dye formed was solubilized with sds / dmf solution ( 20% sds in a 50% solution of dmf , ph 4.7 ) overnight at 37 c . the absorbance of the colored solution was then measured in a microplate reader ( biotek instruments inc . the concentration of compounds that inhibited 50% of lymphocyte proliferation ( ic50 ) was calculated by the curve obtained ( % inhibition versus concentration ) . seven serial concentrations ( 0.0014 ; 0.01 ; 0.04 ; 0.18 ; 0.89 ; 4.44 and 22.20 m ) were studied in the same manner . mcf-7 cells growing exponentially in 96-well plates were treated with a range of concentrations of kuanoniamine a ( 0.04 ; 0.08 ; 0.17 ; 0.34 ; 0.68 ; 1.35 ; 2.70 ; 5.41 ; 10.81 ; 21.63 ; 43.26 and 86.51 m ) and of kuanoniamine c ( 0.03 ; 0.07 ; 0.13 ; 0.26 ; 0.52 ; 1.04 ; 2.09 ; 4.18 ; 8.36 ; 16.71 ; 33.42 and 66.84 m ) for 6 , 12 , 24 and 48 h. following this incubation period cell growth was assessed by [ h]-thymidine incorporation , mtt and srb assays . in the [ h]-thymidine incorporation assay cells were further incubated with 1 ci of [ h ] thymidine for 4 h. pulsed cells were then harvested on a glass filter 102 x 256 mm ( skatron , norway ) using a semiautomatic cell harvester ( skatron instruments , norway ) and allowed to dry . incorporation of radioactive thymidine was determined in a scintillation counter ( ls 6500 , beckman instruments , ca and usa ) and defined by comparing the arithmetic mean of counts per minute ( cpm ) of treated cells with that of untreated control cells . in the mtt assay , mtt was added ( 5 mg / ml ) and cells were incubated for a further 4 h. formazan product were solubilized with sds / dmf solution overnight at 37c . the percentage of mtt reduction was calculated comparing the absorbance of treated cells to that of untreated control cells . in the srb assay adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with the protein binding dye ( 0.4% srb ) solubilized in 1% acetic acid , and then the bound stain was solubilized in 10 mm tris , as described . the bound stain was solubilized and the absorbance was measure at 492 nm in the microplate reader . mcf-7 cells growing exponentially in 25 cm flasks were treated with 0.08 , 0.17 , 0.34 and 0.68 m of kuanoniamine a for 24 h and 4.18 and 8.35 m of kuanoniamine c for 24 and 48 h. after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were centrifuged , washed twice with pbs and fixed with 70% ice - cold ethanol . fixed cells ( 1 x 10 cells / ml ) were resuspended in the dna staining solution containing 50 g / ml propidium iodide , 0.5 mg / ml rnase in 10 mm tris and 5 mm mgcl2 . dna cellular content was analyzed with a facscalibur flow cytometer ( becton dickinson , mountain view , ca ) with excitation at 488 nm . data was acquired in a listmode data file , gated to 30.000 events in cell cycle , using the cellquest pro software , version 4.0.2 ( becton dickinson mountain view , ca ) included in the system . cell cycle was analysed using the modfit lt 3.0 software program ( verity software topsham ) with an activated gate to eliminate aggregates ( fl2-a / fl2-w ) . fragmentation of the cellular dna was evaluated using the in situ cell death detection kit ( tunel ) fluorescein ( boehringer mannheim , germany ) . exponential mcf-7 cells growing on coverglasses in 6-well plates were exposed to 0.5 and 1.0 m of kuanoniamine a and 1.0 and 2.5 m of kuanoniamine c for 24 h. after treatment , plates were centrifuged at 2000 rpm for 5 min at room temperature in order to sediment non - adherent cells and fixed with 4% paraformaldehyde ( in pbs ) for 1 hour at room temperature . fixed cells were washed 4 times for 5 minutes and permeabilized ( 0.1% triton x-100 , 0.1% sodium citrate in distillated water ) for 2 min on ice ( 4c ) and washed with pbs 4 times for 5 minutes . coverglasses were mounted in vectashield containing 50 g / ml pi and 0.5 g / ml rnase . all preparations were observed under the fluorescence microscope ( eclipse e400 , nikon , japan ) . apoptotic cells were quantified by counting a minimum of 400 cells from at least five different random areas of the slide . exponential mcf-7 cells growing in 6-well plates were treated with 0.5 , 1.0 and 2.5 m of kuanoniamine a for 24h . after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were stained with 0.2% trypan blue and viable and non - viable cells were counted in a hemocytometer . results were expressed in terms of percentage of viable cells in the total cell number . si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck . oceanapia sagittaria ( sollas , 1902 ) , order haplosclerida , family oceanapiidae , was collected by scuba diving in the gulf of thailand near koh samed ( 3 meter depth ) in april 2005 and frozen immediately at 20c prior to extraction . sumaitt putchakarn and the voucher registered as bims - i 2004 was deposited at bangsaen institute of marine science , burapha university , thailand . the sample ( 3.5 kg net weight ) was thawed , homogenized with etoh ( 7l ) , allowed to stand for 24 h in the dark chamber and filtered . the residue on the filter paper was again extracted with etoh ( 2x7l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca . the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 13.1 g ) . the crude etoac extract ( 13.1 g ) was applied over a flash chromatography column of si gel 60 ( 150 g ) and eluted with a solvent gradient system of petrol - chcl3 , chcl3 , chcl3,-meoh , etoac and meoh , with 200 ml fractions being collected as follows : frs . 12 ( petrol - chcl3,1:4 ) , 37 ( chcl3 ) , 817 ( chcl3,-meoh , 49:1 ) , 1822 chcl3,-meoh , 19:1 ) , 2324 ( chcl3,-meoh , 9:1 ) , 2526 ( etoac ) , 2730 ( meoh ) . 34 were combined ( 2.6 g ) and recrystallized from a mixture of petrol and chcl3 to give white crystal of 24 -methylcholestanol ( 1 , 436 mg ) . 67 were combined ( 870 mg ) and chromatographed over si gel 60 ( 20 g ) and eluted with petrol - chcl3 , chcl3-me2co , and chcl3-meoh , 200 ml subfractions were collected as follows : sfrs . 17 ( petrol - chcl3 , 2:3 ) , 815 ( petrol - chcl3 , 1:4 ) , 1624 ( chcl3 ) , 2530 ( chcl3-me2co , 19:1 ) , 3134 ( chcl3-me2co , 9:1 ) , 3538 ( chcl3-me2co , 4:1 ) , 3943 ( chcl3-me2co , 3:2 ) , 4445 ( meoh ) . 36 were combined ( 156 mg ) and purified by tlc ( si gel , chcl3 ) to give 33.2 mg of a mixture of p - hydroxybenzoic acid ( 3 ) and phenylacetic acid ( 4 ) . 715 were combined and purified by tlc ( si gel , chcl3-me2co - hco2h , 18:1:0.1 ) to give kuanoniamine a ( 6 , 20 mg ) . 810 were combined ( 390 mg ) and purified by tlc ( si gel , chcl3 , me2co , hco2h , 95:5:0.1 ) to give p - hydroxybenzaldehyde ( 2 , 15.7 mg ) and 3-formylindole ( 5 , 18.6 mg ) . 1113 were combined ( 346 mg ) and purified by tlc ( si gel , chcl3 , meoh , hco2h , 98:2:0.1 ) to give kuanoniamine a ( 6 , 32 mg ) . 2022 were combined ( 355 mg ) and applied over a flash chromatography column of ods gel ( 10 g ) and eluted with a gradient system of meoh - h2o and meoh - chcl3-h2o , 100 ml fractions being eluted as follows : sfrs 12 ( meoh - h2o , 7:3 ) , 34 ( meoh - h2o , 4:1 ) , 56 ( meoh - h2o , 9:1 ) , 710 ( meoh ) , 1114 meoh - chcl3-h2o , 7:3:0.5 ) . 56 were combined ( 81 mg ) and recrystallized from a mixture of chcl3 and me2co to give yellow crystals of kuanonamine c ( 7 , 44 mg ) . fetal bovine serum ( fbs ) , l - glutamine , phosphate buffered saline ( pbs ) and trypsin were from gibco invitrogen co. ( scotland , uk ) . acetic acid , cyclosporin a , dimethyl sulfoxide ( dmso ) , doxorubicin , ethylenediaminetetraacetic acid ( edta ) , histopaque-1077 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , penicillin , phytohemagglutinin ( pha ) , propidium iodide ( pi ) , rnase , streptomycin , sulforhodamine b ( srb ) , triton x-100 , trypan blue were from sigmachemical co ( saint louis , usa ) . magnesium chloride , paraformaldehyde , tricloroacetic acid ( tca ) , tris , sodium citrate and sodium dodecyl sulphate ( sds ) were sourced from merck ( darmstadt , germany ) . vectashield , vectashield containing dapi ( 0.5 g / ml ) were from vector laboratories ( uk ) . facscalibur flow was from becton dickinson ( mountain view , ca ) , [ h]-thymidine from amersham ( illinois , usa ) and scintillation liquid from perkinelmer ( boston , usa ) . stocks solutions of kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) were prepared in dmso and kept at 20c . five human tumour cell lines , mcf-7 ( breast adenocarcinoma , estrogen - dependent er ( + ) ) , mda - mb-231 ( breast adenocarcinoma , estrogen - independent er ( ) ) , nci - h460 ( non - small cell lung cancer ) , sf-268 ( glioma ) and uacc-62 ( melanoma ) and one non - tumour cell line , mrc-5 ( diploid embryonic lung fibroblast ) were used . mcf-7 , mda - mb-231 and mrc-5 were obtained from the european collection of cell cultures ( ecacc , salisbury , uk ) while nci - h460 , sf-268 and uacc-62 cell lines were provided by the national cancer institute ( nci , bethesda , u.s.a . ) . cells , growing as monolayer , were routinely maintained in rpmi-1640 medium supplemented with 5% heat - inactivated fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) , at 37c in a humidified atmosphere containing 5% co2 . exponentially growing cells were obtained by plating 1.510cells / ml for mcf-7 , mda - mb-231 , sf-268 and mrc-5 , 1.0 10cells / ml for uacc-62 and 0.75 10cells / ml for nci - h460 , followed by 24 h incubation . the effect of the vehicle solvent ( dmso ) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration ( 0.5% ) of dmso used in each assay . the effects of the kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) on the growth of human tumour and non - tumour cell lines were evaluated according to the procedure adopted by the national cancer institute ( nci , usa ) in the in vitro anticancer drug discovery screen that uses the protein - binding dye sulforhodamine b to assess cell growth [ 18 , 19 ] . exponentially growing cells were then exposed for 48 h to five serial concentrations of kuanoniamine a ( 0.14 ; 0.69 ; 3.46 ; 17.30 and 86.51 m ) and kuanoniamine c ( 0.11 ; 0.53 ; 2.67 ; 13.37 and 66.84 m ) that were starting from a maximum of 25 g / ml ( 86.51 m and 66.84 m , respectively ) . following this exposure period adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with 0.4% srb solubilized in 1% acetic acid . the bound stain was solubilized in 10 mm tris and the absorbance was measured at 492 nm in a microplate reader ( biotek instruments inc . , powerwave xs , wincoski , usa ) . for each cell line a dose - response curve was obtained and the growth inhibition of 50% ( gi50 ) , corresponding to the concentration of kuanoniamine a and kuanoniamine c that inhibited 50% of the net cell growth was calculated as described elsewhere . the effect of kuanoniamine a and kuanoniamine c on the mitogenic response of human lymphocytes to pha ( 10 g / ml ) was evaluated using a modified version of the colorimetric mtt - assay . briefly , mononuclear cells , isolated from heparinized peripheral blood of healthy volunteers by histopaque-1077 density centrifugation , were adjusted to 23 x 10 cells / ml in rpmi-1640 supplemented with 10% fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) . they were then harvested in 96-well plates and exposed for 4 days to seven serial concentrations of kuanoniamine a ( 0.12 ; 0.36 ; 1.07 ; 3.20 ; 9.61 ; 28.84 and 86.51 m ) and of kuanoniamine c ( 0.09 ; 0.28 ; 0.83 ; 2.48 ; 7.43 ; 22.28 and 66.84 m ) at 37 c . following this incubation period , the mtt solution ( 1 mg / ml ) was added and plates were incubated for more 4 h. the water insoluble formazan dye formed was solubilized with sds / dmf solution ( 20% sds in a 50% solution of dmf , ph 4.7 ) overnight at 37 c . the absorbance of the colored solution was then measured in a microplate reader ( biotek instruments inc . the concentration of compounds that inhibited 50% of lymphocyte proliferation ( ic50 ) was calculated by the curve obtained ( % inhibition versus concentration ) . seven serial concentrations ( 0.0014 ; 0.01 ; 0.04 ; 0.18 ; 0.89 ; 4.44 and 22.20 m ) were studied in the same manner . mcf-7 cells growing exponentially in 96-well plates were treated with a range of concentrations of kuanoniamine a ( 0.04 ; 0.08 ; 0.17 ; 0.34 ; 0.68 ; 1.35 ; 2.70 ; 5.41 ; 10.81 ; 21.63 ; 43.26 and 86.51 m ) and of kuanoniamine c ( 0.03 ; 0.07 ; 0.13 ; 0.26 ; 0.52 ; 1.04 ; 2.09 ; 4.18 ; 8.36 ; 16.71 ; 33.42 and 66.84 m ) for 6 , 12 , 24 and 48 h. following this incubation period cell growth was assessed by [ h]-thymidine incorporation , mtt and srb assays . in the [ h]-thymidine incorporation assay cells were further incubated with 1 ci of [ h ] thymidine for 4 h. pulsed cells were then harvested on a glass filter 102 x 256 mm ( skatron , norway ) using a semiautomatic cell harvester ( skatron instruments , norway ) and allowed to dry . incorporation of radioactive thymidine was determined in a scintillation counter ( ls 6500 , beckman instruments , ca and usa ) and defined by comparing the arithmetic mean of counts per minute ( cpm ) of treated cells with that of untreated control cells . in the mtt assay , mtt was added ( 5 mg / ml ) and cells were incubated for a further 4 h. formazan product were solubilized with sds / dmf solution overnight at 37c . the percentage of mtt reduction was calculated comparing the absorbance of treated cells to that of untreated control cells . in the srb assay adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with the protein binding dye ( 0.4% srb ) solubilized in 1% acetic acid , and then the bound stain was solubilized in 10 mm tris , as described . the bound stain was solubilized and the absorbance was measure at 492 nm in the microplate reader . mcf-7 cells growing exponentially in 25 cm flasks were treated with 0.08 , 0.17 , 0.34 and 0.68 m of kuanoniamine a for 24 h and 4.18 and 8.35 m of kuanoniamine c for 24 and 48 h. after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were centrifuged , washed twice with pbs and fixed with 70% ice - cold ethanol . fixed cells ( 1 x 10 cells / ml ) were resuspended in the dna staining solution containing 50 g / ml propidium iodide , 0.5 mg / ml rnase in 10 mm tris and 5 mm mgcl2 . dna cellular content was analyzed with a facscalibur flow cytometer ( becton dickinson , mountain view , ca ) with excitation at 488 nm . data was acquired in a listmode data file , gated to 30.000 events in cell cycle , using the cellquest pro software , version 4.0.2 ( becton dickinson mountain view , ca ) included in the system . cell cycle was analysed using the modfit lt 3.0 software program ( verity software topsham ) with an activated gate to eliminate aggregates ( fl2-a / fl2-w ) . fragmentation of the cellular dna was evaluated using the in situ cell death detection kit ( tunel ) fluorescein ( boehringer mannheim , germany ) . exponential mcf-7 cells growing on coverglasses in 6-well plates were exposed to 0.5 and 1.0 m of kuanoniamine a and 1.0 and 2.5 m of kuanoniamine c for 24 h. after treatment , plates were centrifuged at 2000 rpm for 5 min at room temperature in order to sediment non - adherent cells and fixed with 4% paraformaldehyde ( in pbs ) for 1 hour at room temperature . fixed cells were washed 4 times for 5 minutes and permeabilized ( 0.1% triton x-100 , 0.1% sodium citrate in distillated water ) for 2 min on ice ( 4c ) and washed with pbs 4 times for 5 minutes . coverglasses were mounted in vectashield containing 50 g / ml pi and 0.5 g / ml rnase . all preparations were observed under the fluorescence microscope ( eclipse e400 , nikon , japan ) . apoptotic cells were quantified by counting a minimum of 400 cells from at least five different random areas of the slide . exponential mcf-7 cells growing in 6-well plates were treated with 0.5 , 1.0 and 2.5 m of kuanoniamine a for 24h . after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were stained with 0.2% trypan blue and viable and non - viable cells were counted in a hemocytometer . results were expressed in terms of percentage of viable cells in the total cell number . fetal bovine serum ( fbs ) , l - glutamine , phosphate buffered saline ( pbs ) and trypsin were from gibco invitrogen co. ( scotland , uk ) . acetic acid , cyclosporin a , dimethyl sulfoxide ( dmso ) , doxorubicin , ethylenediaminetetraacetic acid ( edta ) , histopaque-1077 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , penicillin , phytohemagglutinin ( pha ) , propidium iodide ( pi ) , rnase , streptomycin , sulforhodamine b ( srb ) , triton x-100 , trypan blue were from sigmachemical co ( saint louis , usa ) . magnesium chloride , paraformaldehyde , tricloroacetic acid ( tca ) , tris , sodium citrate and sodium dodecyl sulphate ( sds ) were sourced from merck ( darmstadt , germany ) . vectashield , vectashield containing dapi ( 0.5 g / ml ) were from vector laboratories ( uk ) . facscalibur flow was from becton dickinson ( mountain view , ca ) , [ h]-thymidine from amersham ( illinois , usa ) and scintillation liquid from perkinelmer ( boston , usa ) . stocks solutions of kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) were prepared in dmso and kept at 20c . five human tumour cell lines , mcf-7 ( breast adenocarcinoma , estrogen - dependent er ( + ) ) , mda - mb-231 ( breast adenocarcinoma , estrogen - independent er ( ) ) , nci - h460 ( non - small cell lung cancer ) , sf-268 ( glioma ) and uacc-62 ( melanoma ) and one non - tumour cell line , mrc-5 ( diploid embryonic lung fibroblast ) were used . mcf-7 , mda - mb-231 and mrc-5 were obtained from the european collection of cell cultures ( ecacc , salisbury , uk ) while nci - h460 , sf-268 and uacc-62 cell lines were provided by the national cancer institute ( nci , bethesda , u.s.a . ) . cells , growing as monolayer , were routinely maintained in rpmi-1640 medium supplemented with 5% heat - inactivated fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) , at 37c in a humidified atmosphere containing 5% co2 . exponentially growing cells were obtained by plating 1.510cells / ml for mcf-7 , mda - mb-231 , sf-268 and mrc-5 , 1.0 10cells / ml for uacc-62 and 0.75 10cells / ml for nci - h460 , followed by 24 h incubation . the effect of the vehicle solvent ( dmso ) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration ( 0.5% ) of dmso used in each assay . the effects of the kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) on the growth of human tumour and non - tumour cell lines were evaluated according to the procedure adopted by the national cancer institute ( nci , usa ) in the in vitro anticancer drug discovery screen that uses the protein - binding dye sulforhodamine b to assess cell growth [ 18 , 19 ] . exponentially growing cells were then exposed for 48 h to five serial concentrations of kuanoniamine a ( 0.14 ; 0.69 ; 3.46 ; 17.30 and 86.51 m ) and kuanoniamine c ( 0.11 ; 0.53 ; 2.67 ; 13.37 and 66.84 m ) that were starting from a maximum of 25 g / ml ( 86.51 m and 66.84 m , respectively ) . following this exposure period adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with 0.4% srb solubilized in 1% acetic acid . the bound stain was solubilized in 10 mm tris and the absorbance was measured at 492 nm in a microplate reader ( biotek instruments inc . , powerwave xs , wincoski , usa ) . for each cell line a dose - response curve was obtained and the growth inhibition of 50% ( gi50 ) , corresponding to the concentration of kuanoniamine a and kuanoniamine c that inhibited 50% of the net cell growth was calculated as described elsewhere . the effect of kuanoniamine a and kuanoniamine c on the mitogenic response of human lymphocytes to pha ( 10 g / ml ) was evaluated using a modified version of the colorimetric mtt - assay . briefly , mononuclear cells , isolated from heparinized peripheral blood of healthy volunteers by histopaque-1077 density centrifugation , were adjusted to 23 x 10 cells / ml in rpmi-1640 supplemented with 10% fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) . they were then harvested in 96-well plates and exposed for 4 days to seven serial concentrations of kuanoniamine a ( 0.12 ; 0.36 ; 1.07 ; 3.20 ; 9.61 ; 28.84 and 86.51 m ) and of kuanoniamine c ( 0.09 ; 0.28 ; 0.83 ; 2.48 ; 7.43 ; 22.28 and 66.84 m ) at 37 c . following this incubation period , the mtt solution ( 1 mg / ml ) was added and plates were incubated for more 4 h. the water insoluble formazan dye formed was solubilized with sds / dmf solution ( 20% sds in a 50% solution of dmf , ph 4.7 ) overnight at 37 c . the absorbance of the colored solution was then measured in a microplate reader ( biotek instruments inc . the concentration of compounds that inhibited 50% of lymphocyte proliferation ( ic50 ) was calculated by the curve obtained ( % inhibition versus concentration ) . seven serial concentrations ( 0.0014 ; 0.01 ; 0.04 ; 0.18 ; 0.89 ; 4.44 and 22.20 m ) were studied in the same manner . mcf-7 cells growing exponentially in 96-well plates were treated with a range of concentrations of kuanoniamine a ( 0.04 ; 0.08 ; 0.17 ; 0.34 ; 0.68 ; 1.35 ; 2.70 ; 5.41 ; 10.81 ; 21.63 ; 43.26 and 86.51 m ) and of kuanoniamine c ( 0.03 ; 0.07 ; 0.13 ; 0.26 ; 0.52 ; 1.04 ; 2.09 ; 4.18 ; 8.36 ; 16.71 ; 33.42 and 66.84 m ) for 6 , 12 , 24 and 48 h. following this incubation period cell growth was assessed by [ h]-thymidine incorporation , mtt and srb assays . in the [ h]-thymidine incorporation assay cells were further incubated with 1 ci of [ h ] thymidine for 4 h. pulsed cells were then harvested on a glass filter 102 x 256 mm ( skatron , norway ) using a semiautomatic cell harvester ( skatron instruments , norway ) and allowed to dry . incorporation of radioactive thymidine was determined in a scintillation counter ( ls 6500 , beckman instruments , ca and usa ) and defined by comparing the arithmetic mean of counts per minute ( cpm ) of treated cells with that of untreated control cells . in the mtt assay , mtt was added ( 5 mg / ml ) and cells were incubated for a further 4 h. formazan product were solubilized with sds / dmf solution overnight at 37c . the percentage of mtt reduction was calculated comparing the absorbance of treated cells to that of untreated control cells . in the srb assay adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with the protein binding dye ( 0.4% srb ) solubilized in 1% acetic acid , and then the bound stain was solubilized in 10 mm tris , as described . the bound stain was solubilized and the absorbance was measure at 492 nm in the microplate reader . mcf-7 cells growing exponentially in 25 cm flasks were treated with 0.08 , 0.17 , 0.34 and 0.68 m of kuanoniamine a for 24 h and 4.18 and 8.35 m of kuanoniamine c for 24 and 48 h. after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were centrifuged , washed twice with pbs and fixed with 70% ice - cold ethanol . fixed cells ( 1 x 10 cells / ml ) were resuspended in the dna staining solution containing 50 g / ml propidium iodide , 0.5 mg / ml rnase in 10 mm tris and 5 mm mgcl2 . dna cellular content was analyzed with a facscalibur flow cytometer ( becton dickinson , mountain view , ca ) with excitation at 488 nm . data was acquired in a listmode data file , gated to 30.000 events in cell cycle , using the cellquest pro software , version 4.0.2 ( becton dickinson mountain view , ca ) included in the system . cell cycle was analysed using the modfit lt 3.0 software program ( verity software topsham ) with an activated gate to eliminate aggregates ( fl2-a / fl2-w ) . fragmentation of the cellular dna was evaluated using the in situ cell death detection kit ( tunel ) fluorescein ( boehringer mannheim , germany ) . exponential mcf-7 cells growing on coverglasses in 6-well plates were exposed to 0.5 and 1.0 m of kuanoniamine a and 1.0 and 2.5 m of kuanoniamine c for 24 h. after treatment , plates were centrifuged at 2000 rpm for 5 min at room temperature in order to sediment non - adherent cells and fixed with 4% paraformaldehyde ( in pbs ) for 1 hour at room temperature . fixed cells were washed 4 times for 5 minutes and permeabilized ( 0.1% triton x-100 , 0.1% sodium citrate in distillated water ) for 2 min on ice ( 4c ) and washed with pbs 4 times for 5 minutes . coverglasses were mounted in vectashield containing 50 g / ml pi and 0.5 g / ml rnase . all preparations were observed under the fluorescence microscope ( eclipse e400 , nikon , japan ) . apoptotic cells were quantified by counting a minimum of 400 cells from at least five different random areas of the slide . exponential mcf-7 cells growing in 6-well plates were treated with 0.5 , 1.0 and 2.5 m of kuanoniamine a for 24h . after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were stained with 0.2% trypan blue and viable and non - viable cells were counted in a hemocytometer . results were expressed in terms of percentage of viable cells in the total cell number . fetal bovine serum ( fbs ) , l - glutamine , phosphate buffered saline ( pbs ) and trypsin were from gibco invitrogen co. ( scotland , uk ) . acetic acid , cyclosporin a , dimethyl sulfoxide ( dmso ) , doxorubicin , ethylenediaminetetraacetic acid ( edta ) , histopaque-1077 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , penicillin , phytohemagglutinin ( pha ) , propidium iodide ( pi ) , rnase , streptomycin , sulforhodamine b ( srb ) , triton x-100 , trypan blue were from sigmachemical co ( saint louis , usa ) . magnesium chloride , paraformaldehyde , tricloroacetic acid ( tca ) , tris , sodium citrate and sodium dodecyl sulphate ( sds ) were sourced from merck ( darmstadt , germany ) . vectashield , vectashield containing dapi ( 0.5 g / ml ) were from vector laboratories ( uk ) . facscalibur flow was from becton dickinson ( mountain view , ca ) , [ h]-thymidine from amersham ( illinois , usa ) and scintillation liquid from perkinelmer ( boston , usa ) . stocks solutions of kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) were prepared in dmso and kept at 20c . five human tumour cell lines , mcf-7 ( breast adenocarcinoma , estrogen - dependent er ( + ) ) , mda - mb-231 ( breast adenocarcinoma , estrogen - independent er ( ) ) , nci - h460 ( non - small cell lung cancer ) , sf-268 ( glioma ) and uacc-62 ( melanoma ) and one non - tumour cell line , mrc-5 ( diploid embryonic lung fibroblast ) were used . mcf-7 , mda - mb-231 and mrc-5 were obtained from the european collection of cell cultures ( ecacc , salisbury , uk ) while nci - h460 , sf-268 and uacc-62 cell lines were provided by the national cancer institute ( nci , bethesda , u.s.a . ) . cells , growing as monolayer , were routinely maintained in rpmi-1640 medium supplemented with 5% heat - inactivated fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) , at 37c in a humidified atmosphere containing 5% co2 . exponentially growing cells were obtained by plating 1.510cells / ml for mcf-7 , mda - mb-231 , sf-268 and mrc-5 , 1.0 10cells / ml for uacc-62 and 0.75 10cells / ml for nci - h460 , followed by 24 h incubation . the effect of the vehicle solvent ( dmso ) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration ( 0.5% ) of dmso used in each assay . the effects of the kuanoniamine a ( 6 ) and kuanoniamine c ( 7 ) on the growth of human tumour and non - tumour cell lines were evaluated according to the procedure adopted by the national cancer institute ( nci , usa ) in the in vitro anticancer drug discovery screen that uses the protein - binding dye sulforhodamine b to assess cell growth [ 18 , 19 ] . exponentially growing cells were then exposed for 48 h to five serial concentrations of kuanoniamine a ( 0.14 ; 0.69 ; 3.46 ; 17.30 and 86.51 m ) and kuanoniamine c ( 0.11 ; 0.53 ; 2.67 ; 13.37 and 66.84 m ) that were starting from a maximum of 25 g / ml ( 86.51 m and 66.84 m , respectively ) . following this exposure period adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with 0.4% srb solubilized in 1% acetic acid . the bound stain was solubilized in 10 mm tris and the absorbance was measured at 492 nm in a microplate reader ( biotek instruments inc . for each cell line a dose - response curve was obtained and the growth inhibition of 50% ( gi50 ) , corresponding to the concentration of kuanoniamine a and kuanoniamine c that inhibited 50% of the net cell growth was calculated as described elsewhere . the effect of kuanoniamine a and kuanoniamine c on the mitogenic response of human lymphocytes to pha ( 10 g / ml ) was evaluated using a modified version of the colorimetric mtt - assay . briefly , mononuclear cells , isolated from heparinized peripheral blood of healthy volunteers by histopaque-1077 density centrifugation , were adjusted to 23 x 10 cells / ml in rpmi-1640 supplemented with 10% fbs , 2 mm glutamine and antibiotics ( penicillin 100 u / ml , streptomycin 100 g / ml ) . they were then harvested in 96-well plates and exposed for 4 days to seven serial concentrations of kuanoniamine a ( 0.12 ; 0.36 ; 1.07 ; 3.20 ; 9.61 ; 28.84 and 86.51 m ) and of kuanoniamine c ( 0.09 ; 0.28 ; 0.83 ; 2.48 ; 7.43 ; 22.28 and 66.84 m ) at 37 c . following this incubation period , the mtt solution ( 1 mg / ml ) was added and plates were incubated for more 4 h. the water insoluble formazan dye formed was solubilized with sds / dmf solution ( 20% sds in a 50% solution of dmf , ph 4.7 ) overnight at 37 c . the absorbance of the colored solution was then measured in a microplate reader ( biotek instruments inc . the concentration of compounds that inhibited 50% of lymphocyte proliferation ( ic50 ) was calculated by the curve obtained ( % inhibition versus concentration ) . seven serial concentrations ( 0.0014 ; 0.01 ; 0.04 ; 0.18 ; 0.89 ; 4.44 and 22.20 m ) were studied in the same manner . mcf-7 cells growing exponentially in 96-well plates were treated with a range of concentrations of kuanoniamine a ( 0.04 ; 0.08 ; 0.17 ; 0.34 ; 0.68 ; 1.35 ; 2.70 ; 5.41 ; 10.81 ; 21.63 ; 43.26 and 86.51 m ) and of kuanoniamine c ( 0.03 ; 0.07 ; 0.13 ; 0.26 ; 0.52 ; 1.04 ; 2.09 ; 4.18 ; 8.36 ; 16.71 ; 33.42 and 66.84 m ) for 6 , 12 , 24 and 48 h. following this incubation period cell growth was assessed by [ h]-thymidine incorporation , mtt and srb assays . in the [ h]-thymidine incorporation assay cells were further incubated with 1 ci of [ h ] thymidine for 4 h. pulsed cells were then harvested on a glass filter 102 x 256 mm ( skatron , norway ) using a semiautomatic cell harvester ( skatron instruments , norway ) and allowed to dry . incorporation of radioactive thymidine was determined in a scintillation counter ( ls 6500 , beckman instruments , ca and usa ) and defined by comparing the arithmetic mean of counts per minute ( cpm ) of treated cells with that of untreated control cells . in the mtt assay , mtt was added ( 5 mg / ml ) and cells were incubated for a further 4 h. formazan product were solubilized with sds / dmf solution overnight at 37c . the percentage of mtt reduction was calculated comparing the absorbance of treated cells to that of untreated control cells . in the srb assay adherent cells were fixed in situ with 50% tca , washed with distillated water and stained with the protein binding dye ( 0.4% srb ) solubilized in 1% acetic acid , and then the bound stain was solubilized in 10 mm tris , as described . the bound stain was solubilized and the absorbance was measure at 492 nm in the microplate reader . mcf-7 cells growing exponentially in 25 cm flasks were treated with 0.08 , 0.17 , 0.34 and 0.68 m of kuanoniamine a for 24 h and 4.18 and 8.35 m of kuanoniamine c for 24 and 48 h. after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were centrifuged , washed twice with pbs and fixed with 70% ice - cold ethanol . fixed cells ( 1 x 10 cells / ml ) were resuspended in the dna staining solution containing 50 g / ml propidium iodide , 0.5 mg / ml rnase in 10 mm tris and 5 mm mgcl2 . dna cellular content was analyzed with a facscalibur flow cytometer ( becton dickinson , mountain view , ca ) with excitation at 488 nm . data was acquired in a listmode data file , gated to 30.000 events in cell cycle , using the cellquest pro software , version 4.0.2 ( becton dickinson mountain view , ca ) included in the system . cell cycle was analysed using the modfit lt 3.0 software program ( verity software topsham ) with an activated gate to eliminate aggregates ( fl2-a / fl2-w ) . fragmentation of the cellular dna was evaluated using the in situ cell death detection kit ( tunel ) fluorescein ( boehringer mannheim , germany ) . exponential mcf-7 cells growing on coverglasses in 6-well plates were exposed to 0.5 and 1.0 m of kuanoniamine a and 1.0 and 2.5 m of kuanoniamine c for 24 h. after treatment , plates were centrifuged at 2000 rpm for 5 min at room temperature in order to sediment non - adherent cells and fixed with 4% paraformaldehyde ( in pbs ) for 1 hour at room temperature . fixed cells were washed 4 times for 5 minutes and permeabilized ( 0.1% triton x-100 , 0.1% sodium citrate in distillated water ) for 2 min on ice ( 4c ) and washed with pbs 4 times for 5 minutes . coverglasses were mounted in vectashield containing 50 g / ml pi and 0.5 g / ml rnase . all preparations were observed under the fluorescence microscope ( eclipse e400 , nikon , japan ) . apoptotic cells were quantified by counting a minimum of 400 cells from at least five different random areas of the slide . exponential mcf-7 cells growing in 6-well plates were treated with 0.5 , 1.0 and 2.5 m of kuanoniamine a for 24h . after treatment , attached cells were released by trypsinization and mixed with non - adherent cells . cells were stained with 0.2% trypan blue and viable and non - viable cells were counted in a hemocytometer . results were expressed in terms of percentage of viable cells in the total cell number .	the pyridoacridine alkaloids kuanoniamines a and c were isolated together with 24 -methylcholestanol , p - hydroxybenzaldehyde , p - hydroxybenzoic acid , phenylacetic acid and 3-formylindole from the ethyl acetate extract of the marine sponge oceanapia sagittaria ( sollas ) , collected from the gulf of thailand . kuanoniamines a and c were evaluated for their effect on the growth of five human tumour and a non - tumour cell lines , as well as on the proliferation of human lymphocytes . kuanoniamine a was found to be a potent growth inhibitor of all the tumour and a non - tumour cell lines while kuanoniamine c was less potent but showed high selectivity toward the estrogen dependent ( er+ ) breast cancer cell line . kuanoniamine a has shown to be a more potent inhibitor of dna synthesis than kuanoniamine c. kuanoniamine a was also found to cause an extensive reduction of the mcf-7 cells in g2/m phase as well as an increase in the apoptotic cells .
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Proceed to summarize the following text: patients with the involvement of the brachial or lumbosacral plexus or other causes of neuropathic pain often need escalating doses of opioids and adjuvants . despite this , some patients may experience refractory pain as well as the side effects of multiple analgesics . palliative radiotherapy is therefore often used for pain relief in malignant conditions such as pancoast tumor and metastatic spinal cord compression . this case report illustrates the role of antineoplastic treatment in a locally aggressive but nonmalignant tumor , where it was possible to relieve pain , reduce analgesics , and shrink the tumor , even though surgical excision was not possible . a 45-year - old man presented with right - sided chest pain and pain in the right upper limb since 2 years , with a recent onset of paraesthesia in the medial two fingers . imaging revealed a superior sulcus mass measuring 6 6 11.5 cm , infiltrating the chest wall and eroding the posterior ends of the first three ribs . since it was an inoperable desmoid tumor , the patient was treated with radiation therapy , 45 gy administered in 25 fractions . on the first follow - up , 3 months after radiotherapy , the patient was referred to the palliative care unit with persistent pain . he was treated with diclofenac 50 mg three times daily , amitriptyline 50 mg hsod and dextropropoxyphene 1 q6h and prn . three days later , because of persistent pain he was given a single dose of dexamethasone 8 mg , and the weak opioids , adjuvants , diclofenac and tamoxifen , were continued . it was planned to increase amitriptyline , but the patient was from a distant part of the country and returned only after 6 months . during this period , he had taken imatinib for 3 months , and analgesics and tamoxifen for 6 months . on the second follow - up 8 months after radiotherapy , his ptosis had improved ; the doses of opioids and antidepressants were therefore reduced and tamoxifen was continued . since the pain was very mild and the tumor had reduced by more than 50% [ figure 1 ] , analgesics were further reduced to paracetamol 500 mg three times daily and one capsule of dextropropoxyphene at night . desmoid tumors are histologically benign neoplasms , being a monoclonal proliferation of cytologically benign fibrocytes arising from musculoaponeurotic structures . desmoid tumors do not metastasize , but since they lack a pseudocapsule , they can infiltrate local structures and have a high risk of local recurrence , even after major surgical resections . recurrences occur due to ill - defined margins of infiltration along the septal planes , and it is difficult to estimate adequate surgical margins . radiotherapy can permit less mutilating surgery , and can also serve as the primary mode of treatment in unresectable tumors . in a survey of 110 patients with a median follow - up of 6 years ( range 144 years ) , baumert et al . reported that the addition of radiotherapy significantly reduced local recurrences compared to surgery alone ( hazard ratio 0.19 ) . with surgery and postoperative radiotherapy , the progression - free survival was 95% at 5 years and 93% at 10 years . patients are likely to survive many years , and short courses of hypofractionated palliative radiotherapy could result in significant late toxicities . appropriate fractionation is necessary and a radiotherapy dose of more than 50 gy can provide better tumor control . to date , there are no randomized trials of systemic therapy but a retrospective analysis by the memorial sloan kettering center studied 68 patients . although a variety of systemic therapies had been used including nsaids , chemotherapy , and imatinib , the best response rates were observed with anthracyclines and hormonal therapy , with only 12% of tumors showing disease progression . reported the complete response of a recurrent pelvic desmoid tumor to 7 months of tamoxifen with the response sustained at a follow - up of 6 years . tamoxifen was first used by kinzbrunner in recurrent , large painful desmoid lesions , based on the observation that desmoid tumors were common in women in their childbearing years and reduced after menopause . he observed a complete relief of pain within a week and 50% reduction in tumor size . in a systematic review of pharmacological therapies , janinis et al . responses to systemic therapy and radiotherapy are often delayed by many months , as in our patient , where we did not see much benefit at 3 months , but the tumor continued to shrink at 1-year and 2-year follow - ups . hormonal therapies and/or nsaids are recommended as first - line systemic therapy since toxicity is less than that with chemotherapy . if the tumor is not causing significant problems , and is stable in size , it may be appropriate to continue current therapies or watch and wait rather than embark on heroic surgeries , or toxic or expensive systemic therapies . wilcken et al . have reported benefit with the use of progesterones as second - line hormonal therapy . desmoid tumors are histologically benign neoplasms , being a monoclonal proliferation of cytologically benign fibrocytes arising from musculoaponeurotic structures . desmoid tumors do not metastasize , but since they lack a pseudocapsule , they can infiltrate local structures and have a high risk of local recurrence , even after major surgical resections . recurrences occur due to ill - defined margins of infiltration along the septal planes , and it is difficult to estimate adequate surgical margins . radiotherapy can permit less mutilating surgery , and can also serve as the primary mode of treatment in unresectable tumors . in a survey of 110 patients with a median follow - up of 6 years ( range 144 years ) , baumert et al . reported that the addition of radiotherapy significantly reduced local recurrences compared to surgery alone ( hazard ratio 0.19 ) . with surgery and postoperative radiotherapy , the progression - free survival was 95% at 5 years and 93% at 10 years . patients are likely to survive many years , and short courses of hypofractionated palliative radiotherapy could result in significant late toxicities . appropriate fractionation is necessary and a radiotherapy dose of more than 50 gy can provide better tumor control . to date , there are no randomized trials of systemic therapy but a retrospective analysis by the memorial sloan kettering center studied 68 patients . although a variety of systemic therapies had been used including nsaids , chemotherapy , and imatinib , the best response rates were observed with anthracyclines and hormonal therapy , with only 12% of tumors showing disease progression . reported the complete response of a recurrent pelvic desmoid tumor to 7 months of tamoxifen with the response sustained at a follow - up of 6 years . tamoxifen was first used by kinzbrunner in recurrent , large painful desmoid lesions , based on the observation that desmoid tumors were common in women in their childbearing years and reduced after menopause . he observed a complete relief of pain within a week and 50% reduction in tumor size . in a systematic review of pharmacological therapies , janinis et al . responses to systemic therapy and radiotherapy are often delayed by many months , as in our patient , where we did not see much benefit at 3 months , but the tumor continued to shrink at 1-year and 2-year follow - ups . hormonal therapies and/or nsaids are recommended as first - line systemic therapy since toxicity is less than that with chemotherapy . if the tumor is not causing significant problems , and is stable in size , it may be appropriate to continue current therapies or watch and wait rather than embark on heroic surgeries , or toxic or expensive systemic therapies . wilcken et al . have reported benefit with the use of progesterones as second - line hormonal therapy . brachial plexopathy and other types of difficult neuropathic pain are a challenge in the palliative care setting . radiation therapy for achieving good symptom relief can be worthwhile in locally aggressive but nonmetastasizing neoplasms such as desmoid fibromatosis . affordable targeted systemic therapies including hormonal treatment may have a role even in patients who are frail and who have relapsed on earlier anticancer therapies .	desmoid fibromatosis , although histologically benign , infiltrates local structures . the involvement of neural structures can lead to difficult neuropathic pain and the escalating use of analgesics . we report a patient with desmoid fibromatosis of the chest wall causing brachial plexus infiltration . as the tumor was locally invasive and unresectable , he was treated with radiation therapy and oral tamoxifen . on follow - up , there was significant pain relief , sustained reduction in the tumor size , and reduced analgesic requirement . antineoplastic treatments like local radiation therapy and targeted systemic therapy with hormones or other agents can be considered in the management of selected unresectable desmoid fibromatosis to improve symptom control and reduce polypharmacy .
You are an expert at summarizing long articles. Proceed to summarize the following text: though gastrointestinal symptoms are relatively common in bd , ulcerative change of the intestine is not that frequent . these include rectovaginal fistula , vesicovaginal fistula , aortoatrial fistula and postoperative complication such as aortoenteric fistula and enterocutaneus fistula . our patient who presented with massive bloody diarrhea and severe pain in rla had a large deep cavitating ileocecal ulcer along with a cecocecal fistula around the previous appendectomy site . a 38-year - old man presented with massive , bloody diarrhea and colicky pain in rla . he had been having recurrent oral ulcer , genital ulcer and erythema nodosum - like lesion for the past ten years . six years ago , he had an appendectomy done at a primary clinic . at that time , the multiple ulcers were noted in the ileocecal region and histology showed minimal inflammation in the appendix . he was referred to the university medical center and diagnosed as having an intestinal bd . after surgery , intermittent severe abdominal pain has occurred while he has been on medication on and off . on examination , he had a regular pulse of 120 beats / min , blood pressure of 70/40 mmhg and body temperature of 36.5c . the lungs were clear to auscultation . though his abdomen was soft , the abdominal examination revealed tenderness and rebound tenderness in rla . he had a hematocrit of 33.5% , a white blood cell count of 11800//mm and a platelet count of 185000/mm . even with enough saline and transfusion of packed red blood cells , he had persistent hypotension and severe abdominal pain . emergency ileocecal segmental resection was performed . pathologic examination revealed large cavitating ulcers(figure 1 ) , lymphocyte aggregates and lymphocytic vasculitis ( figure 2 ) in the ileocecal region . there was cecocecal fistula ( figures 3 & 4 ) presumably around the previous appendectomy site . he was started on oral prednisolone 10 mg / day , sulfasalazine 2 g / day and cyclophosphamide 100 mg / day in the sixth postoperative day . bd is a multisystemic disorder characterized by oral ulcer , genital ulcer , uveitis and skin lesions , most likely occurring with the underlying vasculitis . there is other organ involvement of joints , heart and lungs , as well as neurologic and gastrointestinal involvement . our case fulfilled the diagnostic criteria of the intestinal study group for behcet s disease . many patients complained of gastrointestinal symptoms such as nausea , vomiting and abdominal pain , but the ulcerative changes in the intestine were found in 1% or less of all patients with bd . the commonest sites for the ulcerative changes of intestinal bd were terminal ileum in 44% , followed by the ileocecal region in 34% and the cecum in 12% . the clinical manifestations of intestinal bd are similar to inflammatory bowel disease , especially crohn s disease . but lymphoid aggregates , submucosal fibrosis , no granuloma , and deep penetrating , easily perforating ulcer in behcet s colitis help to distinguish it from crohn s disease . histology in our case revealed lymphoid aggregates , no granuloma and deep penetrating ulcers in the ileocecal region . about 22% of the patients with intestinal bd developed symptoms mimicking appendicitis during the clinical course . because the most common sites of intestinal bd are the terminal ileum and the ileocecal region , some cases can be diagnosed as an appendicitis . we could speculate that vasculitis and ulcer necrosis , along with pathergy reaction by surgical trauma in our case , might have contributed to the fistula formation .	we describe a case of behcet s disease ( bd ) which showed the ileocecal ulcer and cecocecal fistula . this 38-year - old man had appendectomy six years ago because of colicky pain in the right lower abdomen ( rla ) . there are some reports on fistula formation in bd . in those , some are related to surgery and others are not . bd with cecocecal fistula , possibly associated with a past operation , has not been reported in the literature .
You are an expert at summarizing long articles. Proceed to summarize the following text: with the increasing prevalence of dementia , waiting lists for memory disorder clinics are growing . time constraints , location , and poor weather conditions all impact on the ability of both patients and providers to attend rural clinics . telemedicine is increasingly being used to provide the follow - up care that will help elderly people and those with chronic conditions maintain their independence and continue living in their own homes . home telecare has been found effective for improving clinical indicators and reducing health service use . a recent review found veterans health administration telemedicine interventions to be advantageous when ongoing monitoring of patient symptoms is needed and noted that telemedicine also appeared to enhance patient access to healthcare professionals . telemedicine is the use of telecommunication technology for medical diagnosis and patient care . from its beginnings telemedicine has been used in a variety of health - care fields . while technological aspects of the interventions appear reliable and well - accepted by patients , the clinical benefits are less well - established . in addition , issues relating to satisfaction with this care modality require further exploration from both client and provider perspectives . this study explored the feasibility of introducing a telemedicine memory disorder clinic in a rural community , and the results of a four - year evaluation are described . the specific goal of the clinic was to provide timely access to follow - up care for patients who have been previously assessed at the champlain ccac memory disorder clinic in cornwall , ontario by a geriatrician . issues related to cognition , medication management , driving safety assessment , future planning , ongoing support , and other related health concerns were addressed through the telemedicine clinic . the evaluation of the clinic was based on feedback from patients , the ccac assessors who managed the clinic and were at the site with the patient during the telemedicine follow - up , and the geriatricians . additional details , such as the reason for the telemedicine appointment , were also captured . information about the time commitment involved in setting up and conducting the telemedicine clinic was provided by the ccac assessors and the geriatricians . this program evaluation study involved the prospective enrollment of patients who were initially assessed in person by a geriatrician at a memory disorder clinic and referred for follow - up at a telemedicine clinic . the partners were the regional geriatric program of eastern ontario ( rgpeo ) , the ottawa hospital ( host site ) , the champlain ccac memory disorder clinic and tri - county mental health in cornwall ( patient site ) , and the ontario telemedicine network . to be included in the study , patients needed first to be identified by the geriatrician as suitable for telemedicine follow - up care . they had to be medically stable with non - complex conditions , and present with mild cognitive impairment ( cognitive change but with no impact on functional level ) , or mild dementia ( a score of 2024 on the mmse ) . referrals were also accepted for patients who could potentially benefit from anti - dementia medication review . patients were excluded if they were new consultations , clinically unstable , or had severe cognitive impairment . patients who did not wish to be seen through telemedicine had the option of being seen at the regular memory disorder clinic . approximately 15% of patients were flagged for telemedicine follow - up during the initial office visit by the geriatrician . in consultation with the family physician and geriatrician , a ccac case manager / assessor created a list of potential patients for referral which was faxed to the geriatrician two weeks prior to the clinic . the case managers / assessors had all participated in the geriatric assessor training program of the rgpeo . the patient site was responsible for notifying the patient s family / caregiver of the date and time of the appointment , and advising them to bring the patient s medications , as well as to arrive 30 minutes in advance of the appointment . the additional 30 minutes allowed the geriatric assessor time to explain the telemedicine process to patients and to obtain consent . during this time , the assessor also gathered pertinent medical history , administered cognitive tests such as the 3ms or mmse , measured vital signs , gathered results from adl and iadl functional assessments , obtained the medication list , and interviewed the primary caregiver . the geriatrician and ccac case managers / assessors independently rated each video - conference clinic . the geriatrician provided details on the specific issues being addressed for each patient , and the assessors indicated why the client was a good candidate for telemedicine . the total time required for the video conference was recorded on the clinic questionnaire completed by the physician , and the assessor questionnaire provided details regarding time needed to set up / take down equipment , explain or reassure the patient , and any other required activities . physicians also recorded whether the clinic might have been cancelled had it not been held by video conference . all surveys involved respondents rating their agreement with a number of statements on a 5-point likert scale , in which 1 indicated they strongly disagreed with the statement and 5 indicated they strongly agreed . physicians and assessors rated each clinic on a number of items , such as how smoothly the clinic ran . patients completed an anonymous survey in which they were asked to include some personal information and indicate their level of agreement with 19 statements related to their telemedicine appointment . although there was no caregiver survey , caregivers occasionally provided feedback in the comments section of the patient survey . completed questionnaires were sent to the rgpeo program evaluator for analysis using spss paws 17.0 . the geriatrician and ccac case managers / assessors independently rated each video - conference clinic . the geriatrician provided details on the specific issues being addressed for each patient , and the assessors indicated why the client was a good candidate for telemedicine . the total time required for the video conference was recorded on the clinic questionnaire completed by the physician , and the assessor questionnaire provided details regarding time needed to set up / take down equipment , explain or reassure the patient , and any other required activities . physicians also recorded whether the clinic might have been cancelled had it not been held by video conference . all surveys involved respondents rating their agreement with a number of statements on a 5-point likert scale , in which 1 indicated they strongly disagreed with the statement and 5 indicated they strongly agreed . physicians and assessors rated each clinic on a number of items , such as how smoothly the clinic ran . patients completed an anonymous survey in which they were asked to include some personal information and indicate their level of agreement with 19 statements related to their telemedicine appointment . although there was no caregiver survey , caregivers occasionally provided feedback in the comments section of the patient survey . completed questionnaires were sent to the rgpeo program evaluator for analysis using spss paws 17.0 . mean and median scores were calculated for each item . results were collected for 32 clinics held between nov 2006 and nov 2010 involving a total of 99 patients . the most frequent reasons for the telemedicine follow - up appointment were : assessment / reassessment for cognitive decline assessment of patient s suitability for initiating new medication follow - up of patient s response to dementia therapy home safety and future planning other medical concerns : nutrition , falls , chf , osteoporosis , continence in almost all cases ( 91% ) , the physician identified more than one reason for the appointment . completed surveys were received from 50 of 99 patients ( 51% ) and 48 respondents provided some personal background information . given the many challenges faced by this group of patients , this was considered a good response rate . more than one - half ( 58% ) were between 7584 years of age , 29% were between 6574 years , and 13% were 85 years or older . it was the first video session for over two - thirds of the patients ( 69% ) , and their satisfaction ratings were consistently positive . overall , 92% of the respondents were satisfied with the session , and almost all indicated that they would be willing to use video conferencing again ( 94% ) . one patient commented , this is a great way to serve the people of cornwall neither they nor the doctor need to drive to ottawa for a follow - up . i would like to see more of this type of work and innovative ideas . i thank all of you for helping me get back to my old self again . on a few occasions , the otn video link did nt work or the screen froze , which impacted on a patient s ability to see the doctor . this likely resulted in slightly lower satisfaction rating given to this item ( 88% ) . clients felt they were understood by the on - site nurse ( 96% ) and the physician who was being teleconferenced ( 94% ) . almost all patients agreed that their privacy and confidentiality were respected ( 98% ) , although a small number stated they would have preferred to see their doctor alone ( 16% ) . in terms of their sense of involvement in their care , most patients felt that their questions were answered by the doctor ( 98% ) , that the video conference session ran smoothly ( 98% ) , and that the appointment provided enough time to deal with everything that needed to be covered ( 92% ) . items with the greatest variability in responses were those related to patient preferences . while most felt that they were able to present the same information they would have provided in person ( 92% ) , a slightly smaller number said that they felt as confident about the doctor s assessment through video as they would an in - person assessment ( 88% ) . thirty percent stated that they were more anxious with the video session than if they had seen the doctor in person , and over one - quarter ( 28% ) felt that they would have preferred to see their doctor in person . details related to the clinic processes were captured through the ccac assessor and the physician clinic summary surveys . usually , physicians saw 34 patients and spent about two hours video conferencing , per clinic . ccac case managers spent an average of 51 minutes : setting up / taking down equipment ( 12 min ) ; explaining video conferencing to patients and providing reassurance ( 22 min ) ; debriefing patients at the end of their session ( 13 min ) ; and 4 minutes on other clinic activities . three clinics encountered technical problems that required significantly more time on the part of the ccac assessor . the ccac assessors were generally more conservative than patients in their rankings of the clinic experience , with 79% agreeing or strongly agreeing that they were satisfied with the sessions . it was not surprising to find a strong correlation between the assessors satisfaction with the clinic and their perception as to whether it ran smoothly . physicians provided feedback on 30 of the 32 clinics , and for the most part felt that the clinic ran smoothly ( 88% ) . they indicated that 17 of the 30 clinics would likely have been cancelled had the geriatrician needed to travel to the clinic site . this is an important finding , given that the objective of providing telemedicine to the target population was to provide more timely access to care . minor technical problems were identified by the geriatrician for 7 of the 30 clinics . on two occasions the video link did not work at all and cases were discussed by phone instead . in addition to rating the clinic experience , physicians also provided feedback on their session with each patient , for 91 of the 99 patients . as figure 3 shows , physicians agreed or strongly agreed that video conferencing provided them with opportunities to ask the patient / ccac assessor questions ( 100% ) . they generally felt that the appointments met their needs ( 96% ) , and that they were able to get enough relevant information to assist in clinical decision - making ( 96% ) . ( 96% ) , and that the patient was comfortable talking about his / her problems using video conferencing perhaps he wanted to ask more questions in private . another significant challenge , especially with this older population , was the issue of hearing loss , which was identified as a problem for seven patients . completed surveys were received from 50 of 99 patients ( 51% ) and 48 respondents provided some personal background information . given the many challenges faced by this group of patients , this was considered a good response rate . more than one - half ( 58% ) were between 7584 years of age , 29% were between 6574 years , and 13% were 85 years or older . it was the first video session for over two - thirds of the patients ( 69% ) , and their satisfaction ratings were consistently positive . overall , 92% of the respondents were satisfied with the session , and almost all indicated that they would be willing to use video conferencing again ( 94% ) . one patient commented , this is a great way to serve the people of cornwall neither they nor the doctor need to drive to ottawa for a follow - up . i would like to see more of this type of work and innovative ideas . i thank all of you for helping me get back to my old self again . on a few occasions , the otn video link did nt work or the screen froze , which impacted on a patient s ability to see the doctor . this likely resulted in slightly lower satisfaction rating given to this item ( 88% ) . clients felt they were understood by the on - site nurse ( 96% ) and the physician who was being teleconferenced ( 94% ) . almost all patients agreed that their privacy and confidentiality were respected ( 98% ) , although a small number stated they would have preferred to see their doctor alone ( 16% ) . in terms of their sense of involvement in their care , most patients felt that their questions were answered by the doctor ( 98% ) , that the video conference session ran smoothly ( 98% ) , and that the appointment provided enough time to deal with everything that needed to be covered ( 92% ) . items with the greatest variability in responses were those related to patient preferences . while most felt that they were able to present the same information they would have provided in person ( 92% ) , a slightly smaller number said that they felt as confident about the doctor s assessment through video as they would an in - person assessment ( 88% ) . thirty percent stated that they were more anxious with the video session than if they had seen the doctor in person , and over one - quarter ( 28% ) felt that they would have preferred to see their doctor in person . details related to the clinic processes were captured through the ccac assessor and the physician clinic summary surveys . usually , physicians saw 34 patients and spent about two hours video conferencing , per clinic . ccac case managers spent an average of 51 minutes : setting up / taking down equipment ( 12 min ) ; explaining video conferencing to patients and providing reassurance ( 22 min ) ; debriefing patients at the end of their session ( 13 min ) ; and 4 minutes on other clinic activities . three clinics encountered technical problems that required significantly more time on the part of the ccac assessor . the ccac assessors were generally more conservative than patients in their rankings of the clinic experience , with 79% agreeing or strongly agreeing that they were satisfied with the sessions . it was not surprising to find a strong correlation between the assessors satisfaction with the clinic and their perception as to whether it ran smoothly . physicians provided feedback on 30 of the 32 clinics , and for the most part felt that the clinic ran smoothly ( 88% ) . they indicated that 17 of the 30 clinics would likely have been cancelled had the geriatrician needed to travel to the clinic site . this is an important finding , given that the objective of providing telemedicine to the target population was to provide more timely access to care . minor technical problems were identified by the geriatrician for 7 of the 30 clinics . on two occasions the video link did not work at all and cases were discussed by phone instead . in addition to rating the clinic experience , physicians also provided feedback on their session with each patient , for 91 of the 99 patients . as figure 3 shows , physicians agreed or strongly agreed that video conferencing provided them with opportunities to ask the patient / ccac assessor questions ( 100% ) . they generally felt that the appointments met their needs ( 96% ) , and that they were able to get enough relevant information to assist in clinical decision - making ( 96% ) . ( 96% ) , and that the patient was comfortable talking about his / her problems using video conferencing another significant challenge , especially with this older population , was the issue of hearing loss , which was identified as a problem for seven patients . as a result of scarce geriatric health - care human resources within small urban and rural communities , there is the need to explore viable clinical options such as telemedicine . the use of telemedicine provides an extension of specialized geriatric services into communities that previously would not have had access to these resources unless the patient and family travelled to larger urban sites . the champlain local health integration network ( of which cornwall is a part ) , reports that one - third of its 1.2 million population live in smaller cities , towns , and rural areas . as a result many have challenges accessing health - care services , either due to lack of proximity or lack of transportation . the lhin notes also that a growing seniors population and increasing rates of chronic diseases are putting pressure on current health - care resources . the eastern counties ( the catchment area for the study population ) clinical services distribution plan identified building geriatric capacity as one of two clinical priorities . to date , the strategy has had limited success and , at present , there is not a geriatric primary care physician champion within the community . the geriatric services action plan includes a recommendation to continue developing the telemedicine initiative and expand its scope of service . the challenges are further compounded when the targeted population is patients who have been assessed and identified with some degree of cognitive impairment . the burden is multiplied when major life - altering issues , such as driving safety , home safety , living at risk , future planning , caregiver and/or family stress , need to be addressed in a timely and responsive manner . telemedicine , in conjunction with the support of case managers / geriatric assessors can improve care transitions between the specialist and primary care physicians . although the expectation was that the incorporation of telemedicine as a follow - up care strategy for this patient population would pose challenges given the patients complex health status and limited exposure to this type of technology , this was not found to be the case : 94% of the patients involved with the initiative agreed that they would use video conferencing again . the majority of patients agreed that they felt understood by the doctor during the video session and felt there was adequate time to deal with everything that needed to be covered . almost all the patients felt that their privacy and confidentiality were respected throughout the video conferencing session . nine out of 10 patients reported that they felt confident that their assessment by the doctor through video conferencing was comparable to an assessment being delivered in person . however , three out 10 patients acknowledged that they felt more anxious with video conferencing than an in - person session . technical limitations such as connection difficulty , images freezing , delays in establishing connections , and connections being lost did impact on overall satisfaction . however , these issues arose infrequently and are continually being addressed as the technology advances . this study has a number of limitations : the sample size is small and data were obtained from a single venue , using questionnaires developed specifically for the study rather than standardized satisfaction surveys . the intent was to assess the receptivity among patients , physicians , and case managers / assessors to using telemedicine for follow - up in a memory disorder clinic . further research is needed to determine the generalizability of this approach and its cost - effectiveness . in summary , there are many reasons to develop telemedicine clinics for assessment and management of dementia . for instance : long wait times for appointments at rural clinics to see a specialist ; commuting distances for rural populations ; the stresses of driving to and in an unfamiliar city or location ; and financial constraints for many older persons who are on a fixed income . the utility of telemedicine in the diagnosis of dementia and follow - up in the literature appears to be very promising . the findings do show that the telemedicine follow - up clinic provided a number of favourable results such as timely access to a follow - up clinic in the patient s own community , fewer cancelled clinics , access to a geriatric specialist , and enhanced care transitions between the follow - up clinic and primary care with the support of the case manager / assessor . the technology ensured that specialist medical follow - up was available to this complex , geographically dispersed , patient population . the high satisfaction rates amongst all key stakeholders ( patients , physicians , and case managers / geriatric assessors ) affirm that telemedicine is a viable option and worth continued shaping and developing , particularly in regions where local physician specialists are a scare resource .	backgroundthere are many reasons to develop telemedicine clinics for assessment and management of dementia . time constraints , location , and poor weather conditions can all impact on the ability of patients and providers to attend rural clinics . the utility of telemedicine in the diagnosis of dementia and subsequent follow - up appears promising in the literature , as it provides a viable means of assessing cognition in patients in remote areas with limited access to medical specialists.methods & resultsthis study explored the feasibility of introducing a telemedicine memory disorder follow - up clinic in a rural community . the evaluation of 32 clinic sessions found high levels of satisfaction , with over 90% of physicians and patients indicating that they d be willing to use video conferencing again . physicians overwhelmingly felt the sessions provided enough information to assist in clinical decision - making ( 96% ) , and patients and ccac case managers / geriatric assessors felt able to present the same information by video conferencing as in person ( 92% for both groups ) . the telemedicine clinic provided a number of favourable results such as : timely access to specialist care in the patient s own community ; fewer cancelled clinics ; enhanced care transitions between the follow - up clinic and primary care with the support of a case manager / geriatric assessor ; and enhanced follow - up for a complex patient population . in addition , the telemedicine initiative freed up spaces for in - person clinics . this allowed them to focus on new patient assessments.conclusionsthe high satisfaction rates amongst all key stakeholders affirm that telemedicine is a viable option and worth continued efforts at shaping and developing , particularly in regions where local physician specialists are a scare resource .
You are an expert at summarizing long articles. Proceed to summarize the following text: the national confidential enquiry into patient outcome and death ( ncepod)1 highlighted issues concerning safety in endoscopic procedures including endoscopic retrograde cholangiopancreatography ( ercp ) . although it did not identify a relationship between the numbers of procedures performed and performance , it found that in 11% of procedures the senior endoscopist performed fewer than 50 ercp per year.24 published studies have shown a variation in ercp workload in different institutions both in the uk1 57 and internationally.810 varadarajula et al8 analysing the outcome of nearly 200 000 ercp performed in the usa over a 3-year period between 1998 and 2001 found the median number of procedures performed in institutions over that period was only 49 , with only 5% performing more than 200 per year . in a large swedish study enochsson et al9 reviewed over 10 000 ercp performed by 177 endoscopists , noting that 55 performed 10 or fewer procedures over a 2-year period . cote et al,10 in an assessment of ercp practice in the usa , found that 40% of more than 1000 american society for gastrointestinal endoscopy members were performing fewer than 50 procedures per year . cotton11 has recently highlighted the ongoing problem of low volume ercp endoscopists in america , stating that now is the time for improvements in practice to be made . perhaps reassuringly within the uk , green et al3 noted in a british society of gastroenterology survey of ercp practice that 84% of uk ercp endoscopists were performing 75 or more procedures per year . the british society of gastroenterology ercp stakeholders group4 recommended a combination of audit , standards and strategy to improve the outcomes of ercp practice and these were adopted in full by the joint advisory group ( jag ) in gastrointestinal endoscopy.12 jag has provided guidelines for trainees wishing to obtain specialist accreditation in ercp , and confirmed that these criteria should be the same for those continuing in practice and include : a complication rate of less than 5% ; a satisfactory completion of the intended procedure for grade 1 cases of over 80% ( defined as all standard biliary procedures);13 14 and that individuals should perform more than 75 cases per 12 months . green et al4 also proposed that units performing fewer than 150 procedures per annum should join a network of local hospitals to allow high standards to be maintained . these guidelines have led to local units , in part associated with hospital trust mergers , rationalising their service . local units have joined together , some experienced colleagues have given up their ercp practice to concentrate on other clinical demands such as bowel cancer screening , with the remaining specialists performing an increasing number of procedures . for an ercp service to remain local for its population , units and individual endoscopists need to audit their performance to enable them to provide evidence to commissioners that these procedures can be performed safely and efficiently with a low complication rate and in a timely manner . varadarajula et al8 demonstrated a relationship between the volume of procedures and outcome both with respect to length of stay and procedural failure . enochsson et al9 found that although the overall rate of complications was low , there did appear to be an increased peri - operative complication rate in low volume when compared to intermediate volume ( 200500 procedures / year ) and high volume hospitals ( > 500 procedures / year ) , although interestingly , the study also highlighted a higher rate of pancreatitis in the high volume hospitals perhaps as a reflection of case mix . by contrast williams et al,15 examining complications of ercp prospectively in five english regions , found a higher risk of pancreatitis in district as opposed to university hospitals . those studies found no relationship between procedures performed and mortality , although thankfully the numbers of such cases were small . bodger et al2 found the mortality risk for these procedures was comparable across english hospitals . further evidence of a better outcome in units performing higher volumes of procedures is supported by kapral et al,16 who demonstrated greater technical success and fewer complications by endoscopists performing more than 50 procedures per year , and loperfido et al17 when more than 200 procedures per year were performed . within the uk , a number of studies has reported on the success , safety and complication rates of ercp , showing that they meet the standards set by jag and that they compared favourably to outcomes from international centres.57 18 district hospitals generally have all of the facilities to provide a therapeutic ercp service , including access to a specialist radiological diagnostic ( such as magnetic resonance cholangiopancreatography ) and biliary intervention and to endoscopic ultrasound . local units should be able to perform most level 1 and 2 procedures ( including large bile duct stone extraction , treating hilar strictures and benign biliary strictures),13 whereas most grade 3 procedures should be referred to tertiary centres . individual endoscopists and units should audit their performance to confirm they are meeting the recommended standards . the evidence from ncepod1 and the recommendations by jag12 have resulted in a significant change in ercp practice in local hospitals . trust mergers , together with a rationalisation of ercp provision have already resulted in a concentration of local services , which is likely to continue to evolve . low volume ercp endoscopists should no longer be providing a service . if local units are able to provide a timely ercp service with sufficient volume of procedures that meet or improve on the recommended national standards , the need for further centralisation may be unnecessary .	more than 48 000 endoscopic retrograde cholangiopancreatographies ( ercp ) are performed in the uk per annum ; the majority within district general hospitals . the proposal for centralisation of ercp services is based on evidence that technical success , length of stay and complication rates are related to the numbers of procedures performed . local units wishing to continue their ercp practice , must demonstrate that they are performing sufficient numbers of procedures in a safe , timely and competent fashion .
You are an expert at summarizing long articles. Proceed to summarize the following text: apoptosis , which protects organisms by removing potentially damaged cells and unnecessary cells after differentiation , is a widely occurring phenomenon in animal development . apoptosis occurs in blastocyst cavitation and continues during gastrulation in mammal and chicken cells [ 2 , 3 ] . increased caspase - like activity and dna fragmentation occurs in xenopus laevis ( x. laevis ) embryos at the gastrula stage [ 4 , 5 ] . moreover , studies have revealed that apoptosis may contribute to development of a variety of organs such as brain , heart , lungs , and kidneys [ 710 ] . x. laevis is considered as one of the most important model organisms for studying vertebrate development and diseases . in contrast with mouse embryos , the large and robust x. laevis embryos are accessible in the stages during which most important decisions of development are taken so that a large variety of methodologies can be used to analyze the genetic regulation of many different developmental processes . pnas-4 was previously identified as a novel apoptosis - related protein in human acute promyelocytic leukemia nb4 cells . recently , a report showed that the human pnas-4 gene was activated during the early response to dna damage . in our previous studies , we proved that the overexpression of pnas-4 induced cancer cell apoptosis via the activation of a mitochondrial pathway . the potential antitumor effects of human pnas-4 were also demonstrated [ 13 , 14 ] . to understand the biological function of the pnas-4 gene in development , we first cloned the homologous pnas-4 gene from x. laevis based on pnas-4 bioinformatics analysis . in the present work , we addressed the preliminary functional annotation of x. laevis pnas-4 ( xpnas-4 ) including protein sequence characterization , subcellular localization , gene expression profiles in developing embryos , and the impacts of microinjection of xpnas-4 mrna or antisense morpholino oligonucleotides ( mos ) on embryonic development . our primary studies on xpnas-4 showed that xpnas-4 may play important roles in embryo development . the hek ( human embryonic kidney ) 293 epithelial cell line was purchased from atcc . female xenopus laevis were injected with 300500 iu human chorionic gonadotropin ( hcg ) on the evening before egg collection . nine to 12 hours after injection , eggs were collected and transferred into a fresh petri dish with culture medium . the xenopus homolog of pnas-4 ( xpnas-4 ) was identified in silico by blasting the x. laevis database with the full - length sequence of the human pnas-4 protein ( genbank protein_id np_057160 ) . bc087412 ) was amplified using the primer combination 5-ggatccatggccaaccagcccatcatc-3 and 5-ctcgagctatagttttgtgtggcgcccagg-3. rt - pcr amplification was carried out in a mycycler thermal cycler ( bio - rad ) using the following program : reverse transcription at 50c for 40 minutes , and denaturing at 94c for 2 minutes , followed by a standard touchdown pcr regime of 94c for 30 seconds , 60c for 30 seconds , and 72c for 40 seconds ( 15 cycles , 1c / cycle ) , and 94c for 30 seconds , 48c for 30 seconds , and 72c for 40 seconds ( 35 cycles ) . the pcr product was cloned directly into the pgem - t easy vector ( promega ) to obtain pgem - t - xpnas-4 plasmid . dna sequencing was performed by bigdye terminator cycle sequencing and the sequences were obtained with a 3730 dna analyzer ( applied biosystems ) . in order to construct the pegfp - n1-xpnas-4 plasmid , the following primers were used , the sense primer : 5-ccgctcgagatggccaaccagcccatca-3 and the antisense primer : 5-cgggatcccgtagttttgtgtggcgcccag-3 , containing the xho i and bamh i restriction sites ( underlined ) , respectively . the termination codon was removed to be in - frame with the enhanced green fluorescent protein ( egfp ) sequence . the following amplification procedure was applied : initial pcr activation step at 94c for 4 minutes , 35 pcr amplification cycles at 94c for 30 seconds , 55c for 30 seconds , 72c for 40 seconds and a final extension cycle of 72c for 10 minutes . nucleotide sequence and conserved domain analysis were carried out using blast programs ( http://www.ncbi.nlm.nih.gov/blast/ ) . alignment of amino acid sequences was constructed using the clustal w method ( http://www.ebi.ac.uk/clustalw/ ) . homology analysis and molecular evolutionary analysis were generated using lasergene software and the megalign program ( dnastar ) . semiquantitative rt - pcr analysis of xpnas-4 expression was performed using the above - mentioned primers . as an internal control for rna quality , the housekeeping gene ornithine decarboxylase ( odc ) was also amplified with sense primer 5-gtcaatgatggagtgtatggatc-3 and antisense primer 5-tccattccgctctcctgagcac-3 according to published document . both reactions were amplified by using the same number of extension cycles ( 30 cycles ) . the samples were resolved on a 1.5% agarose gel with 1 g / ml of ethidium bromide , and analyzed in a uv - transilluminator mini darkroom using quantity one software ( bio - rad ) . digoxigenin - rutp - labeled antisense probes for xpnas-4 were synthesized from pgem - t - xpnas-4 plasmid via sp6 rna polymerase . hybridized transcripts were visualized in situ with antidigoxigenin antibodies and photographed using a nikon dissecting microscope with an attached camera . 5 10 hek293 cells which had been grown in dmem culture medium containing 10% fbs , 100 units / ml penicillin , and 100 mg / ml streptomycin were seeded on 6-well plates in a 37c incubator with 5% co2 . transfections were performed according to the manufacturer 's recommendations by using 10 l of lipofectamine 2000 ( invitrogen ) and either 4 g of pegfp - n1 or pegfp - n1-xpnas-4 plasmid dna . the culture medium was changed after 6 hours with growth medium and maintained for another 24 hours to allow expression of the fusion protein . the transfected cells were examined under a zeiss axiophot fluorescence microscope . capped mrnas used for microinjection in vitro were produced using an mmessage mmachine kit ( ambion ) . the reaction was set up in a total volume of 50 l containing 2.5 g xho i linearized pgem - t - xpnas-4 plasmid , 1transcription buffer , 0.5 mm dntps , 2.5 mm rna cap structure analogue , 10 mm dtt , 20 u rnasin , and 40 u t7 rna polymerase , and then incubated for 2 hours at 37c . the templates were digested with 10 u of rnase free dnase i by incubating the samples for 30 minutes at 37c . the volume was increased with rnase free water to 100 l , and then the samples were applied on quick spin columns ( ambion ) to purify the products . published procedures for the whole - mount tunel staining of embryos were followed . in brief , embryos were fixed overnight at 4c in a 4% solution of formaldehyde in pbs . the samples were washed twice in 100 mm tris - hcl buffer , ph 7.5 , containing 150 mm sodium chloride and 0.1% tween 20 ( tbst ) , and fixed in methanol at 20c for an additional 24 hours . the samples were washed three times at room temperature for 15 minutes in tbst to rehydrate the embryos following methanol fixation . the samples were transferred to tunel buffer ( 25 mm tris - hcl , ph 6.6 , containing 200 mm sodium cacodylate , 5 mm cobalt chloride , and 0.25% bovine serum albumin ) , and washed for 30 minutes . fluorescent labeling of fragmented dna in the samples was carried out for 1 hour at 37c in tunel buffer containing 1 mm fluorescein - dutp and 50 units / ml terminal deoxynucleotidyl transferase . the reaction was stopped by washing the sample in tbst buffer five times for 5 minutes at room temperature . the fluorescein - labeled tunel - positive cells were photographed using an olympus microscope with excitation at 460490 nm and emission at 510 nm . all antisense morpholino oligonucleotides ( mos ) were designed and supplied by gene tools co. of america . mos used in this study were as follows : xpnas-4 mo ( 5-tctcctcctcctcctgagaatatcc-3 ) , and control mo ( 5-cctcttacctcagttacaatttata-3 ) . both xpnas-4 mo and control mo were diluted to be 5 ng / nl and stored at 80c in aliquots mos were microinjected into the animal blastomere of stage-2 embryos . for rescue experiments , rna and mos co - injection was performed at two different ratios : 20 ng xpnas-4 mo plus 450 pg xpnas-4 mrna and 20 ng xpnas-4 mo plus 900 pg xpnas-4 mrna . by blasting the x. laevis database with a human full - length pnas-4 protein sequence ( genbank protein_id : np_057160 ) , we found an x. laevis pnas-4 homologue ( genbank protein_id : aah87412 ) which we named xpnas-4 . the xpnas-4 cdna sequence contains an open reading frame of 579 bp encoding 192 amino acids . just like the human pnas-4 protein , xpnas-4 contains a putative duf862 conserved domain corresponding to amino acid residues 4 to 131 ( figure 1(a ) ) . the sequence of xpnas-4 protein was compared with those of other species including bos taurus ( genbank protein_id . xp_597874 ) , pongo pygmaeus ( genbank protein_id : q5r456 ) , homo sapiens ( genbank protein_id : np_057160 ) , gallus gallus ( genbank protein_id : np_001008460 ) , danio rerio ( genbank protein_id : np_001003532 ) , rattus norvegicus ( genbank protein_id : aah83584 ) , and mus musculus ( genbank protein_id : q9d291 ) , the identity was 88.1% , 87.6% , 87.6% , 87.0% , 86.0% , 85.5% , and 85.5% , respectively . based on the homology analysis , a phylogenetic tree of pnas-4 proteins was constructed ( figure 1(b ) ) . to examine xpnas-4 cellular localization , we fused xpnas-4 to egfp . expression of xpnas-4-egfp in hek293 cells appeared in the cytoplasm and was closely located around the nuclei , but the control ( egfp ) protein was evenly distributed throughout the whole cell without any compartmentalization ( figure 2(a ) ) . thus , xpnas-4 was a cytoplasmic protein which was localized in the perinuclear region of the cytoplasm . in order to determine the temporal expression pattern of xpnas-4 , the mrna level at different developmental stages was detected . semiquantitative rt - pcr analysis showed that xpnas-4 mrna could be detected at all stages of development , from the unfertilized egg to the tadpole stage ( figure 2(b ) ) . the lowest expression level of xpnas-4 appeared at stage 8 ( the mid - blastula transition ) . before the mid - blastula transition , the expression level of xpnas-4 decreased along with the rate of cell division . after the mid - blastula transition , however , the expression level of xpnas-4 increased rapidly . this result suggests that there is xpnas-4 message of maternal origin , since zygotic transcription starts at the mid - blastula transition ( mbt ) . indeed , many other apoptosis - related genes showed similar transcription patterns [ 22 , 23 ] . consequently , xpnas-4 may play an important role in xenopus embryo development . for a better understanding of the expression of xpnas-4 mrna in embryonic tissues , whole mount in situ hybridization up to the gastrula stage ( stage 10 ) , the expression of xpnas-4 was present ubiquitously in the embryo . by the tail bud stage ( stage 35 ) , furthermore , a weak expression of xpnas-4 could also be observed in the prospective heart of the tail bud embryo . its transcripts are present ubiquitously in gastrula stage embryos , while tissue specific expression can be detected in later embryogenesis . to explore the role of xpnas-4 during embryonic development , the synthesized xpnas-4 mrna was injected into xenopus embryos at the one - cell stage . as shown in figures 3(a ) , 3(e ) , and 3(f ) , the eyes were poorly developed and were smaller than normal . about 26% small eye embryos were produced by injection of xpnas-4 mrna at 450 pg / embryo ; while about 38% small eye embryos were observed when xpnas-4 mrna was injected at 900 pg / embryo . taking into consideration the high conservation of xpnas-4 protein with that of zebrafish , we hypothesized that xpnas-4 would act in a similar manner in zebrafish , another developmental system used to study eye development . to test this postulation the resulting phenotype was quite similar to what occurred with xpnas-4 mrna injection in xenopus . following injection of 300 pg xpnas-4 mrna 38% of embryos had abnormal eyes , and after injection of 600 pg mrna 86% of embryos exhibited abnormal eye development . unlike xenopus embryos , however , zebrafish embryos with eye defects could be divided into two groups : the less affected embryos had reduced eye size , while the more strongly affected embryos exhibited a one - eye phenotype ( figures 4(d ) , 4(e ) , and 4(h ) ) . in addition to the eye defect , some strongly affected embryos had a bent axis ( figures 4(f ) and 4(i ) ) . injection of 300 pg of xpnas-4 mrna resulted in 34% reduced eye embryos and 4% one - eye embryos , while injection of 600 pg of xpnas-4 mrna resulted in 24% reduced eye embryos and 62% one - eye embryos ( figures 4(j)4(l ) ) . all of the uninjected embryos were normal ( figures 4(a)4(c ) , and 4(g ) ) . to confirm that the developmental defects caused by heterologous injection of xpnas-4 resulted from apoptosis , whole - mount tunel ( terminal deoxynucleotidyl transferase - mediated nicked - end labeling ) staining , a highly sensitive indicator of dna fragmentation in situ was employed to detect the presence of apoptotic cells . tunel - positive cells were present at high frequency on the side of zebrafish embryos on which eye development was affected ( figure 4(n ) ) when compared with the control embryos ( figure 4(m ) ) . it is widely accepted that apoptosis is involved in many embryological processes and plays a significant role in animal development . the tumor suppressor protein p53 , a widely known trigger of apoptosis , has been implicated as an important protein in embryonic development . inappropriate overexpression or underexpression of p53 can lead to embryonic lethality or increased risk of malformations . inactivation of p53 function by gene knock - out or through lens specific promoter - driven expression of the viral gene e6 , interrupts normal apoptosis in the lens leading to formation of a cataract [ 28 , 29 ] . the caspase family including caspase-1 , -2 , -3 , -4 , -6 , and -9 , is involved in eye differentiation in different species of vertebrates . during development of the chicken lens , members of the bcl-2 family , such as bcl-2 , bax , bad , and bcl - xs / l , are abundantly expressed between stages e12 and e16 . in our experiment , we found a considerable number of apoptotic cells appeared in abnormal embryos by whole mount in situ hybridization , suggesting that abnormal eye development resulted from the overexpression or heterologous expression of xpnas-4 . this further confirmed that xpnas-4 is involved in apoptosis during embryonic development . to further define the function of xpnas-4 during embryogenesis , we performed translational inhibition of xpnas-4 by microinjecting antisense morpholino oligonucleotides ( mos ) . control and xpnas-4 mos ( 20 ng ) were microinjected into the animal blastomere of stage-2 embryos . as development proceeded , 86.7% of embryos ( n = 98 ) microinjected with xpnas-4 mo had a failure of head development and a shortened axis ( figures 5(a ) and 5(c ) ) . embryos with failed head development died in the late tailbud stage , while control mo microinjected embryos ( 98.4% , n = 128 ) developed to the next stages normally . in order to confirm that the phenotypes were due to translational inhibition of xpnas-4 , as expected , the failure of head development and the shortened axis were rescued to some extent after co - injection of 20 ng xpnas-4 mo plus 450 pg xpnas-4 mrna . about 56.5% of embryos ( n = 92 ) exhibited a failure of head development . the defective embryos had longer axes than those microinjected with xpnas-4 mo alone . a greater extent of rescue was observed after co - injection of 20 ng xpnas-4 mo plus 900 pg xpnas-4 mrna . the defective embryos had longer axes than those microinjected with xpnas-4 mo plus 450 pg xpnas-4 mrna ( figures 5(b ) and 5(c ) ) . these results revealed that the failure of head development and shortened axis caused by xpnas-4 mo microinjection were due to depletion of xpnas-4 . we also found similar defects using zebrafish as a model system in our previous studies . loss of function of the pnas-4 gene caused gastrulation defects with a shorter and broader axis , as well as a posteriorly mis - positioned prechordal plate , due to the defective convergence and extension movement . in the present work , we found that loss of function of the pnas-4 gene in x. laevis embryos resulted in the failure of head development and a shortened axis . the phenotypic differences were probably due to the different species or differences in the inhibition rate of morpholino oligonucleotides . here we identified the pnas-4 homolog from x. laevis based on the amino acid sequence of human pnas-4 protein . the high similarity suggested that the human and x. laevis proteins might have analogous functions . cellular localization suggested that xpnas-4 is in the cytoplasm and is located around the nucleus . investigation of the developmental expression pattern showed that xpnas-4 is present throughout embryogenesis and undergoes pronounced changes in its level of mrna during embryo development . in addition , microinjections of xpnas-4 mrna into xenopus laevis embryos resulted in embryonic developmental defects including a small eye phenotype in developing x. laevis embryos , and a reduced eye size or one - eye phenotype in the developing zebrafish embryos . whole mount in situ hybridization further confirmed that the developmental defects resulted from apoptosis during embryonic development . furthermore , embryos microinjected with xpnas-4 mo had a failure of head development and shortened axis . in conclusion , our results provide important clues about the molecular mechanisms by which xpnas-4 regulates developmental processes .	apoptosis plays an important role in embryonic development . pnas-4 has been demonstrated to induce apoptosis in several cancer cells . in this study , we cloned xenopus laevis pnas-4 ( xpnas-4 ) , which is homologous to the human pnas-4 gene . bioinformatics analysis for pnas-4 indicated that xpnas-4 shared 87.6% identity with human pnas-4 and 85.5% with mouse pnas-4 . the phylogenetic tree of pnas-4 protein was also summarized . an analysis of cellular localization using an egfp - fused protein demonstrated that xpnas-4 was localized in the perinuclear region of the cytoplasm . rt - pcr analysis revealed that xpnas-4 , as a maternally expressed gene , was present in all stages of early embryo development . whole - mount in situ hybridization showed that xpnas-4 was mainly expressed in ectoderm and mesoderm . furthermore , microinjection of xpnas-4 mrna in vivo caused developmental defects manifesting as a small eye phenotype in the xenopous embryos , and as a small eye or one - eye phenotype in developing zebrafish embryos . in addition , embryos microinjected with xpnas-4 antisense morpholino oligonucleotides ( mos ) exhibited a failure of head development and shortened axis .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 14-year - old boy presented with asymptomatic swelling of the chin that had been occurring over 18 months . there was no history of similar lesions elsewhere in the patient or his family members . there was no history of pain , alteration in speech or difficulty in speaking . on examination , there was a skin colored , non - tender , firm and irregular swelling over the left mentolabial groove extending onto the lower lip ( fig . 1 ) . there was bulging of the left part of the lower lip with distortion of the lip architecture even on occlusion of the lips . the mass produced an irregular , smooth protrusion on the mucosal surface of the lower lip without any discoloration . there was no evidence of freckles or caf au lait macules and no bruit over the lesion . clinically a diagnosis of neurofibroma was made and excisional biopsy under local anesthesia and aseptic conditions was performed . gross observation showed that the tumor consisted of a skin covered nodule measuring 10.50.5 cm . the tumor was ill defined and consisted of fascicles of mature nerve tissue arranged in a multinodular pattern separated by fibrocollagenous septa . the nerve fascicles showed an intricate admixture , with mature striated skeletal muscle bundles of varying sizes ranging from 15 to 20 m with some being up to 50 m ( figs . 2 , 3 ) . a histopathology diagnosis of btt involving the mental branch of the trigeminal nerve was made . a wide variety of benign soft tissue tumors occur in the head and neck region . these include tumors arising from adipocytes , fibrohistiocytes , as well as vascular and neural tissue . the latter include neurofibroma , schwannoma , neuromas and other uncommon tumors like meningioma , granular cell tumor , ganglioneuroma , and very rarely , neuromuscular hamartoma or triton tumors.1,6 triton tumors are neoplasms composed of neural and skeletal muscle cells of which fewer than 20 cases have been reported in the english language literature.4 the name triton has been derived from the amphibian triton salamander , in which the normal nerve was believed to induce regeneration of skeleton muscle.7 tumors included under these categories are btt , neurofibroma with rhabdomyomatous differentiation , medulloblastoma with rhabdomyosarcoma elements , rhabdomyosarcoma with ganglion cells ( ectomesenchymoma ) and malignant peripheral nerve sheath tumor with rhabdomyosarcoma , the latter being the most commonly recognized entity.1 btts commonly occur in infants and children , though it has been rarely reported in adults.2,8 the most common sites of predilection are the large peripheral nerves , especially the brachial and sciatic nerves.1 very rarely have these cases been reported in the head and neck region.2 - 5 btts of the head and neck fall into two categories . first , are the aggressive central types involving large cranial nerve trunks ( v , vii , and xii ) with occasional aggressive behavior . the other variant is the nonaggressive peripheral type which presents as asymptomatic slow growing skin nodules and is known to occur in adults.5 in the peripheral category , only four cases have been reported including two cases occurring in the tongue , both affecting adults.2,5 two other cases were in a 3-month - old infant in the submandibular skin and a 17-year - old female in the left chin.3,4 all four of these cases in the peripheral category were cured by simple surgical excision except in one case report by demir et al.,4 which had a local recurrence . our case belongs to the peripheral type , involving the mental nerve , which is the terminal branch of the inferior alveolar branch of the trigeminal nerve . though the histogenesis of these unusual tumors has initiated much discussion , the exact mechanism is not known . some authors believe that the cell lines are derived from primitive neural crest cells , while others consider it to represent a muscle spindle . recently , daley et al.5 put forth an alternative theory that an epigenetic or sporadic alteration of the motor end - plate leads to reactive proliferation of neural and skeletal muscle tissue in an organoid pattern.1 clinically , patients are usually asymptomatic , except for the presence of a palpable mass , which may be solitary or multinodular . however , depending on the site of involvement , pain , neurologic dysfunction in the form of muscle weakness , parasthesia and headache including oculomotor ophthalmoplegic migraine , has been reported.6,8,9 computed tomography scans provide a good guide to suggest the diagnosis and serve to define the entire scope of the lesion , especially in the central type of tumors.6 due to the rarity of these lesions , clinical diagnosis is difficult and histopathology is required . histopathologically these tumors are multinodular , subdivided by collagenous connective tissue into smaller nodules and fascicles . the fascicles are comprised of mature striated muscle fibers intimately associated with nerve fibers and enclosed within the same perimysial - like fibrous sheath . sometimes the fibrous tissue predominates , rendering the diagnosis difficult.7 treatment involves excision which is usually more aggressive in cases of the central type and conservative for the peripheral tumors . post - resection nerve palsies are always a risk , and hence , nerve sparing is advocated.1,5,7,10 though prognosis is excellent , recurrence has been reported rarely , which mandates follow - up.4 an important generalization to bear in mind is that , though rare , btts should be considered in the differential diagnosis of head and neck tumors . since treatment of some of these tumors results in residual nerve dysfunction on attempted surgical resection , while others spontaneously regress , recognition of this entity is important for planning appropriate treatment .	benign triton tumors ( btt ) are very rare lesions composed of mature skeletal muscle and neural tissue . we report a case of a 14-year - old boy who presented with asymptomatic swelling of the chin over an 18-month duration which increased gradually to involve the left side of the lower lip . clinically , a diagnosis of neurofibroma was made . excisional biopsy confirmed the diagnosis of a btt . having an affinity for large nerve trunks like the brachial and sciatic , these tumors rarely occur in the head and neck region . when they do , they may involve the large central cranial nerve trunk and present as intracranial masses or involve the smaller peripheral nerve twigs and present as asymptomatic skin nodules , of which only four cases involving peripheral nerves are reported in the english literature . here , we report the fifth documented case of a btt involving the mental branch of the trigeminal nerve . a brief review of the literature is also provided .
You are an expert at summarizing long articles. Proceed to summarize the following text: fogo selvagem ( fs ) has been classically defined as a b cell - mediated disease due to the presence of both 1 ) spontaneously appearing , intraepidermal clinical blisters as well as 2 ) epidermal - specific autoantibodies belonging to the igg class[13 ] , predominantly the igg4 subclass . the role of b cells in endemic pemphigus foliaceus ( epf ) is also demonstrated by the demonstration that igg and the f ( ab ) 2 fab fragments of fs igg are pathogenic when passively transferred into neonatal mice and rabbits[47 ] . in 1937 , a model of fs in rabbits was first developed by injecting the serum of pf patients . these animals developed intra - epidermal blisters , which clinically , histologically , and immunologically duplicated the human disease . the role of b cells in the disease is also highlighted by the passive transfer of maternal pregnancy igg autoantibodies ( pregnant fs patients with high levels of autoantibodies ) . the antibody transfer has been reported in less than five cases , with temporary compromise of the fetus . moreover , the materno - fetal passive transfer epf disease consistently clears over the first two months of life . thus , this disease is present only temporarily , causing difficulties with any comparison to the natural , chronic course of epf in adults . no neonatal cases of the el bagre endemic pemphigus foliaceus ( el bagre - epf ) or the tunisian variant ( tunisian epf ) have been described , since the disease in these variants does not affect women of childbearing age[911 ] . although el bagre - epf intercellular keratinocyte autoantibodies are primarily from the igg4 subclass , and their iif titers correlate with clinical activity and disease severity ( as also reported in fs ) , el bagre epf sera contains autoantibodies of the igg3 subclass[911 ] that recognize an intracytoplasmic antigen , whose nature remains unknown . in addition to this potential antigen(s ) , many others likely remain unknown in el bagre - epf disease . moreover , in addition to plakins , desmogleins and bullous pemphigoid antigens 1 and 3 in el bagre epf , studies have shown emerging autoantigens in fs such e - cadherin . the role of classical cadherins as immunological targets of fs was well documented by 1 ) immunoprecipitation coupled with immunoblotting ( ip - ib ) and 2 ) elisa techniques utilizing a baculovirus - expressed ectodomain of e - cadherin . by ip - ib , the immunoreactivity of fs sera with e - cadherin was also demonstrated by ip - ib using human epidermal extracts . however , immunofluorescence staining of a431de cells ( e - cadherin positive , dsg1 negative ) with pemphigus sera showed negative results . immunoadsorption and competitive elisa analysis suggest that most of the anti - e - cadherin antibodies cross - react with desmoglein 1 ( dsg1 ) , whereas others may represent independent antibodies that do not cross - react with dsg1 . the functional relevance of these anti - e - cadherin igg autoantibodies detected in these pemphigus sera remains to be defined . the baculovirus heterologous gene expression system has also been utilized to attempt to explain intramolecular epitope spreading on fs another autoantigen described in fs is an ubiquitin carrier protein . the authors screened the autoantibodies from a fs patient utilizing a lambda gt11 human keratinocyte cdna library . one immunoreactive cdna clone ( lambda epf5 ) containing a 900-base pair insert was isolated , and subjected to further analysis . eight of 25 fs sera were then shown to react with an epf5 fusion protein on immunoblots ( ib ) . the epf5 cdna insert hybridized with a 1.2-kilobase epidermal rna transcript on a northern blot . sequence analysis revealed that lambda epf5 contained a complete coding sequence for a 24-kda polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins , or e2s , which are an essential component of the ubiquitin - protein conjugation system . the relevance of the cloned epf epitope in the pathogenesis of this autoimmune disorder remains to be determined . the same group of researchers also identified a novel igg anti - keratin autoantibody in the serum of a fs patient ( cascas-42 ) . the cascas-42 antibody was shown to be specific for a 59 kda acidic murine keratin , as well as its 56.5 kda human counterpart ( moll classification cytokeratin 10 ) ; it was also distinct from previously described pemphigus antibodies . anti - keratin autoantibodies present in the cascas-42 serum were purified by affinity chromatography with a 59 kda murine keratin - agarose column . in 80% of el bagre - epf patients , the presence of a lupus - like band is seen on direct immunofluorescence ( dif ) . the identity of the individual autoantigens , and deposition of a possible immune - complex at the basement membrane zone ( bmz ) are not yet characterized . selected epf patients have developed 1 ) glomerulonephritis with chronic renal failure , and 2 ) sudden death syndrome ; however , correlation of these clinical sequelae has not been demonstrated to be associated with epf . in addition to the lupus band , preliminary studies have revealed the presence of epf autoantibodies against urinary bladder , parietal cells of the stomach , kidney , neural , vascular , and skeletal , cardiac and smooth muscle cells ; however , their role in pathogenicity needs to be clarified ( abreu et . al . , manuscript in preparation ) . a recent study utilizing fs sera searched for the presence of a broad spectrum of autoantibodies and possible association with other autoimmune diseases in 120 patients with fs and 200 healthy controls from brazil in contrast to el - bagre epf , no differences were found between the two groups ; however , 92.5% of the patients were undergoing steroid therapy . other studies have also been reviewed ; for example , 196 patients with fs from mato grosso do sul and 20 fs cases from gois ( states of brazil ) were analyzed for the presence of antinuclear antibodies ( ana ) by indirect immunofluorescence ( iif ) , with no positive results . in 28 patients with fs , 21.4% were positive for anti - gastric parietal cell ( gpca ) antibodies , 7.1% for anti - mitochondrial antibodies ( amas ) , and 3.6% for anti - smooth muscle ( sma ) antibodies . however , the autoantibody positivity in these studies was not conclusively diagnostic of other , concurrent autoimmune disease . the 1 ) use of nonspecific methods , 2 ) small number of patients investigated and 3 ) concomitant steroid therapy could contribute to variations in the results obtained . desmoglein 1 ( dsg1 ) is reported to be one of the antigens recognized by autoantibodies in fs , tunisian epf and el bagre - epf[9111725 ] . however , we recently reported some differences between fs and el bagre - epf by examining autoantigens in both diseases by various biochemical and molecular biological techniques , including immunoblotting ( ib ) , immunoprecipitation ( ip ) and elisa ; we utilized various antigen sources including baculovirus - expressed proteins . in order to confirm the reactivity of el bagre epf sera with plakin family proteins ( also known as paraneoplastic pemphigus antigens ) , we examined sera from both el bagre - epf and fs patients by ib using various recombinant , domain - specific peptides . these studies indicated that a considerable number of el bagre - epf sera reacted with various domains of periplakin , while only a few fs sera reacted with some of them . in addition , the number of el bagre - epf sera reactive with recombinant periplakin proteins was significantly higher than the fs sera , indicating that preferential reactivity with periplakin may be characteristic of el bagre - epf . in contrast , the sera of both types of epf rarely reacted with bp230 recombinant proteins . therefore , it seems likely that the 230 kda protein band detected by some el - bagre - epf sera in our previous studies[911 ] may , in fact , represent a different protein with a molecular weight of approximately 230 kda different than bp230 . we also developed an elisa for detecting a heterogeneous antibody population in serum from people suffering from el bagre - epf that is suitable for detection of plakins . another interesting finding is that el - bagre - epf sera preferentially reacted with paraneoplastic pemphigus ( pnp ) antigens , and there may be a similar pathogenic mechanism in autoantibody production in el bagre - epf and pnp , although the two diseases are quite different clinically and epidemiologically . these results lead us to speculate that , in conjunction with genetic predisposition , specific triggers in each endemic focus may induce a highly immunogenic state in the patients , resulting in the production of antibodies to many self - antigens . in addition , we speculate that some of these self - antigens ( specifically in el bagre - epf cases ) are associated with the antigenic response seen in paraneoplastic syndromes . further , multiple other proteins of different molecular weights are specifically recognized by both fs and el bagre - epf patient sera . one possible explanation for these findings might involve an epitope spreading mechanism leading to the production of multiple autoantibodies , although the amino acid sequences of the desmogleins and plakins display marked differences . sera from fs patients in the preclinical stage have been shown to recognize epitopes on the cooh - terminal ec5 domain of dsg1 . actual clinical disease onset is associated with the emergence of specific antibodies for epitopes on the nh2-terminal ec1 and ec2 domains ; all sera from fs patients with active disease recognize the ec1 and/or ec2 domains of dsg1[2125 ] . these results suggest that anti - dsg1 autoantibodies in fs are initially raised against the cooh - terminal ec5 domain in individuals without skin disease . in genetically predisposed subjects , the autoimmune response may then undergo intramolecular epitope spreading toward epitopes on the nh2-terminal ec1 and ec2 domains of dsg1 , leading to disease onset . moreover , intramolecular epitope spreading could also modulate remissions and relapses of fs[2125 ] . fs is endemic in limo verde ( lv ) , state of mato grosso , brazil . in one study , anti - dsg1 igm antibodies were detected in sera from 58% of fs lv patients ( n=31 ) , in contradistinction to sera from other geographic areas , including 19% of fs patients from hospital - campo grande ( n=57 ) , 19% from hospital - goiania ( n=42 ) , 12% from hospital - so paulo ( n=56 ) , 10% of pf patients from united states ( n=20 ) , and 0% of pf patients from japan ( n=20 ) . sera from pemphigus vulgaris patients ( pv ) ( n=40 , usa and japan ) , bullous pemphigoid patients ( bp ) ( n=40 , usa ) , and healthy donors ( n=55 , usa ) demonstrated negligible percentages of positivity . high percentages of positive anti - dsg1 igm antibodies were found in healthy donors from four rural , brazilian amerindian populations ( 42% of 243 ) as compared with healthy urban donors ( 14% of 81 ; p<0.001 ) . more than 50% of healthy donors from lv ( n=99 , age 5 - 20 years ) possessed igm anti - dsg1 across a wide age range , whereas igg - anti - dsg1 was differentially detected in age specific ranges : 2.9% at ages 5 - 10 years , 7.3% at ages 11 - 15 years , and 29% of donors above age 16 . anti - dsg1 igm epitopes were found to be ca2 + and carbohydrate - independent . based on the above data , the authors suggested the possibility that anti - dsg1 igm antibodies are common in fs patients in their native environment , and uncommon in other pemphigus phenotypes as well as in fs patients who migrate to urban hospitals . thus , recurrent environmental antigenic exposure may lead to both igg and igm responses that trigger fs . another fs study utilized fs hybridomas that secreted either igg or igm ( predominantly igg1 subclass ) autoantibodies from the b cells of 1 ) eight current fs patients , as well as 2 ) one individual taken 4 years before fs onset ; the h and l chain v genes of anti - dsg1 autoantibodies were analyzed . first , clonally related sets of anti - dsg1 hybridomas characterize the response in individual fs patients . second , h and l chain v gene use seems to be biased , particularly among igg hybridomas , and third , most hybridomas are mutants and exhibit a bias in favor of cdr ( complementary determining region ) amino acid replacement(aar ) mutations . pre - fs hybridomas also exhibit evidence of antigen selection , including an overlap in v ( h ) gene use and shared multiple aar mutations with anti - dsg1 fs hybridomas , suggesting selection by the same or similar antigen . the authors concluded that the anti - dsg1 response in fs is antigen driven , and that selection for mutant anti - dsg1 b cells begins well before the onset of disease . the prevalence of anti - dsg1 antibodies in healthy subjects and their distribution in the different regions of tunisia was recently investigated , to better identify endemic areas of pemphigus foliaceus ( pf ) . the authors tested , by elisa , sera from 270 normal subjects recruited from different tunisian areas , 90 tunisian epf patients and 203 healthy relatives these patients . seventy - six patients ( 84.4% ) , 20 healthy controls ( 7.4% ) , and 32 relatives ( 15.76% ) possessed anti - dsg1 antibodies . in southern tunisian regions , where epf is associated with a significant sex ratio imbalance ( 9 females : 1 male in the south vs. 2.3 : 1 in the north ) and a lower mean age of disease onset ( 33.5 years in the south vs. 45 years in the north ) , a higher prevalence of anti - dsg1 antibodies in healthy controls was observed ( 9.23% vs. 5.71% in the north ) . the highest prevalence of anti - dsg1 antibodies in healthy relatives ( up to 22% ) was observed in the most rural southern localities . more than half anti - dsg1 positive healthy controls were living in rural conditions with a farming occupation , which suggests that this activity may expose the subjects to predilecting environmental conditions . in addition , desmocollins i and ii have been reported as autoantigens recognized by sera from some patients with fs . desmocollins are transmembranous glycoproteins that serve as components of desmosomal junctions , and occur in three different isoforms ( desmocollins 1 , 2 and 3 ) ; all isoforms are represented in the epidermis . in this study , the authors examined sera of various types of pemphigus by immunoblotting ( ib ) with purified bovine desmosomes and bovine desmocollin 1 , 2 and 3 fusion proteins , expressed in pgex vectors . six of 15 ( 40.0% ) fs sera , two of 18 ( 11.1% ) non - endemic pemphigus foliaceus sera , eight of 39 ( 20.5% ) pemphigus vulgaris ( pv ) sera , and two of 11 ( 18.2% ) normal sera , demonstrated reactivity with desmocollins from desmosomes . experiments with the fusion proteins showed that no desmocollin isoform was specifically recognized by sera of any individual type of pemphigus . the authors concluded that the pathogenesis of pemphigus might thus be more complex than previously believed . exposure of uninvolved skin to uvb induces acantholysis , with in vivo binding of igg and c3 to the epidermal intercellular spaces . nevertheless , it is also known that complement activation per se is not necessary for pemphigus bulla formation . recently , the role of proteases in the pathology of acantholysis in pemphigus has been addressed . since 1983 , the plasminogen - plasmin system has been implicated in the pathophysiology of epf . however , mitigating against the participation of this system in acantholysis , pemphigus vulgaris and pemphigus foliaceus autoantibodies were shown to be pathogenic in plasminogen activator knockout mice . nevertheless , other work has suggested that regulation of the expression of the urokinase receptor and junctional proteins ( including plasminogen , its activators , and the kininogen - kallikrein - kinin system ) contribute to acantholysis as part of the mechanisms mediating pemphigus . the role of urokinase type plasminogen activator ( upa ) has been well documented in the pathogenesis of pemphigus vulgaris ( pv ) . activation of plasminogen into the active serine protease plasmin initiates extracellular proteolysis , leading to acantholysis ; however , the mechanisms underlying this process are not clearly understood . in addition , the expression of p63 and the induction of apoptosis have been documented in healthy and lesional skin from fs patients , when compared to normal subjects . in a separate study , an igg passive transfer mouse model of pf tunel - positive ( apoptosis positive ) epidermal cells and increased oligonucleosomes in epidermal cytosolic fractions were detected in diseased mice . a time course analysis revealed that the tunel - positive epidermal cells appeared prior to the formation of intra - epidermal blisters . moreover , after pf igg injection , the pro - apoptotic factor bax was upregulated at early time points ( 2 and 4 h ) , and the anti - apoptotic factor bcl - xl was downregulated at later time points ( 6 , 8 , and 20 h ) ( determined by western blot analysis ) . the active forms of caspase-3 and -6(a putative initiator of acantholysis ) were detected in the later time period ( 6 , 8 , and 20 h ) . in addition , administration of ac - devd - cmk , a peptide - based caspase-3/7 inhibitor , protected mice from developing intra - epidermal blisters and clinical disease induced by pf igg . the same protective effect was also observed utilizing a broad - spectrum caspase inhibitor , bok - d - fmk . collectively , these findings show that biochemical events leading to apoptosis are provoked in the epidermis of mice injected with pf autoantibodies . other studies in fs have shown that mannose - binding lectin ( mbl ) and mbl - associated serine protease ( masp-2 ) play a key role in innate immunity ; their deficiency has also been related to both increased susceptibility to infection and development of autoimmune diseases not related to fs . mbl and masp-2 serum levels were measured in 114 patients with fs and in 100 healthy individuals in brazil . mbl and masp-2 levels were measured by sandwich assays ( time - resolved immunofluorimetic assays ) utilizing monoclonal antibodies . no differences were observed in mbl levels in patients with fs compared with controls [ mean sem 1230.07 , + /- 132.18 ng / ml ( median 789.0 ng / ml ) vs. mean 1036.98 , + /- 117.99 ng / ml ( median 559.5 ng / ml ) , p = 0.32 ] . non - significant , lower masp-2 levels were observed in epf patients , compared with controls [ mean 274.34 , + /- 15.66 ng / ml ( median 239.5 ng / ml ) , vs. mean 304.72 , + /- mbl deficiency ( < 10 ng / ml ) and masp-2 deficiency ( < 100 ng / ml ) did not differ significantly between patients and controls . these data indicate that mbl and masp-2 deficiency are not associated with susceptibility to fs . few studies have been performed testing immune cells other than b cells in epf . in recent studies , b and t lymphocytes were quantified in the peripheral blood of 30 fs patients according to their ability to form rosettes with 1 ) sheep erythrocytes , or 2 ) sheep erythrocytes sensitized with antibody and complement . the total t lymphocyte count and the functional t cell count were significantly lower ( by student 's t test ) as detected by the active rosette test in fs versus normal patients . in addition , peripheral lymph nodes from three patients showed a decrease in the number of t cells in the paracortical areas . paradoxically , it had been thought that b cells represented the main immunological mediators in the pathogenesis epf . however , multiple complications of epf are associated with non - b - cell immunity , including secondary infections by 1 ) poxvirus ( molluscum contagiosum ) , 2 ) mycobacterium tuberculosis , 3 ) parasites ( strongyloidiasis and entamoebiasis ) , 4 ) fungi and 5 ) herpes virus , including the relapsing kaposi 's varicelliform eruption . thus , these studies suggest that fs is not solely a b - cell - mediated disease . it is important to keep in mind that susceptibility to these secondary infections may or may not be related to concurrent corticoid therapy . further , an understanding of the immune response of patients to molluscum virus may also help to explain the susceptibility to smallpox seen in some individuals . an additional study was conducted on 16 patients with fs , ten of them with the localized form ( group g1 ) and six with the disseminated form ( group g2 ) . these patients underwent complete blood counts , quantitation of mononuclear cell subpopulations by monoclonal antibodies , studies of blastic lymphocyte transformation , and quantitation of circulating fs antibodies by iif testing . the test profile was created order to correlate their clinical signs , symptoms and laboratory data with their immunological profiles and to determine the relationship between circulating autoantibody titers , lesion intensity and course of lesions under treatment . all patients showed decreased relative cd3 + and cd4 + t cell values , and a tendency towards decreased relative values of the cd8 + t cell subpopulation . blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients of groups g1 and g2 than in controls . the fs iif testing was positive in 100% of g2 patients and in 80% of g1 patients . the median value for the iif antibody titers was higher in group g2 than in group g1 . analyzing the results , the authors concluded that cell immunity was preserved , and that there was a direct relationship between antibody titers detected by the iif test and dissemination of skin lesions . to elucidate the role of pathogenic t cells in fs autoimmunity , t cell receptors ( tcrs ) were characterized utilizing complementary dna isolated from 17 dsg1-specific t cell clones , generated from fs patients by clonal expansion in vitro . to analyze the t cell repertoire , a panel of primers , collectively specific for the known human t cell receptor variable region ( tcrbv ) family , was paired with a constant region primer in order to amplify one distinct t cell receptor variable region allele for each t cell clone . polymerase chain reaction ( pcr ) products were then sequenced to determine the precise chain gene usages . in the 17 clones tested , 10 distinct t cell receptor variable region usages and nine tcr joining gene segment usages were identified . furthermore , tcr variable region and joining usage did not appear to be random , but were oligoclonal in nature , with some preference shown for t cell receptor variable regions 5s1 and tcr bj2s5 . in addition , another study characterizing the autoimmune t cell response associated with fs indicated that the great majority of fs patients have circulating t lymphocytes that specifically proliferate in response to the extracellular domain of dsg1 . long - term t cell cultures developed from these patients also responded to dsg1 , and this antigen - specific response was shown to be restricted to hla - dr molecules . these dsg1-reactive fs t cells exhibited a cd4-positive memory t - cell phenotype , and produced a t helper 2(th2)-like cytokine profile . other studies have shown that if tissue damage is mediated by anti - dsg1 antibodies in epf , an initial t cell response is a likely requirement for autoantibody generation . further studies have described attempts to determine the number of dendritic langerhans cells in fs . since serum il-12 is increased in patients with fs and langerhans cells ( lc ) produce il-12 , serum il-12 autoantibodies were titrated by elisa in another study , resulting in increased level in pf sera . biopsies of blistering lesions were obtained from 22 fs patients , 13 of whom were submitted to biopsy of both lesional and apparently healthy skin . the control groups consisted of skin from 8 cadavers , and from 12 unaffected women presenting for breast plastic surgery . lcs and dcs were also identified with anti - cd1a antibodies , and quantified by morphometric analysis . lc numbers in the lesional and non - lesional skin from fs patients were similar to that of both control groups . dc numbers in the lesional skin ( median=0.94 dc / mm basement membrane ) were higher than those of the cadaver group ( median=0.13 dc / mm basement membrane ) . in the 13 fs patients with biopsies of both lesional and non - lesional skin , lcs and dcs were present in larger numbers in the lesions . a direct correlation existed between dc numbers in the lesions of the fs group and serum autoantibody titers ; this correlation was not observed for lc numbers . the increased number of dcs in the lesion , as well as the direct correlation with serum autoantibody titers suggests the participation of dcs in the pathogenesis of pf . the relationship between increased dc number and il-12 in pf needs to be further clarified . some attention has been given to the role of dendritic cells in the mechanism of epf acantholysis . a predominance of cd4 + t cells , a decrease in langherhans cells ( lcs ) , an increase of cd1a+ dermal dendritic cells , and a lack of keratinocyte icam-1/cd54 and hla - dr molecules in lesional skin was observed in fs . twenty biopsy specimens of lesional and perilesional skin of fs patients were analyzed by immunohistochemical techniques . the panel of monoclonal antibodies included cd1a , cd4 , cd8 , hla - dr , il-2r , lfa-1/cd11a , icam-1/cd54 , and pan - b . a semiquantitative analysis of the cell populations revealed a predominance of cd4 + t lymphocytes in the tissue in perilesional and lesional skin . the population of lcs was decreased in lesional skin when compared with the perilesional skin , whereas cd1a+ dermal dendritic cells predominated in lesional skin . keratinocyte expression of icam-1/cd54 and hla - dr was negative in both lesional and perilesional skin . these results suggest the involvement of cell - mediated immunity in fs . the lack of keratinocyte icam-1/cd54 expression may be related to the pattern of cytokines secreted by the cd4 + t cells of the tissue in fs . utilizing morphometric analysis , increased dermal dendritic cells in lesional skin from epf patients was directly correlated with igg autoantibody titration by iif . these results may reflect the possibility that impairment in the cellular , and/or more complex immune response might play an important role in fs disease . other authors had reported paucicellularity of s-100 positive cells in perilesional and lesional skin from patients with fs . in 36 fs patients , the presence of s-100 protein antigens identified by immunohistochemistry showed a marked reduction or even disappearance of the cell population bearing the s-100 antigen in lesional skin . these results are in agreement with the findings discussed above , and might provide an indirect insight into the possible peripheral anergy that may be part of the loss of peripheral tolerance in epf disease . a recent study evaluated the expression of the proinflammatory cytokines interleukin 1 , interferon gamma and tumor necrosis factor alpha ; the pro - apoptotic inducers fas and inducible nitric oxide synthase ; and the apoptosis inhibitor bcl-2 to evaluate the presence of apoptosis in fs . skin biopsies from 13 patients with fs and controls were evaluated by immunohistochemistry , and apoptosis was determined by a terminal deoxynucleotidyl transferase - mediated dutp nick - end labeling assay . the results showed that proinflammatory cytokines were only detected in cells of the inflammatory exudate . inducible nitric oxide synthase , fas , and bcl-2 were all expressed by both epithelial and inflammatory cells . epithelial apoptosis was observed in 12 cases ( 92.3% ) , and subepithelial apoptosis in 11 cases ( 85% ) . the study suggests that apoptosis , as well as a local production of proinflammatory cytokines are associated with fs lesions . twenty - five patients with fs and a control group consisting of 10 healthy individuals were studied . serum il-2 , il-4 , il-5 , il-10 , il-12 and ifn - gamma were measured in the two groups by elisa . the median concentration of il-2 was lower in pf patients compared to the control group ( 0.45 and 9.50 pg / ml , respectively ) , as was the concentration of il-4 ( 0.26 and 10.16 pg / ml , respectively ) . the same was observed for il-5 ( 7.94 and 15.74 pg / ml , respectively ) and for ifn - gamma ( 5.90 and 8.58 pg / ml , respectively ) . for il-10 and il-12 , higher concentrations were observed in fs compared to the control group ( il-10 : 24.76 and 20.92 ; il-12 : 2.92 and 1.17 pg / ml , respectively ) . the authors concluded that considering the th1/th2 paradigm , it seems that a th2 profile , largely manifested by il-10 , predominates in fs . another study reported that supernatants of peripheral blood mononuclear cell populations from patients affected by fs produced significantly more interleukin-1beta ( il-1beta ) than those from stimulated healthy controls . furthermore , a th2 bias was observed in fs patients when the il-5/gamma interferon ratio was analyzed . one group of researchers studied the association of routine laboratory tests in fs , and screened 20 patients for antinuclear antibodies ( anas ) , rheumatoid factor ( rf ) , c - reactive protein ( crp ) , and changes in the erythrocyte sedimentation rate ( esr ) , serum proteins electrophoresis ( spep ) and total leukocyte count . the crp was found to be elevated in 60% of fs cases , a leukocytosis in 85% , and an elevated esr and mild alterations in the serum protein electrophoretic analysis in all of the patients . although widely accepted as nonspecific tests , the authors believe that an association of these routine laboratory tests with the clinical findings can prove to be helpful in the follow - up care of fs patients . in addition to the immunological alterations mentioned above , skin hypersensitivity mediated by ige has been thought to play a role in the pathogenesis of fs . the concept is based on studies utilizing a reverse reaginic test in patients with fs ( 16 patients ) , and an equal number of epidemiologically matched controls living in the same endemic area . in both groups , skin tests were performed using house dust , air fungi , and dermatophagoid sp . the ige levels in patients with fs were significantly increased when compared with the control group . another study also showed that patients with el bagre - epf exhibited increased ige levels compared with controls . we recently reported an elevation of ige levels in serum of patients with el bagre - epf utilizing a case controlled study , and this increase correlated with high levels of mercury in the hair and nails of el bagre - epf patients . the complement system represents one of the major mediators of inflammation and the humoral immune response , which when activated evokes multiple biological effects . multiple studies have shown that complement may be an important mediator of skin lesions in pemphigus[5666 ] . although complement does not seem to be essential for acantholysis , its activation appears to amplify the pathogenicity of pemphigus autoantibodies . specifically , in fs , it has been shown that autoantibodies activate complement components in vitro , and that complement activation in vivo is strongly associated with disease activity . one of our authors has demonstrated by dif that fs patients display simultaneous intercellular deposition of c3 and igg autoantibodies ; and that 57% of the patients in one study presented c3 deposition in the skin basement membrane zone . circulating pemphigus autoantibodies displayed titers ranging from 1:10 to more than 1:1280 , and the titers were drastically decreased during treatment . although complement may not be absolutely necessary for the development of skin lesions in epf , these longitudinal studies showed that significant activation of complement is observed during the active phase of the disease . fogo selvagem ( fs ) has been classically defined as a b cell - mediated disease due to the presence of both 1 ) spontaneously appearing , intraepidermal clinical blisters as well as 2 ) epidermal - specific autoantibodies belonging to the igg class[13 ] , predominantly the igg4 subclass . the role of b cells in endemic pemphigus foliaceus ( epf ) is also demonstrated by the demonstration that igg and the f ( ab ) 2 fab fragments of fs igg are pathogenic when passively transferred into neonatal mice and rabbits[47 ] . in 1937 , a model of fs in rabbits was first developed by injecting the serum of pf patients . these animals developed intra - epidermal blisters , which clinically , histologically , and immunologically duplicated the human disease . the role of b cells in the disease is also highlighted by the passive transfer of maternal pregnancy igg autoantibodies ( pregnant fs patients with high levels of autoantibodies ) . the antibody transfer has been reported in less than five cases , with temporary compromise of the fetus . moreover , the materno - fetal passive transfer epf disease consistently clears over the first two months of life . thus , this disease is present only temporarily , causing difficulties with any comparison to the natural , chronic course of epf in adults . no neonatal cases of the el bagre endemic pemphigus foliaceus ( el bagre - epf ) or the tunisian variant ( tunisian epf ) have been described , since the disease in these variants does not affect women of childbearing age[911 ] . although el bagre - epf intercellular keratinocyte autoantibodies are primarily from the igg4 subclass , and their iif titers correlate with clinical activity and disease severity ( as also reported in fs ) , el bagre epf sera contains autoantibodies of the igg3 subclass[911 ] that recognize an intracytoplasmic antigen , whose nature remains unknown . in addition to this potential antigen(s ) , many others likely remain unknown in el bagre - epf disease . moreover , in addition to plakins , desmogleins and bullous pemphigoid antigens 1 and 3 in el bagre epf , studies have shown emerging autoantigens in fs such e - cadherin . the role of classical cadherins as immunological targets of fs was well documented by 1 ) immunoprecipitation coupled with immunoblotting ( ip - ib ) and 2 ) elisa techniques utilizing a baculovirus - expressed ectodomain of e - cadherin . by ip - ib , the immunoreactivity of fs sera with e - cadherin was also demonstrated by ip - ib using human epidermal extracts . however , immunofluorescence staining of a431de cells ( e - cadherin positive , dsg1 negative ) with pemphigus sera showed negative results . immunoadsorption and competitive elisa analysis suggest that most of the anti - e - cadherin antibodies cross - react with desmoglein 1 ( dsg1 ) , whereas others may represent independent antibodies that do not cross - react with dsg1 . the functional relevance of these anti - e - cadherin igg autoantibodies detected in these pemphigus sera remains to be defined . the baculovirus heterologous gene expression system has also been utilized to attempt to explain intramolecular epitope spreading on fs another autoantigen described in fs is an ubiquitin carrier protein . the authors screened the autoantibodies from a fs patient utilizing a lambda gt11 human keratinocyte cdna library . one immunoreactive cdna clone ( lambda epf5 ) containing a 900-base pair insert was isolated , and subjected to further analysis . eight of 25 fs sera were then shown to react with an epf5 fusion protein on immunoblots ( ib ) . the epf5 cdna insert hybridized with a 1.2-kilobase epidermal rna transcript on a northern blot . sequence analysis revealed that lambda epf5 contained a complete coding sequence for a 24-kda polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins , or e2s , which are an essential component of the ubiquitin - protein conjugation system . the relevance of the cloned epf epitope in the pathogenesis of this autoimmune disorder remains to be determined . the same group of researchers also identified a novel igg anti - keratin autoantibody in the serum of a fs patient ( cascas-42 ) . the cascas-42 antibody was shown to be specific for a 59 kda acidic murine keratin , as well as its 56.5 kda human counterpart ( moll classification cytokeratin 10 ) ; it was also distinct from previously described pemphigus antibodies . anti - keratin autoantibodies present in the cascas-42 serum were purified by affinity chromatography with a 59 kda murine keratin - agarose column . in 80% of el bagre - epf patients , the presence of a lupus - like band is seen on direct immunofluorescence ( dif ) . the identity of the individual autoantigens , and deposition of a possible immune - complex at the basement membrane zone ( bmz ) are not yet characterized . selected epf patients have developed 1 ) glomerulonephritis with chronic renal failure , and 2 ) sudden death syndrome ; however , correlation of these clinical sequelae has not been demonstrated to be associated with epf . in addition to the lupus band , preliminary studies have revealed the presence of epf autoantibodies against urinary bladder , parietal cells of the stomach , kidney , neural , vascular , and skeletal , cardiac and smooth muscle cells ; however , their role in pathogenicity needs to be clarified ( abreu et . a recent study utilizing fs sera searched for the presence of a broad spectrum of autoantibodies and possible association with other autoimmune diseases in 120 patients with fs and 200 healthy controls from brazil . in contrast to el - bagre epf , no differences were found between the two groups ; however , 92.5% of the patients were undergoing steroid therapy . other studies have also been reviewed ; for example , 196 patients with fs from mato grosso do sul and 20 fs cases from gois ( states of brazil ) were analyzed for the presence of antinuclear antibodies ( ana ) by indirect immunofluorescence ( iif ) , with no positive results . in 28 patients with fs , 21.4% were positive for anti - gastric parietal cell ( gpca ) antibodies , 7.1% for anti - mitochondrial antibodies ( amas ) , and 3.6% for anti - smooth muscle ( sma ) antibodies . however , the autoantibody positivity in these studies was not conclusively diagnostic of other , concurrent autoimmune disease . the 1 ) use of nonspecific methods , 2 ) small number of patients investigated and 3 ) concomitant steroid therapy could contribute to variations in the results obtained . desmoglein 1 ( dsg1 ) is reported to be one of the antigens recognized by autoantibodies in fs , tunisian epf and el bagre - epf[9111725 ] . however , we recently reported some differences between fs and el bagre - epf by examining autoantigens in both diseases by various biochemical and molecular biological techniques , including immunoblotting ( ib ) , immunoprecipitation ( ip ) and elisa ; we utilized various antigen sources including baculovirus - expressed proteins . in order to confirm the reactivity of el bagre epf sera with plakin family proteins ( also known as paraneoplastic pemphigus antigens ) , we examined sera from both el bagre - epf and fs patients by ib using various recombinant , domain - specific peptides . these studies indicated that a considerable number of el bagre - epf sera reacted with various domains of periplakin , while only a few fs sera reacted with some of them . in addition , the number of el bagre - epf sera reactive with recombinant periplakin proteins was significantly higher than the fs sera , indicating that preferential reactivity with periplakin may be characteristic of el bagre - epf . in contrast , the sera of both types of epf rarely reacted with bp230 recombinant proteins . therefore , it seems likely that the 230 kda protein band detected by some el - bagre - epf sera in our previous studies[911 ] may , in fact , represent a different protein with a molecular weight of approximately 230 kda different than bp230 . we also developed an elisa for detecting a heterogeneous antibody population in serum from people suffering from el bagre - epf that is suitable for detection of plakins . another interesting finding is that el - bagre - epf sera preferentially reacted with paraneoplastic pemphigus ( pnp ) antigens , and there may be a similar pathogenic mechanism in autoantibody production in el bagre - epf and pnp , although the two diseases are quite different clinically and epidemiologically . these results lead us to speculate that , in conjunction with genetic predisposition , specific triggers in each endemic focus may induce a highly immunogenic state in the patients , resulting in the production of antibodies to many self - antigens . in addition , we speculate that some of these self - antigens ( specifically in el bagre - epf cases ) are associated with the antigenic response seen in paraneoplastic syndromes . further , multiple other proteins of different molecular weights are specifically recognized by both fs and el bagre - epf patient sera . one possible explanation for these findings might involve an epitope spreading mechanism leading to the production of multiple autoantibodies , although the amino acid sequences of the desmogleins and plakins display marked differences . sera from fs patients in the preclinical stage have been shown to recognize epitopes on the cooh - terminal ec5 domain of dsg1 . actual clinical disease onset is associated with the emergence of specific antibodies for epitopes on the nh2-terminal ec1 and ec2 domains ; all sera from fs patients with active disease recognize the ec1 and/or ec2 domains of dsg1[2125 ] . these results suggest that anti - dsg1 autoantibodies in fs are initially raised against the cooh - terminal ec5 domain in individuals without skin disease . in genetically predisposed subjects , the autoimmune response may then undergo intramolecular epitope spreading toward epitopes on the nh2-terminal ec1 and ec2 domains of dsg1 , leading to disease onset . moreover , intramolecular epitope spreading could also modulate remissions and relapses of fs[2125 ] . fs is endemic in limo verde ( lv ) , state of mato grosso , brazil . in one study , anti - dsg1 igm antibodies were detected in sera from 58% of fs lv patients ( n=31 ) , in contradistinction to sera from other geographic areas , including 19% of fs patients from hospital - campo grande ( n=57 ) , 19% from hospital - goiania ( n=42 ) , 12% from hospital - so paulo ( n=56 ) , 10% of pf patients from united states ( n=20 ) , and 0% of pf patients from japan ( n=20 ) . sera from pemphigus vulgaris patients ( pv ) ( n=40 , usa and japan ) , bullous pemphigoid patients ( bp ) ( n=40 , usa ) , and healthy donors ( n=55 , usa ) demonstrated negligible percentages of positivity . high percentages of positive anti - dsg1 igm antibodies were found in healthy donors from four rural , brazilian amerindian populations ( 42% of 243 ) as compared with healthy urban donors ( 14% of 81 ; p<0.001 ) . more than 50% of healthy donors from lv ( n=99 , age 5 - 20 years ) possessed igm anti - dsg1 across a wide age range , whereas igg - anti - dsg1 was differentially detected in age specific ranges : 2.9% at ages 5 - 10 years , 7.3% at ages 11 - 15 years , and 29% of donors above age 16 . anti - dsg1 igm epitopes were found to be ca2 + and carbohydrate - independent . based on the above data , the authors suggested the possibility that anti - dsg1 igm antibodies are common in fs patients in their native environment , and uncommon in other pemphigus phenotypes as well as in fs patients who migrate to urban hospitals . thus , recurrent environmental antigenic exposure may lead to both igg and igm responses that trigger fs . another fs study utilized fs hybridomas that secreted either igg or igm ( predominantly igg1 subclass ) autoantibodies from the b cells of 1 ) eight current fs patients , as well as 2 ) one individual taken 4 years before fs onset ; the h and l chain v genes of anti - dsg1 autoantibodies were analyzed . first , clonally related sets of anti - dsg1 hybridomas characterize the response in individual fs patients . second , h and l chain v gene use seems to be biased , particularly among igg hybridomas , and third , most hybridomas are mutants and exhibit a bias in favor of cdr ( complementary determining region ) amino acid replacement(aar ) mutations . pre - fs hybridomas also exhibit evidence of antigen selection , including an overlap in v ( h ) gene use and shared multiple aar mutations with anti - dsg1 fs hybridomas , suggesting selection by the same or similar antigen . the authors concluded that the anti - dsg1 response in fs is antigen driven , and that selection for mutant anti - dsg1 b cells begins well before the onset of disease . the prevalence of anti - dsg1 antibodies in healthy subjects and their distribution in the different regions of tunisia was recently investigated , to better identify endemic areas of pemphigus foliaceus ( pf ) . the authors tested , by elisa , sera from 270 normal subjects recruited from different tunisian areas , 90 tunisian epf patients and 203 healthy relatives these patients . seventy - six patients ( 84.4% ) , 20 healthy controls ( 7.4% ) , and 32 relatives ( 15.76% ) possessed anti - dsg1 antibodies . in southern tunisian regions , where epf is associated with a significant sex ratio imbalance ( 9 females : 1 male in the south vs. 2.3 : 1 in the north ) and a lower mean age of disease onset ( 33.5 years in the south vs. 45 years in the north ) , a higher prevalence of anti - dsg1 antibodies in healthy controls was observed ( 9.23% vs. 5.71% in the north ) . the highest prevalence of anti - dsg1 antibodies in healthy relatives ( up to 22% ) was observed in the most rural southern localities . more than half anti - dsg1 positive healthy controls were living in rural conditions with a farming occupation , which suggests that this activity may expose the subjects to predilecting environmental conditions . in addition , desmocollins i and ii have been reported as autoantigens recognized by sera from some patients with fs . desmocollins are transmembranous glycoproteins that serve as components of desmosomal junctions , and occur in three different isoforms ( desmocollins 1 , 2 and 3 ) ; all isoforms are represented in the epidermis . in this study , the authors examined sera of various types of pemphigus by immunoblotting ( ib ) with purified bovine desmosomes and bovine desmocollin 1 , 2 and 3 fusion proteins , expressed in pgex vectors . six of 15 ( 40.0% ) fs sera , two of 18 ( 11.1% ) non - endemic pemphigus foliaceus sera , eight of 39 ( 20.5% ) pemphigus vulgaris ( pv ) sera , and two of 11 ( 18.2% ) normal sera , demonstrated reactivity with desmocollins from desmosomes . experiments with the fusion proteins showed that no desmocollin isoform was specifically recognized by sera of any individual type of pemphigus . the authors concluded that the pathogenesis of pemphigus might thus be more complex than previously believed . exposure of uninvolved skin to uvb induces acantholysis , with in vivo binding of igg and c3 to the epidermal intercellular spaces . nevertheless , it is also known that complement activation per se is not necessary for pemphigus bulla formation . recently , the role of proteases in the pathology of acantholysis in pemphigus has been addressed . since 1983 , the plasminogen - plasmin system has been implicated in the pathophysiology of epf . however , mitigating against the participation of this system in acantholysis , pemphigus vulgaris and pemphigus foliaceus autoantibodies were shown to be pathogenic in plasminogen activator knockout mice . nevertheless , other work has suggested that regulation of the expression of the urokinase receptor and junctional proteins ( including plasminogen , its activators , and the kininogen - kallikrein - kinin system ) contribute to acantholysis as part of the mechanisms mediating pemphigus . the role of urokinase type plasminogen activator ( upa ) has been well documented in the pathogenesis of pemphigus vulgaris ( pv ) . activation of plasminogen into the active serine protease plasmin initiates extracellular proteolysis , leading to acantholysis ; however , the mechanisms underlying this process are not clearly understood . in addition , the expression of p63 and the induction of apoptosis have been documented in healthy and lesional skin from fs patients , when compared to normal subjects . in a separate study , an igg passive transfer mouse model of pf tunel - positive ( apoptosis positive ) epidermal cells and increased oligonucleosomes in epidermal cytosolic fractions were detected in diseased mice . a time course analysis revealed that the tunel - positive epidermal cells appeared prior to the formation of intra - epidermal blisters . moreover , after pf igg injection , the pro - apoptotic factor bax was upregulated at early time points ( 2 and 4 h ) , and the anti - apoptotic factor bcl - xl was downregulated at later time points ( 6 , 8 , and 20 h ) ( determined by western blot analysis ) . the active forms of caspase-3 and -6(a putative initiator of acantholysis ) were detected in the later time period ( 6 , 8 , and 20 h ) . in addition , administration of ac - devd - cmk , a peptide - based caspase-3/7 inhibitor , protected mice from developing intra - epidermal blisters and clinical disease induced by pf igg . the same protective effect was also observed utilizing a broad - spectrum caspase inhibitor , bok - d - fmk . collectively , these findings show that biochemical events leading to apoptosis are provoked in the epidermis of mice injected with pf autoantibodies . other studies in fs have shown that mannose - binding lectin ( mbl ) and mbl - associated serine protease ( masp-2 ) play a key role in innate immunity ; their deficiency has also been related to both increased susceptibility to infection and development of autoimmune diseases not related to fs . mbl and masp-2 serum levels were measured in 114 patients with fs and in 100 healthy individuals in brazil . mbl and masp-2 levels were measured by sandwich assays ( time - resolved immunofluorimetic assays ) utilizing monoclonal antibodies . no differences were observed in mbl levels in patients with fs compared with controls [ mean sem 1230.07 , + /- 132.18 ng / ml ( median 789.0 ng / ml ) vs. mean 1036.98 , + /- 117.99 ng / ml ( median 559.5 ng / ml ) , p = 0.32 ] . non - significant , lower masp-2 levels were observed in epf patients , compared with controls [ mean 274.34 , + /- 15.66 ng / ml ( median 239.5 ng / ml ) , vs. mean 304.72 , + /- mbl deficiency ( < 10 ng / ml ) and masp-2 deficiency ( < 100 ng / ml ) did not differ significantly between patients and controls . these data indicate that mbl and masp-2 deficiency are not associated with susceptibility to fs . few studies have been performed testing immune cells other than b cells in epf . in recent studies , b and t lymphocytes were quantified in the peripheral blood of 30 fs patients according to their ability to form rosettes with 1 ) sheep erythrocytes , or 2 ) sheep erythrocytes sensitized with antibody and complement . the total t lymphocyte count and the functional t cell count were significantly lower ( by student 's t test ) as detected by the active rosette test in fs versus normal patients . in addition , peripheral lymph nodes from three patients showed a decrease in the number of t cells in the paracortical areas . paradoxically , it had been thought that b cells represented the main immunological mediators in the pathogenesis epf . however , multiple complications of epf are associated with non - b - cell immunity , including secondary infections by 1 ) poxvirus ( molluscum contagiosum ) , 2 ) mycobacterium tuberculosis , 3 ) parasites ( strongyloidiasis and entamoebiasis ) , 4 ) fungi and 5 ) herpes virus , including the relapsing kaposi 's varicelliform eruption . thus , these studies suggest that fs is not solely a b - cell - mediated disease . it is important to keep in mind that susceptibility to these secondary infections may or may not be related to concurrent corticoid therapy . further , an understanding of the immune response of patients to molluscum virus may also help to explain the susceptibility to smallpox seen in some individuals . an additional study was conducted on 16 patients with fs , ten of them with the localized form ( group g1 ) and six with the disseminated form ( group g2 ) . these patients underwent complete blood counts , quantitation of mononuclear cell subpopulations by monoclonal antibodies , studies of blastic lymphocyte transformation , and quantitation of circulating fs antibodies by iif testing . the test profile was created order to correlate their clinical signs , symptoms and laboratory data with their immunological profiles and to determine the relationship between circulating autoantibody titers , lesion intensity and course of lesions under treatment . all patients showed decreased relative cd3 + and cd4 + t cell values , and a tendency towards decreased relative values of the cd8 + t cell subpopulation . blastic lymphocyte transformation indices in the presence of phytohemagglutinin were higher in patients of groups g1 and g2 than in controls . the fs iif testing was positive in 100% of g2 patients and in 80% of g1 patients . the median value for the iif antibody titers was higher in group g2 than in group g1 . analyzing the results , the authors concluded that cell immunity was preserved , and that there was a direct relationship between antibody titers detected by the iif test and dissemination of skin lesions . other authors have reported that the immune response is preserved in fs . to elucidate the role of pathogenic t cells in fs autoimmunity , t cell receptors ( tcrs ) were characterized utilizing complementary dna isolated from 17 dsg1-specific t cell clones , generated from fs patients by clonal expansion in vitro . to analyze the t cell repertoire , a panel of primers , collectively specific for the known human t cell receptor variable region ( tcrbv ) family , was paired with a constant region primer in order to amplify one distinct t cell receptor variable region allele for each t cell clone . polymerase chain reaction ( pcr ) products were then sequenced to determine the precise chain gene usages . in the 17 clones tested , 10 distinct t cell receptor variable region usages and nine tcr joining gene segment usages were identified . furthermore , tcr variable region and joining usage did not appear to be random , but were oligoclonal in nature , with some preference shown for t cell receptor variable regions 5s1 and tcr bj2s5 . in addition , another study characterizing the autoimmune t cell response associated with fs indicated that the great majority of fs patients have circulating t lymphocytes that specifically proliferate in response to the extracellular domain of dsg1 . long - term t cell cultures developed from these patients also responded to dsg1 , and this antigen - specific response was shown to be restricted to hla - dr molecules . these dsg1-reactive fs t cells exhibited a cd4-positive memory t - cell phenotype , and produced a t helper 2(th2)-like cytokine profile . other studies have shown that if tissue damage is mediated by anti - dsg1 antibodies in epf , an initial t cell response is a likely requirement for autoantibody generation . further studies have described attempts to determine the number of dendritic langerhans cells in fs . since serum il-12 is increased in patients with fs and langerhans cells ( lc ) produce il-12 , serum il-12 autoantibodies were titrated by elisa in another study , resulting in increased level in pf sera . biopsies of blistering lesions were obtained from 22 fs patients , 13 of whom were submitted to biopsy of both lesional and apparently healthy skin . the control groups consisted of skin from 8 cadavers , and from 12 unaffected women presenting for breast plastic surgery . lcs and dcs were also identified with anti - cd1a antibodies , and quantified by morphometric analysis . lc numbers in the lesional and non - lesional skin from fs patients were similar to that of both control groups . dc numbers in the lesional skin ( median=0.94 dc / mm basement membrane ) were higher than those of the cadaver group ( median=0.13 dc / mm basement membrane ) . in the 13 fs patients with biopsies of both lesional and non - lesional skin , lcs and dcs were present in larger numbers in the lesions . a direct correlation existed between dc numbers in the lesions of the fs group and serum autoantibody titers ; this correlation was not observed for lc numbers . the increased number of dcs in the lesion , as well as the direct correlation with serum autoantibody titers suggests the participation of dcs in the pathogenesis of pf . the relationship between increased dc number and il-12 in pf needs to be further clarified . some attention has been given to the role of dendritic cells in the mechanism of epf acantholysis . a predominance of cd4 + t cells , a decrease in langherhans cells ( lcs ) , an increase of cd1a+ dermal dendritic cells , and a lack of keratinocyte icam-1/cd54 and hla - dr molecules in lesional skin was observed in fs . twenty biopsy specimens of lesional and perilesional skin of fs patients were analyzed by immunohistochemical techniques . the panel of monoclonal antibodies included cd1a , cd4 , cd8 , hla - dr , il-2r , lfa-1/cd11a , icam-1/cd54 , and pan - b . a semiquantitative analysis of the cell populations revealed a predominance of cd4 + t lymphocytes in the tissue in perilesional and lesional skin . the population of lcs was decreased in lesional skin when compared with the perilesional skin , whereas cd1a+ dermal dendritic cells predominated in lesional skin . keratinocyte expression of icam-1/cd54 and hla - dr was negative in both lesional and perilesional skin . these results suggest the involvement of cell - mediated immunity in fs . the lack of keratinocyte icam-1/cd54 expression may be related to the pattern of cytokines secreted by the cd4 + t cells of the tissue in fs . utilizing morphometric analysis , increased dermal dendritic cells in lesional skin from epf patients was directly correlated with igg autoantibody titration by iif . these results may reflect the possibility that impairment in the cellular , and/or more complex immune response might play an important role in fs disease . other authors had reported paucicellularity of s-100 positive cells in perilesional and lesional skin from patients with fs . in 36 fs patients , the presence of s-100 protein antigens identified by immunohistochemistry showed a marked reduction or even disappearance of the cell population bearing the s-100 antigen in lesional skin . these results are in agreement with the findings discussed above , and might provide an indirect insight into the possible peripheral anergy that may be part of the loss of peripheral tolerance in epf disease . a recent study evaluated the expression of the proinflammatory cytokines interleukin 1 , interferon gamma and tumor necrosis factor alpha ; the pro - apoptotic inducers fas and inducible nitric oxide synthase ; and the apoptosis inhibitor bcl-2 to evaluate the presence of apoptosis in fs . skin biopsies from 13 patients with fs and controls were evaluated by immunohistochemistry , and apoptosis was determined by a terminal deoxynucleotidyl transferase - mediated dutp nick - end labeling assay . the results showed that proinflammatory cytokines were only detected in cells of the inflammatory exudate . inducible nitric oxide synthase , fas , and bcl-2 were all expressed by both epithelial and inflammatory cells . epithelial apoptosis was observed in 12 cases ( 92.3% ) , and subepithelial apoptosis in 11 cases ( 85% ) . the study suggests that apoptosis , as well as a local production of proinflammatory cytokines are associated with fs lesions . twenty - five patients with fs and a control group consisting of 10 healthy individuals were studied . serum il-2 , il-4 , il-5 , il-10 , il-12 and ifn - gamma were measured in the two groups by elisa . the median concentration of il-2 was lower in pf patients compared to the control group ( 0.45 and 9.50 pg / ml , respectively ) , as was the concentration of il-4 ( 0.26 and 10.16 pg / ml , respectively ) . the same was observed for il-5 ( 7.94 and 15.74 pg / ml , respectively ) and for ifn - gamma ( 5.90 and 8.58 pg / ml , respectively ) . for il-10 and il-12 , higher concentrations were observed in fs compared to the control group ( il-10 : 24.76 and 20.92 ; il-12 : 2.92 and 1.17 pg / ml , respectively ) . the authors concluded that considering the th1/th2 paradigm , it seems that a th2 profile , largely manifested by il-10 , predominates in fs . another study reported that supernatants of peripheral blood mononuclear cell populations from patients affected by fs produced significantly more interleukin-1beta ( il-1beta ) than those from stimulated healthy controls . furthermore , a th2 bias was observed in fs patients when the il-5/gamma interferon ratio was analyzed . one group of researchers studied the association of routine laboratory tests in fs , and screened 20 patients for antinuclear antibodies ( anas ) , rheumatoid factor ( rf ) , c - reactive protein ( crp ) , and changes in the erythrocyte sedimentation rate ( esr ) , serum proteins electrophoresis ( spep ) and total leukocyte count . the crp was found to be elevated in 60% of fs cases , a leukocytosis in 85% , and an elevated esr and mild alterations in the serum protein electrophoretic analysis in all of the patients . although widely accepted as nonspecific tests , the authors believe that an association of these routine laboratory tests with the clinical findings can prove to be helpful in the follow - up care of fs patients . in addition to the immunological alterations mentioned above , skin hypersensitivity mediated by ige has been thought to play a role in the pathogenesis of fs . the concept is based on studies utilizing a reverse reaginic test in patients with fs ( 16 patients ) , and an equal number of epidemiologically matched controls living in the same endemic area . in both groups , skin tests were performed using house dust , air fungi , and dermatophagoid sp . the ige levels in patients with fs were significantly increased when compared with the control group . another study also showed that patients with el bagre - epf exhibited increased ige levels compared with controls . we recently reported an elevation of ige levels in serum of patients with el bagre - epf utilizing a case controlled study , and this increase correlated with high levels of mercury in the hair and nails of el bagre - epf patients . the complement system represents one of the major mediators of inflammation and the humoral immune response , which when activated evokes multiple biological effects . multiple studies have shown that complement may be an important mediator of skin lesions in pemphigus[5666 ] . although complement does not seem to be essential for acantholysis , its activation appears to amplify the pathogenicity of pemphigus autoantibodies . specifically , in fs , it has been shown that autoantibodies activate complement components in vitro , and that complement activation in vivo is strongly associated with disease activity . one of our authors has demonstrated by dif that fs patients display simultaneous intercellular deposition of c3 and igg autoantibodies ; and that 57% of the patients in one study presented c3 deposition in the skin basement membrane zone . circulating pemphigus autoantibodies displayed titers ranging from 1:10 to more than 1:1280 , and the titers were drastically decreased during treatment . although complement may not be absolutely necessary for the development of skin lesions in epf , these longitudinal studies showed that significant activation of complement is observed during the active phase of the disease . research utilizing dif in fs has been primarily focused on the study of lesional and perilesional skin , while little attention has been given to uninvolved skin . in a recent study , the authors analyzed the frequency of iga , igm , and igg ( and its subclasses igg1 , igg2 , igg3 and igg4 ) and c3 complement fraction deposition in the intercellular spaces ( ics ) and basal membrane zones ( bmz ) in uninvolved , lesional and perilesional skin from 47 fs patients with dif .. the results showed a predominance of igg and igg4 deposition in all skin samples , followed by c3 and igg1 deposits . the positive response for igg in uninvolved ( 91.48% ) , lesional ( 93.61% ) and perilesional ( 97.87% ) skin was similar to that found for igg4 in the same samples : 95.74% , 95.74% and 97.87% , respectively . regarding igg1 , the uninvolved skin showed lower results ( 14.89% ) than the lesional ( 29.78% ) and perilesional skin ( 29.78% ) . with c3 , the perilesional skin displayed higher results ( 40.42% ) than the uninvolved and lesional skin ( 34.04% for both ) . the results suggest the importance of uninvolved skin for direct immunofluorescence in the diagnosis of fs , and further suggest that any cutaneous region can demonstrate pemphigus antibodies by dif . other authors studied a group of 16 patients with chronic fs under corticosteroid therapy and persistent erythematous , papulous , verrucous , and generally hyperpigmented lesions , which were characterized as corticoid therapy resistant lesions . pathologically , the lesions showed tendencies toward epithelial hyperplasia , and cleavage in variable levels of the epidermis . these findings differ from classic , early lesions of fs , and contain features of chronic fs lesions examined in the pre - corticoid therapy era . the dif of the injured skin was positive for igg in 93.75% of these persistent cases , similar to the early stages of fs ; the igg was negative in a single persistent case in which there was no epidermal cleavage . in addition , in eight of the persistent lesion patients , the dif and iif of the healthy skin were studied . the dif was positive in three of these fs cases , and the iif was negative in all eight . other authors investigated whether ultrastructural changes present in clinically normal mucosa could occur in patients with fs . surgical biopsy specimens were taken from the foreskin of 8 patients with epf and 3 control subjects , from the uterine cervix and vaginal wall of 9 patients with fs and 2 controls , and from the oral mucosa of 5 patients with fs and 4 controls . the patients all had received a clinical and histopathologic diagnosis of epf , and all had clinically normal oral and genital mucosa . in the fs patients , widening of the intercellular spaces and distended , elongated cytoplasmic projections ( the tips of which contained desmosomes that were sometimes disassembled ) , were evident in all 4 regions studied . at the edges of the spinous layer keratinocytes , cytoplasmic vesicles were present , apparently containing intact or fragmented desmosomes , or half - desmosomes : these ultrastructural findings in the mucosa are similar to those previously described in the literature in the oral mucosa of patients with fs . in the current fs study , although the desmosomal changes occurred in all epithelial layers , blisters did not occur in the mucosa , possible due to a coexpression of desmoglein 1 and desmoglein 3 . often in fs , no clinical lesions are seen in the mucosa and non glabrous skin ; however , some additional studies have shown disease autoreactivity in these areas[6973 ] . in el bagre - epf , some of the reactivity is directed towards the sweat glands , the palms , neurovascular areas , the meibomian glands and tarsal muscle . early studies of epf utilzing animal models that were reported in 1937 by lindenberg , and by beutner et al . in 1971 further , multiple studies using passive or active , largely neonatal mouse models have been described in the study of epf[35 ] . it has been noted that the presence of epf autoantibodies is not sufficient to clinically present or maintain the disease , since the passive transfer experiments on animals using epf autoantibodies often resulted in temporary disease if the animals were clinically followed[35 ] . one animal fs passive transfer model has been utilized to examine the possible pathogenic role of fs autoantibodies . igg from the sera of human fs patients was purified , and injected into the peritoneum ( ip ) of neonatal balb / c mice[35 ] . thirty - four of 46 mice ( 74% ) receiving parenteral igg fractions from fs patients developed cutaneous lesions that were identical to the human disease by clinical , histologic , immunologic , and ultrastructural criteria . high - titer fs sera produced lesions more consistently and more rapidly than low - titer sera . when the injections were discontinued , new lesions ceased to appear and old lesions resolved . the extent of murine disease directly correlated with the titer of human anti - epithelial antibodies detected in the mouse serum . similar concentrations of igg fractions obtained from sera of human controls from endemic and non - endemic areas did not induce disease when injected into littermates of the diseased mice . the same researchers subsequently demonstrated that monovalent fab immunoglobulin fragments from fs autoantibodies can reproduce the human disease in neonatal balb / c mice[35 ] . another study has shown that fs acantholysis occurs independently of il-12 , since one of us have shown experimental pemphigus in mutant c57bl/6 animals null for il-12 , in spite of increased levels of serum il-12 found in fs patients . skin and serum were studied at 0 , 1 , 3 , 6 , 12 , 18 , and 24 hours post injection by immunofluorescence ( if ) , electron microscopy , and immuno - em . binding of fs igg in the intercellular spaces ( ics ) of the basal cell layer was seen by if within 1 hour and was strongest at 12 hours . igg binding affected the spinous and granular cell layer by 12 hours , then faded and remain localized only in the basal cell layer at 24 hours . by immuno - em , edema of the ics in the basal cell layer was present at 1 hour by em . at 12 h , there was formation of microvilli , with intact desmosomes at the tip of the microvillous projections . splitting of desmosomes ( forming half desmosomes ) and acantholysis ( primarily affecting the granular cell layer ) were most prominent between 12 and 24 hours . the plaques of the half desmosomes then gradually disappeared , and their tonofilaments retracted into the keratinocyte cytoplasms . detaching keratinocytes demonstrated cytoplasmic vacuolizations , swollen mitochondria , and internalizations of both intact and half desmosomes ( remnants of split desmosomes ) . the investigation showed that the ultrastructural changes observed in the epidermis of patients with fs can be duplicated in experimental animals by ip injection of fs igg . further , fs igg may have direct effects on the assembly and disassembly of desmosomes . fs occurs among rural brazilians living in geographically clustered disease foci , perhaps suggesting that people who carry the proper genetic background develop the disease under the influence of some unknown environmental stimuli . one of the most important features regarding epf is the fact that at least in the fs and el - bagre - epf variants , one third of the patients have relatives affected by the same disorder . in addition , other , unaffected relatives exhibit autoantibodies in their sera that recognize some of the epf antigens ( e.g. , dsg1 ) , but these autoantibodies do not seem to be play a sufficient role in the immunopathogenesis of either fs or el bagre epf . currently , a non - classical mendelian association has been found relative to epf , and more complex genetic segregation studies and studies of allelic interactions must be performed to understand the mechanisms underlying the pathogenesis of this autoimmune disease . one important point is that in both the brazilian areas where fs has been prevailed and the el - bagre area , interracial mating between amerindians and europeans has occurred over a relatively short span of time . multiple tools have been utilized to study the migratory patterns of both the amerindians , as well as historical immigrants into the new world . in fs , familial cases are frequent , and not everyone living in the endemic region develops this disease , suggesting that host factors play a role in determining whether exposed individuals will be affected . previous studies with fs patients indicate the possibility that some hla alleles confer an increased risk for the development of epf disease[7779 ] . brazilian caucasoid fs patients and matched controls were tested for hla - a , b , c ; dr1 to drw8 , and dqw1 to dqw3 . the frequencies of dr1 , dr4 , and b16 were significantly increased , while dr7 was significantly decreased among the fs patients ( 156 - 158 ) . given these findings , it was suggested that at least two mhc - class ii genes are involved in the pathogenesis of fs . two alleles , namely dqw1 ( associated with dr1 ) , and dqw3 ( associated with dr4 ) , confer susceptibility to fs . in contradistinction , at least one allele , dqw2 ( associated with dr7 , dqw2 and dr3 ) confers resistance[7779 ] . the susceptibility gene(s ) ( if allelic ) seem(s ) to be epistatic to or dominant over the resistance gene(s ) . other studies had shown the alleles drb1 ( * ) 0101 , ( * ) 0102 , ( * ) 0103 , ( * ) 0404 , ( * ) 0406 , ( * ) 0410 , ( * ) 1406 and ( * ) 1601 to be significantly more frequent among patients with fs[7779 ] . one genetic study has shown a positive association between fs and selected cytokine genetic variants . the study samples included 168 patients and 189 controls , and were comprised of mostly caucasians and mulattos . the approach consisted of a case - control association study , and the alleles were identified by mismatched pcr - rflp . no associations were found with the cytokine genetic variants il1a , il1b , il1rn , il4r and il10 . there was a weak negative association of the haplotype -1082 g -592c ( or=0.49 ) with the il10 gene in mulattos . in regards to polymorphism -590 of the il4 gene , a positive association with the t / t genotype ( or=2.71 ) and a negative association with the c variant ( or=0.37 ) were found . associations with il6 -174 variants suggest that the c / c genotype has a protective effect ( or=0.13 ) , while carriers of the g allele are more susceptible ( or=7.66 ) to fs . other studies from the same author have shown no association between the polymorphisms of the ctla4 , tumor necrosis factor and lymphotoxin - alpha genes and fs . a recent study reported the presence of epf in two native yanomami children from the venezuelan amazonia ; the children presented with erythroderma , were hospitalized , and subsequent clinical , histologic , and immunofluorescence studies diagnosed epf . an analysis of human leukocyte antigen class ii showed the drb1 * 04 subtype * 0411 , which has not been previously associated with this disease . however , it shares a common epitope with all the hla drb1 alleles that have been involved in this disease among brazilian populations [ 83 . ] . contradictory results have also been reported when studying the pemphigus foliaceus and desmoglein 1 gene polymorphism . other authors determined the hla dr / dq markers of susceptibility and protection in tunisian epf . genomic dna from 90 patients with pf recruited from all parts of the country and matched by age , sex and geographical origin with 270 healthy individuals was genotyped . first , when the whole patient population was studied , drb1 * 03 , dqb1 * 0302 and drb1 * 04 alleles were significantly associated with the disease , while a significant decrease of drb1 * 11 and dqb1 * 0301 were observed in patients compared with controls . drb1 * 0301 was the dominant allele in dr3-positive patients and controls , while drb1 * 0402 was found in 42% of dr4-positive patients . second , when the hla dr / dq allele distribution was studied after dividing patients according to their geographical origin , the southern group ( which consisted exclusively of patients with the endemic form of the disease ) showed the same associations as the whole pf population , particularly with drb1 * 03 . in the northern group , only the drb1 * 04 and dqb1 * 0301 alleles interestingly , anti - desmoglein 1 antibody - positive healthy controls did not carry susceptibility alleles ; in contrast , most carried negatively associated alleles . these observations indicate that a particular genetic background characterizes tunisian epf ; and further , that hla class ii genes control the pathogenic properties of the autoimmune response , rather than the initial breakdown of b - cell tolerance . a recent study of el bagre - epf featured complex segregation analysis ( csa ) and analysis of short tandem repeats ( strs ) to discriminate between environmental and/or genetic factors in this disorder . the csa analysis was carried out according to the unified model ; implemented utilizing the transmission probabilities implemented in the computer program pointer , and evaluated using a software package for population genetic data analysis ( gda ) , namely arlequin . the authors also performed pedigree analyses utilizing cyrillic 2.1 software , with a total of 30 families and 50 probands ( 47 males and 3 females ) tested . in parallel to the csa , the authors also tested for the presence of short tandem repeats from hla class ii , dq alpha 1 , involving the gene locus d6s291 by using the hardy - weinberg- castle law . the results indicate that the best model of inheritance in this disease is a mixed model , with multifactorial effects presenting within a recessive genotype . two types of possible segregation patterns were found : one with strong recessive penetrance , in families whose phenotype is more amerindian - like , and a second of possible somatic mutations . the authors noted that the penetrance of 10% or less in female patients 60 years of age or older indicates that hormones could protect younger females . the greatest risk factor for men being affected by el bagre - epf was the nn genotype . the authors also found a protective role for two genetic loci ( d6s1019 and d6s439 ) in the control group .	background : endemic pemphigus foliaceus ( epf ) is an autoimmune disease , classically occurring in a restricted geographic area . foci of epf have been described in several central and south american countries , often affecting young people and amerindians , with some female predilection . although most american epf cases have been documented in brazil , cases have been reported in peru , paraguay , el salvador and venezuela . an additional variant of epf has been described in el bagre , colombia , ( el bagre - epf ) affecting older men and a few post - menopausal females . finally , one additional type of epf has been described in nomadic tribes affecting females of child bearing age in tunisia , africa.aims:the main aim of this review is to summarize current knowledge about autoantigens , and immunologic and genetic studies in epf.material and methods : we utilized a retrospective review of the literature , aiming to compile and compare the multiple geographic foci of epf.results:the primary autoantigens in epf are still considered to be desmogleins in the case of the tunisian and all american cases , in contradistinction to plakins and desmogleins in el bagre - epf . although several autoantigens are been suggested , their biochemical nature needs further elucidation . current knowledge still supports the concept that an antibody mediated immune response represents the principal pathophysiology in all variants of epf.conclusion:a strong genetic susceptibility appears to contribute to disease development in several people affected by these diseases ; however , no specific genes have been confirmed at present . we conclude that further investigation is necessary to define these disorders immunologically and genetically .
You are an expert at summarizing long articles. Proceed to summarize the following text: polycystic ovary syndrome ( pcos ) is one of the most common endocrine disorders , affecting around 47% of reproductive - aged women . the etiology of pcos remains to be elucidated , although insulin resistance ( ir ) and hyperinsulinemia seem to be the important mechanism for pcos and its metabolic derangements [ 2 , 3 ] , diabetes mellitus ( dm ) in particular [ 4 , 5 ] . it has been reported that pcos women are at high risk of ir as shown by various markers , for example , impaired glucose tolerance of 2531% , and type 2 dm of 7.510% [ 6 , 7 ] . patients with ir , especially those with dm , have a high risk for developing cardiovascular disease in the future [ 8 , 9 ] . although pcos women have a high risk of ir , the rotterdam eshre / asrm - sponsored pcos consensus workshop group does not recommend any test of ir to be routinely used in clinical practice . however , it is suggested that in large - scale clinical or epidemiological studies , simple methods such as fasting plasma glucose ( fpg ) and oral glucose tolerance test ( ogtt ) may provide effective estimates for ir . in our previous report , we found that the pcos women at our clinic were relatively young and thin as compared with those in other reports [ 6 , 7 , 12 ] . the objectives of the present study were to investigate the prevalence and clinical predictors of ir in pcos thai women . the present cross - sectional study was conducted at the gynecologic endocrinology unit , faculty of medicine siriraj hospital , mahidol university . participants were all pcos thai women who registered to the siriraj pcos project from may 2007 to january 2009 . additional exclusion criteria were women who had previous surgery of one or both ovaries , used hormonal treatment , took the medication for dyslipidemia within 3 months , or took steroid within 6 months before registering to the project . after a written informed consent was obtained , a structured medical record form was used to collect the following data : clinical presentation , medical history , age , body weight , height , waist circumference ( wc ) , blood pressure , and skin manifestations . the first sample was drawn at 810 am after overnight fasting for at least 12 hours , and the second samples was drawn two hours after 75 g oral glucose loading . the first blood sample was examined for baseline metabolic profiles ( fasting blood glucose ( fbg ) , cholesterol , triglyceride , high density lipoprotein cholesterol ( hdl - c ) , low density lipoprotein cholesterol ( ldl - c ) ) , and hormonal profiles ( total testosterone , prolactin , thyroid stimulating hormone ( tsh ) , and cortisol ) . obesity was defined as body mass index ( bmi ) 25 kg / m according to the who cutoff points for asian populations . central obesity was defined as wc 80 cm according to the international diabetes federation ( idf ) 2005 . acanthosis nigricans was a cutaneous condition characterized by a velvety darkening of the skin in the posterior region of neck , axillae , antecubital fossa , groin , and other areas . hypertension was defined as systolic blood pressure of 140 mmhg or diastolic blood pressure of 90 mmhg , according to the world health organization / international society of hypertension writing group ( who / ish ) 2003 . dyslipidemia was defined as cholesterol 200 mg / dl , ldl - c 160 mg / dl , hdl - c < 50 mg / dl , or triglyceride 150 mg / dl , according to national cholesterol education program adult treatment panel ( ncep atp ) iii . clinical hyperandrogenism was considered if the participants had at least one of the followings : hirsutism ( a modified ferriman - gallwey score 8 ) , acne , androgenic alopecia , or virilization . hyperandrogenemia was defined as total testosterone > 0.8 ng / dl , free testosterone > 0.006 ng / dl , or dehydroepiandrosterone sulphate ( dheas ) > 350 mcg / dl . abnormal plasma ogtt was classified according to the american diabetes association ( ada ) 2003 criteria as follows : ( i ) impaired fasting glucose ( ifg ) , that is , fasting blood glucose ( fbg ) 100 and < 126 mg / dl , ( ii ) impaired glucose tolerance test ( igt ) , that is , 2-hr glucose 140 and < 200 mg / dl , and ( iii ) type 2 dm , that is , fbg 126 mg / dl or 2-hr glucose 200 mg / dl . in the present study , ir was defined as the presence of ifg , igt , or type 2 dm . all laboratory assays were performed at the laboratory unit of the department of clinical pathology , faculty of medicine siriraj hospital , mahidol university , the central laboratory certified by is0 15189 . all assays were done using automatic analyzers , modular p800 , roche ( for total cholesterol , hdl - c , ldl - c , triglycerides , glucose , and albumin ) and modular e170 , roche ( for insulin , tsh , prolactin , cortisol , total testosterone , and dheas ) . serum - free testosterone was calculated from serum total testosterone , sex hormone - binding globulin ( shbg ) and albumin using the online computer program of international society for the study of the aging male ( issam ) . all techniques had intra- and interassay coefficients of variation ( cv ) less than 5% . when an estimated prevalence of ir = 20% ( from our pilot study ) , precision error of estimation ( 25% of p ) = 0.05 , and = 0.05 , a sample size of at least 246 was needed . the normality of distribution of all continuous variables was checked using kolmogorov - smirnov test . data were presented in mean and standard deviation ( sd ) , number ( n ) and percent ( % ) , or odds ratio ( or ) and 95% confidence interval ( ci ) , as appropriate . univariate analysis using student t - test , mann - whitney u test ( for continuous data ) , or chi - square test or fisher 's exact test ( for categorical data ) was applied to survey potential predictors of ir . all tests were two - sided , and statistical significance was considered to exist when a p value was < 0.05 . table 1 demonstrates clinical and laboratory characteristics of 250 reproductive - aged thai women with pcos . the mean sd of age , body mass index ( bmi ) , and waist circumference ( wc ) were 25.4 5.8 years , 26.2 7.6 kg / m , and 82.3 16.3 cm , respectively . almost all of the participants ( 98.4% ) presented with oligomenorrhea . of all participants , the overall prevalence was 20.0% which included 3.2% of ifg , 13.6% of igt , and 5.6% of type 2 dm ( 3.6% of fbs 126 mg / dl , 5.2% of 2-hr glucose 200 mg / dl , and 3.2% of combined abnormalities ) . table 3 shows clinical predictors for ir in 250 pcos thai women . comparing with the patients without ir , the ones with ir were older , and had a higher bmi , larger wc , and higher prevalence of hypertension , acanthosis nigricans , and dyslipidemia . the results from multiple logistic regression analysis by forward stepwise technique showed that the independent predictors for ir included age of 30 years old , wc of 80 cm , presence of an , and dyslipidemia , with odds ratios of 2.14 ( 95% ci 1.014.52 ) , 3.53 ( 95% ci 1.289.75 ) , 2.63 ( 95% ci 1.175.88 ) , and 3.07 ( 95% ci 1.168.11 ) , respectively . insulin resistance is a metabolic disorder caused by the impairment of insulin function in inducing glucose uptake and utilization . in clinical setting , these techniques , including fpg , ogtt , glucose / insulin ratio , homeostasis model assessment for insulin resistance ( homa - ir ) , and quantitative insulin sensitivity check index ( quicki ) , are shown to correlate well with the gold standard , that is , euglycemic - hyperinsulinemic clamp [ 22 , 23 ] . to date , it is still inconclusive to judge the superiority of one technique over the others [ 24 , 25 ] . in the present study , we used fpg and ogtt as suggested by the rotterdam eshre / asrm - sponsored pcos consensus workshop group . interestingly 88.2% ( 30/34 ) of the patients with igt and 85.7% ( 12/14 ) of those with type 2 dm did not have ifg . our finding was in line with the previous information that fpg alone is not a sensitive indicator for ir in women with pcos [ 6 , 7 , 26 ] . the prevalence of ir in our pcos women was lower than that of many reports [ 6 , 7 , 27 ] which revealed the prevalence up to 75% . the difference in techniques used to define ir was responsible for such large discrepancy . however , even using similar definition , the prevalence of ir in our study was still lower than that in other studies [ 6 , 28 , 29 ] . this was owing to the fact of that our study population was relatively young and thin . previous studies showed that the prevalence of ir increased with age and bmi [ 6 , 7 ] . in the present relatively young and thin pcos women , many factors associating with ir were similar to those with metabolic syndrome [ 17 , 30 ] as these two conditions are closely related [ 31 , 32 ] . these factors included age , central obesity , presence of acanthosis nigricans , and dyslipidemia . hypertension and high bmi were shown to be associated with ir in univariate analysis but regression analysis indicated that these two variables were not independent factors . moreover , the association between ir and central obesity but not bmi suggested the effect of body fat distribution on insulin metabolism . previous studies showed that android fat distribution and upper - half type body fat distribution were linked to insulin resistance in pcos [ 3335 ] . hyperandrogenemia , a diagnostic criterion for pcos , was shown to be significantly associated with ir in our univariate analysis . the discrepancy result between these two analyses indicated that hyperandrogenemia had less impact on ir than other factors did . the association between hyperandrogenemia and ir was also demonstrated in previous reports [ 38 , 39 ] . the pathophysiology of pcos comprising a model of complex interaction between hyperandrogenemia and ir is well accepted . however , the results of some studies suggested that these two conditions might be two separate entities [ 38 , 41 ] . the absence of significant association between hyperandrogenemia and ir in the present study might confirm the concept of two separate entities . however , we could not disregard the important of ethnicity on this issue . to date the reference values used in the present study derived from the us population , the values of which might not be applicable for our population . due to the limitation of a cross - sectional study , the prevalence of ir found at the initial visit would change with time . therefore , a long - term prospective study is needed to reveal the actual prevalence in our population , and the effect of metabolic derangement on future health status . furthermore , the majority of our participants were urbanized women ; therefore , they might not represent the whole picture of pcos thai women . the overall prevalence of abnormal ogtt in reproductive - aged thai women with pcos is 20.0% . clinical predictors are age of 30 years , central obesity , presence of acanthosis nigricans , and dyslipidemia , but not hyperandrogenemia . it is probably beneficial to screen for ir in these specific groups of pcos women in order to provide optimum management for each patient .	objectives . to determine the prevalence of insulin resistance ( ir ) and its predictors in reproductive - aged thai women with polycystic ovary syndrome ( pcos ) . methods . a cross - sectional study was conducted from may 2007 to january 2009 . participants were 250 thai women with pcos . information regarding medical history and physical examination and results of 75 gram ogtt were recorded . results . the overall prevalence of ir was 20.0% , comprising the prevalence of impaired fasting glucose , impaired glucose tolerance , and diabetic mellitus of 3.2% , 13.6% , and 5.6% , respectively . multiple logistic regression analysis showed that the independent predictors for ir were age of 30 years old , waist circumference ( wc ) of 80 cm , presence of acanthosis nigricans ( an ) , and dyslipidemia with odds ratios ( 95% confidence interval ) of 2.14 ( 1.014.52 ) , 3.53 ( 1.289.75 ) , 2.63 ( 1.175.88 ) , and 3.07 ( 1.168.11 ) , respectively . conclusion . the overall prevalence of ir in reproductive - aged thai women with pcos is 20.0% . age 30 years old , wc 80 cm , the presence of an , and dyslipidemia are the significant clinical predictors .
You are an expert at summarizing long articles. Proceed to summarize the following text: treatment - resistant depression ( trd ) is a relatively common condition presenting with substantial challenges to both the clinician and researcher . in fact , despite a progressively higher number of available antidepressant therapies , trd occurs frequently in clinical practice , and is associated with profound psychosocial disability , personal suffering and economic cost burden . between one and two thirds of major depressive disorder ( mdd ) patients will not respond to the first antidepressant prescribed and 15 to 33 percent will resist to multiple interventions , including non - pharmacological therapies . increasing the burden associated with mdd , its high prevalence : world health organization ( who ) estimated that 5 - 10% of the population at any given time is suffering from identifiable depression needing psychiatric or psychosocial intervention , while the life - time risk of developing depression is 10 - 20% in females and slightly less in males . prevalence estimates for trd are available from several sources , including large clinical trials , large meta - analyses , or naturalistic studies [ 6 - 8 ] . for example , in the first level of the sequenced treatment alternatives to relieve depression ( stard ) trial , only about 30% of patients were in remission following up to 12 weeks of therapy with the selective serotonin reuptake inhibitor ( ssri ) citalopram . in addition , 15.8% of patients developed an intolerable adverse event , 38.6% moderate - to - severe impairment due to an adverse event , 8.6% discontinued treatment due to adverse events , and 4% developed a serious adverse event , findings that underscore efficacy and tolerability limitations of treatment with a typical first - line antidepressant agent . papakostas and fava reviewed 163 randomized , double - blind , placebo - controlled trials involving the use of antidepressants for mdd . approximately 53.4% of patients responded following treatment with an antidepressant , compared to 36.6% of patients who responded following the administration of a placebo pill . corey - lisle and colleagues reported that approximately 22% of patients who received treatment for depression by their primary - care physicians remitted following 6 months of treatment , 32% were partial responders , while 45% were non - responders . similarly , rush and colleagues reported an 11% remission rate and 26.3% response rate among depressed outpatients following 12 months of treatment of depression in one of several public - sector community clinics . petersen and colleagues report a 50.4% remission rate among outpatients with mdd enrolled in 1 of 2 hospital - based , academically affiliated depression specialty clinics ( massachusetts general hospital , an affiliate of harvard medical school and rhode island hospital , an affiliate of brown university ) following an average of 25.8 weeks of treatment . finally , it is also worth noting that while partial or non - response are common , residual symptoms among remitters are also highly prevalent [ 12 , 13 ] , being usually associated with poorer psychosocial functioning as well as an increased relapse rates , higher suicidal ideation and attempts , higher number of lifetime hospitalizations , more frequent healthcare resources utilization , general practitioner consultation , job loss and social retirement . attempts at overcoming treatment resistance in major depression begin with the clinical controversies in defining it . currently , there are no universally accepted operational definitions of trd . since more effective treatment approaches are needed for treating trd , regardless on how it is defined , the purpose of this narrative review is to assess current major definitions of trd and to briefly discuss its putative bipolar diathesis for some of the affected patients . actual definition and clinical classification of trd represent debated issues : while some literature evidences tend to progressively suggest to revise its current nosology , others tend to be more conservative , actually making trd a resistant clinical controversy . according to research practice , a lack of response ( not necessarily trd per se ) is usually defined by failure to reduce of at least 50% in the hamilton depression ( ham - d ) total score or as failure in reducing below a specific cut - off while less objective trd clinical definitions include failure in symptoms resolution or the more accepted failure to respond to 2 or more adequate antidepressant trials . the definition of an adequate treatment trial of antidepressant medication varied widely over the years , as the corresponding definitions of treatment resistance did . in actuality , trd patients present with histories of varying degrees of treatment adequacy . a high proportion of cases referred to university settings specifically for evaluation and treatment of " refractory " depressions have not received even a single adequate anti - depressant trial . clinical controversies related to trd refer not only to its definition but also to the way this latter is conceived : the adequacy of a trial as well the definition of non response seem to be misleading concepts . there are 3 major treatment - resistance classification systems : a 5-stages classification ( stages get up depending on the number of previously failed adequate trials , with fifth one proposing bi - temporal electroconvulsive therapy - ect ) , the national institute for clinical excellence ( nice ) guidelines ( providing a short algorithm ) and the multi - level structured massachusetts general hospital ( mgh ) criteria . about the adequacy of a trial , there is no absolutely correct dosage for a specific antidepressant , since dosage requirements vary depending on factors such as age , weight , general health , concomitant medication usage , and tolerance of a particular medication . confirmation of treatment adequacy by more objective means ( e.g. , serial plasma drug levels ) is not the rule in clinical practice , and valid plasma level response relationships are limited to only a subgroup of the tricyclic antidepressants ( tcas ) and lithium salts . conventionally , adequate trials should last at least for 8-weeks , considering full antidepressant doses ( e.g. 20 - 60mg / day for the ssri fluoxetine or 150 - 300mg / day for the tca clomipramine ) when needed . yet , 8-weeks is just the average rcts follow - up , with clinical practice often requiring more prolonged exposure time . remission usually takes up to 6 months of mdd antidepressant therapy , while recovery with substantial symptoms resolution usually requires at least a 12 months follow - up . remarkably , most of antidepressant medications have a lag - phase of at least 3 - 4 weeks prior exhibiting any substantial clinical response , thus making hyper - dosing a rational strategy if response observed by weeks 5 or 6 is insufficient , while a too praecox pharmacological switch should be avoided in all the cases . with respect to psychotherapy , adequacy of treatment may depend on the number of sessions , the expertise of the practitioner , the therapist 's adherence to a particular form of therapy , and/or the interaction of the patient therapist dyad . ect may be gauged by the total number of treatments , the use of bilateral electrode placement , and the verification of seizure time by electroencephalographic monitoring . therefore , the terms " relative " and " absolute " treatment resistance may best describe lesser and greater degrees of certainty about the adequacy of a specific treatment trial [ 19 , 23 ] . the most common response criteria in clinical trials are a rating of at least " much improved " on the clinical global impressions ( cgi ) scale , a pre - specified level of improvement on a depression symptom rating scale ( e.g. , > 50% reduction in hamilton depression rating scale scores ) , a final absolute score on a symptom measure , or some combination of the above . both the use of composite outcome criteria and documentation of persistent improvement ( e.g. , for 2 weeks or longer ) may improve reliability and validity of classification . at least a 50% reduction in depressive symptom severity generally corresponds to the clinician 's global clinical impression of a moderate level of improvement . however , some patients meeting this commonly used response definition continue to have considerable residual symptomatology . residual symptoms convey a higher risk of relapse during continuation treatment and likely contribute to suboptimal restoration of vocational or interpersonal functioning . the term remission describes a response in which a formerly depressed person 's level of residual symptomatology is essentially indistinguishable from someone who has never been depressed . with respect to standardized scales , a score of 6 or less on the 17-item hamilton rating scale for depression is often used to define a remission . as for response , non - response quantification may vary ( ham - d1775%=remission ; 50%-74%=response ; 25%-49%=partial response ; < 25%=non - response ) , with treatment - non - response being also defined as poor response to a single adequate antidepressant trial and treatment - resistant depression and chronic - resistant depression if resistance lasts for at least 12 months despite 2 or more adequate antidepressant trials ( including augmentation strategies ) . as further confounding variable , the fact that trd itself sometimes receives different appellations ( e.g. treatment - refractory or therapy - resistant depression ) as no univocal definition of the adequacy of an antidepressant trial does exist , making a desirable revision of current trd nosology a difficult , resistant , process . first step should be an appropriate anamnesis , eventually integrating validated instruments as the antidepressant treatment history questionnaire ( atrq ) [ 25 , 26 ] . the recognition of depression subtypes ( particularly melancholic , psychotic , atypical , and seasonal ) is an important element in the evaluation and management of trd because individuals with different subtypes of depression may respond in somewhat different ways to the available therapies , or eventually predict a soft bipolar diathesis ( e.g. in case of seasonal or atypical features ) . additionally , resistance to treatment may also be related to differential diagnosis mistakes as misdiagnosis of a unipolar mdd in patients with declared ( full - threshold ) or undeclared ( sub - threshold ) bd . patients with bd present in the depressive phase 2 to 3 times more often than they do in the manic state and it is estimated that bd i is undetected in 35% to 45% of patients . it is important to evaluate patients with trd specifically for a history of manic or hypomanic episodes to rule out bipolar spectrum disorders since depressive episodes may be the ( only ) clinical presentation of bipolar disorders ( bds ) for many years . treatment should become ineffective if too much retarded : only 60% of persons with depression are treated for the disorder . an appropriate evaluation of psychiatric and medical comorbidities as well a careful acknowledgement of current major depressive episode ( mde ) features is mandatory . as mentioned , many of trd cases show - up with psychotic or atypical features , often requiring more personalized therapies [ 29 , 30 ] ; for example , treating with ssris instead of mono - amino - oxidase inhibitors ( mao - i ) or tcas a mde with atypical features may lead to pseudo - resistance instead of a true resistance phenomenon ( 1999 ) reported up to 3.2% ( p<0.001 ) panic disorders , social phobia 2.1% ; ( p<0.008 ) , other anxiety disorders 2.6% ( p<0.001 ) and axis - ii dsm - iv - defined personality disorders 1.7% ( p<0.049 ) . remarkably , early age of onset ( < 18 years ) 1.7% ( p<0.009 ) and current dsm - iv - defined melancholic features 1.5% ( p<0.018 ) were a frequent trd association . also , most trd cases have multiple co - morbidities and this further increases the burden load , reducing the chance of a substantial inter - episodic depressive symptoms resolution , therefore increasing the risk for relapse . one of the most important set of comorbidities that contributes to inadequate treatment response in mdd and other disorders is substance ( including alcohol ) use disorders and it may be carefully considered too when treatment resistance arises in mdd patients since this may require specific treatment approaches and eventually be in favor of a bipolar diathesis . trd comorbid psychiatric disorders are often missed or are sub - optimally treated , and they can confound both the evaluation and the management of depression . trd - associated biological factors include a reduction in frontal cortex and hippocampal volumes , increase in ventricular volume and amygdala hyperactivity with poor inhibition by prefrontal - cortex , contributing in making most trd patients hypersensitive to environmental stressors . nonetheless , currently proposed biomarkers for trd still require further evidences as false positives cases could occur . frequent trd medical co - morbidities include infective diseases ( e.g. hiv and borna virus ) and endocrine disorders as hypothyroidism or hpa - axis imbalances ( up to 50% of trd cases present a non - suppression with the dexamethasone test due to hpa hyperactivity ) . yet , a major medical comorbidities for most trd cases is represented by cardiovascular disease and diabetes [ 37 , 38 ] . the implication for treatment is to address these conditions simultaneously , if possible , to avoid consolidating treatment resistance . what if trd persists despite repetitive adequate trials and accurate considerations of the potential confounding or concomitant factors ? to date , the stard study represents the broadest , multi - centric clinical trial ever conducted on mdd assessment . star*d lasted for 7 years ( oct 1999-sept 2006 ) involving both primary care and psychiatric facilities and adopting minimum exclusion criteria ( real world ) , with remarkably , 2876 patients ( 4041 enrolled ) had at least one axis - i co - morbidity at baseline . unfortunately , despite a multi - level algorithm including switches and augmentation strategies with different antidepressant classes , lithium , thyroid hormones and cognitive behavioral therapy ( cbt ) , remission rates were almost equivalent for stages i vs. ii ( 32.9% vs. 30.6% ) and stages iii vs. iv ( final ) , ( 13.6% vs. 14.7% ) ; authors concluded that no specific antidepressant treatment was superior to any other one , with cbt role remaining a debated issue . while popular clinical augmentation or switch strategies for trd include a broad number of compounds ( e.g. , thyroid hormones , estrogen , lithium , pindolol , atypical antipsychotics , stimulants , inositol , omega-3 fatty acids , da - agonists , herbal supplements , lamotrigine , etc ) most of them are not accounted in official trd guidelines . also , meta - analyses data tend to suggest the switch strategy versus the augmentation one , with lithium and atypical antipsychotic medication as more favored choice [ 5 , 42 , 43 ] . remarkably , lithium and atypical antipsychotics use is much more consolidated for bipolar disorders rather than unipolar depression . also , it is interesting to observe how a considerable number of trd cases show - up with atypical - mdes or have an earlier age of onset compared non - resistant mdds ? ( 2005 ) tried to answer at some of these questions investigating 69 patients diagnosed with treatment resistant - mdd ( failure to respond to 2 or more adequate clinical trials ) at a local mood clinic ; when patients were retested using the structured clinical interview for axis - i disorders ( scid - i ) 35% of them were diagnosed as bipolar . the whole sample , was re - tested 1 year later , showing 41% mdd , 3% bp - i , 43% bp - ii , 13% bipolar - nos . interestingly , most of former - mdd - trds significantly improved in clinical global impression ( cgi ) total score when switched from antidepressants to mood - stabilizers . indeed , trd cases presented a rapid pop - up on antidepressant , frequent motor agitation , somatic symptoms and fatigue and a history of polypharmacy , which may be accounted as a potential iatrogenic phenomenon for mixed states and cyclicity among those with a supposed bipolar diathesis . further evidences suggesting that some trd - mdd patients may have ( or acquire ) a bipolar diathesis , have been also provided by the french national naturalistic epidep study , which major outcomes included the evaluation of bipolar patients using also temperamental instruments ( as the temperament evaluation of the memphis , pisa , paris , and san diego auto - questionnaire temps - a ) . cyclothymic - sensitive and depressive temperaments , along with motor agitation , greater severity of current depressive episode , scored significantly higher among those with bp - trd . the adoption of less restrictive diagnostic criteria ( e.g. 2 days of hypomania vs. the arbitrary dsm - iv 4 days duration one ) and the use of sensitive ( yet acceptable in sensitivity ) instruments ( e.g. hypomania check - list 32-items hcl-32 ) may lead to a prompter recognition of sub - threshold resistance clusters ( e.g. depressive mixed states while no data support the evidence that all trd cases should follow a bipolar diathesis , neither they support the opposite . it is interesting to observe how most recent evidences tend to suggest a bipolar diathesis in a subgroup of dsm - iv - defined unipolar depressed patients and how significantly should the clinical outcome be influenced by different therapeutic implications ( as pointed out by preliminary systematic treatment enhancement program for bipolar disorder finally , when trd lasts for a very long time ( despite repetitive adequate trials ) , it should be prudent to promptly revise diagnosis and/or therapeutic choices . in fact , prolonged antidepressant treatments may induce bipolar phenomena as rapid cyclicity and mixed states as well as antidepressant - resistance in some patients and their over - prescription should therefore be avoided , preferring lithium and atypical antipsychotics , as indicated by the bp - trd algorhythm recently proposed by pacchiarotti et al . . also , while a bipolar hypothesis for some trd cases is definitely not a novel approach , it appears to become a gradually more popular remark , especially considering that further insights are progressively acquired about a potential bipolar diathesis for some of the features sometimes associated with trd too : just to mention one , a history of substance abuse is today almost widely accepted as a strong bipolar feature . in conclusion , directions for future research on trd should nt apart from a substantial revision of the way trd itself is defined . adopting widely accepted criteria for the adequacy of a trial as well for the concept of resistance is mandatory in order to allow researchers to give rise to a hoped international collaborative group . specifically , more attention should be placed on a plausible bipolar diathesis for some of the trd cases as a progressively greater number of latest literature evidences tend to support this view . otherwise , till a consistent revision of current boundaries of trd nosology will be performed , we ll have no opportunity to fully overcome the resistance phenomenon . trd : as treatment resistant depression ; mdd : as major depressive disorder ; bp : as bipolar disorder .	treatment - resistant depression ( trd ) represents a source of ongoing clinical and nosological controversy and confusion . while no univocal consensus on its definition and specific correlation with major mood disorders has been reached to date , a progressively greater number of evidences tend to suggest a revision of current clinical nosology . since a better assessment of trd should be considered mandatory in order to achieve the most appropriate clinical management , this narrative review aims to briefly present current most accepted definitions of the phenomenon , speculating on its putative bipolar diathesis for some of the cases originally assessed as unipolar depression .
You are an expert at summarizing long articles. Proceed to summarize the following text: motor neurone disease ( mnd ) is considered to be a group of generally fatal , progressive neurodegenerative disorders . the disease is characterised pathologically by progressive degeneration and loss of motor neurones in the anterior horn cells of the spinal cord , motor nuclei of the brainstem , and the descending pathways within the corticospinal tracts . it is primarily a condition of middle to late life , with onset of symptoms between the ages of 50 and 70 . when disorders are recent or rapidly progressive , motor neurone loss results in weakness and wasting . in slowly progressive denervating diseases such as mnd , loss of more than 5080% of motor units may occur before weakness becomes clinically apparent . in mnd , needle electromyography may reveal changes of chronic reinnervation , but provides little direct evidence to the extent of motor neurone and axonal loss . the supramaximal compound muscle action potential ( cmap ) amplitude also provides little direct evidence of the extent of motor neurone loss as a normal cmap amplitude might mistakenly be thought to indicate that motor neurone loss has not occurred . motor unit number estimation ( mune ) has been shown to be a more reliable method for following changes in neurogenic disorders than the cmap amplitude . it estimates the number of functioning lower motor neurones innervating a muscle or a group of muscles . it is an indirect measure of motor neurone loss , rather than a measure of pathology . the principle of mune is that the mean single motor unit amplitude ( smup ) can be measured ; it is possible to obtain an estimate of the total number of motor units in the muscle . the results were comparable with estimates of alpha motor fibres obtained by counting axons in specimens of motor nerves . mune has been performed in a number of different ways , each with their advantages and limitations . each method measures both the average smup and the size of the cmap amplitude obtained with maximal stimulation of a motor nerve . there are a number of methods that have been developed to quantify maximal voluntary isometric contraction ( mvic ) more objectively , as the traditional neurological examination is inadequate for documenting motor performance reliably [ 10 , 11 ] . these include an electronic strain - gauge tensiometer and a hand - held jamar hydraulic dynamometer . we used the jamar hand dynamometer to measure isometric grip strength to determine whether this correlated with the number of functional motor neurones in the thenar group of muscles as measured by the statistical mune method in a group of asymptomatic subjects , all of which had mrc grade 5/5 power on clinical neurological examination . 19 were asymptomatic sod 1 mutation carriers , 34 age and sex matched sod-1-negative family controls and 16 normal population controls . numerous studies have shown that mune can be used to monitor change over time in mnd patients when used by an experienced technician , despite evaluator bias . this technique has undergone a number of revisions since its original description to take into account motor unit instability [ 15 , 16 ] . the motor unit number estimate is calculated by : ( 1 ) mune = maximum cmap amplitude ( or area)average single motor unit potential ( smup ) amplitude or area . in this study , we estimated motor unit numbers from abductor pollicis brevis ( apb ) ( thenar mune ) . this muscle predominately relates to thumb abduction , and therefore only partially contributes to grip strength it is a distal muscle that can be used to determine whether loss of functioning motor neurones is occurring distally . the thenar muscles are vulnerable to common disorders like carpal tunnel syndrome , which may result in handgrip weakness . to exclude this , median motor nerve conduction studies were performed before each mune study . the presence of carpal tunnel syndrome was defined as a median distal motor latency of greater than 4.0 ms . self - adhesive surface recording electrodes ( g1 ) were placed transversely across the innervation zone of the right apb , resulting in a simple biphasic negative - positive m wave , with g2 placed over the distal phalangeal joint . this was performed by strapping the stimulating electrode onto the surface of the skin , at the point where the threshold of the nerve to electrical stimulation was at its lowest . a hand - held stimulator was not used , as reproducibility is enhanced when the stimulating electrodes are fixed to the surface of the skin . median nerve stimulation at the wrist was well tolerated by all subjects , as the stimulation intensity required to obtain an adequate response was generally less than 20 ma with duration of 0.1 ms . maximum isometric grip strength was obtained using the jamar hydraulic dynamometer at the standardised , middle handle position with consistent instructions given to the 69 subjects . this technique was used as studies have shown no significant difference in reliability between one attempt , mean score of two or three attempts , or the highest score of 3 attempts . clinical neurological examination was performed on all 69 subjects , with the strength of thumb abduction , finger flexion and finger abduction measured according to the medical research council ( mrc ) grading system . the correlation between hand grip and mune was measured using the pearson product moment correlation , as the pearson 's coefficient reflects the degree of linear relationship between two variables . isometric grip strength tests , thenar mune , and mrc power were performed on 69 asymptomatic subjects . the technique was found to be reproducible , in our previous study indicating that the test - retest correlation of thenar mune in these 69 asymptomatic subjects was high , with a pearson correlation coefficient of 0.93 . the mean difference between mune results on separate occasions on the same individual was 3.6% , with a range of 011.7% ( figure 1 ) . right - hand grip strength correlated with left - hand grip strength , with pearson 's correlation coefficients of 0.945 ( p < 0.001 ) . the ranges for right- and left - grip strength were similar right 20135 lb with a mean on 75.04 lb and left 22132 lb with a mean of 71.14 lb . two - way analyses of variance showed a no - significant difference between the right and left hands , figure 2 . right maximum isometric grip strength using the jamar hydraulic dynamometer correlated poorly with the number of functional motor neurones in thenar group of muscles as measured using the statistical method of mune , with a pearson correlation coefficient of only 0.028 ( figure 3 ) . power right thumb abduction , finger flexion , and finger abduction was measured according to the medical research council ( mrc ) grading system and was found to be of grade 5/5 in all subjects . this study shows that there is a lack of correlation between handgrip mvic and thenar mune , indicating that handgrip strength is not as sensitive as thenar mune for monitoring disease progression , as it is unable to detect early motor neurone loss due to the presence of compensatory mechanisms . the surviving motor neurones enlarge territories , through collateral sprouting ( reinnervation ) until late in the disease , when collateral reinnervation is no longer able to provide full functional compensation and weakness occurs . mune may be reduced because of remodelling of the motor units , but in this study , cmap amplitudes were maintained despite a reduction in mune . this may be because , early in the disease , the rate of cell death is low and not detectable . mune can be used as a noninvasive method of predicting impending decline in motor neurones and estimating the rate of neuronal death in asymptomatic subjects . analysis of the density of the electromyographic interference pattern during maximal effort is not quantitative and requires the full cooperation of the patient . mune has been performed in a number of different ways , each with their advantages and limitations . the choice of technique depends largely on the speed and simplicity of the technique , as well as its accuracy and reproducibility . each method measures the average smup and the size of the cmap obtained with maximal stimulation of a motor nerve . most employ electrical stimulation of the motor nerve to determine the sizes of the smup , but a few techniques use needle emg . the choice of technique largely depends on the speed and simplicity of the technique , as well as its accuracy and reproducibility . the statistical mune technique used in this study has been compared to the multiple point stimulation method and found to be more reproducible ( 7% versus 12% ) and faster to administer . the technique has been greatly modified since its original description , but , despite this , numerous studies have shown that mune can be used to monitor change in mnd patients when used by experienced technicians . there have been studies performed indicating that the statistical mune is unreliable in the presence of clinical weakness due to motor unit instability and have suggested that the multipoint incremental method is more reliable as an outcome measure in als . from our previous study , we found that the statistical method of mune method was fast , noninvasive , and reproducible , so that results obtained could be used as a baseline to follow loss of motor units over time , with estimates having coefficients of variability in the order of 5% . in addition , all subjects had normal medical research council ( mrc ) grade 5/5 power in thumb abduction , finger flexion , and finger abduction , so the criticism of this technique was not a concern . generally , however , manual motor testing used in a standard neurological motor examination does not allow objective documentation of change in performance , as it may be influenced by the patient 's history and progress . major changes are apparent , but subtle changes are difficult to determine with accuracy . to improve this the hand - held jamar hydraulic dynamometer has been proposed as a clinically useful , reliable , reproducible , time efficient , and quantitative measure for monitoring disease progression in mnd . there have also been studies investigating the reliability of isometric grip strength testing , and more recently , normative maximum grip strength data for men and women has been developed to help clinicians interpret results obtained . thumb abduction power correlates better with thenar mune , but the jamar dynamometer is not able to determine strength of isolated functional groups of muscles . also , the thenar mune does not examine all of the motor units involved in handgrip mvic , as forearm flexors and ulnar - innervated muscles are also involved . finally , another explanation for the lack of correlation may be that , in mnd , there is not uniform motor neurone loss in all parts of peripheral motor nerves . in our previous study , we showed that mune was able to identify a deterioration in functional motor units before clinical weakness became apparent . this confirms mccomas ' observation that patients with substantial chronic denervation could maintain normal muscle twitch tension until loss of about 5080% of motor units occurs . felice showed that , in 21 mnd patients , change in thenar mune was the most sensitive outcome measure for following disease progression , when compared to other quantitative tests , such as cmap , isometric grip strength , forced vital capacity , and medical research council manual muscle testing . this study suggests that there is a lack of correlation between handgrip mvic and thenar mune , implying that mune may be a reliable and sensitive method for diagnosing mnd . mune can be used as a noninvasive method of predicting impending decline in motor neurones and estimating the rate of neuronal death in asymptomatic subjects .	in slowly progressive conditions , such as motor neurone disease ( mnd ) , 5080% of motor units may be lost before weakness becomes clinically apparent . despite this , maximal voluntary isometric contraction ( mvic ) has been reported as a clinically useful , reliable , and reproducible measure for monitoring disease progression in mnd . we performed a study on a group of asymptomatic subjects that showed a lack of correlation between isometric grip strength and thenar mune . motor unit number estimation ( mune ) estimates the number of functioning lower motor neurones innervating a muscle or a group of muscles . we used the statistical electrophysiological technique of mune to estimate the number of motor units in thenar group of muscles in 69 subjects : 19 asymptomatic cu , zn superoxide dismutase 1 ( sod 1 ) mutation carriers , 34 family controls , and 16 population controls . the jamar hand dynamometer was used to measure isometric grip strength . this study suggests that mune is more sensitive for monitoring disease progression than maximal voluntary isometric contraction ( mvic ) , as mune correlates with the number of functional motor neurones . this supports the observation that patients with substantial chronic denervation can maintain normal muscle twitch tension until 5080% of motor units are lost and weakness is detectable .
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Proceed to summarize the following text: nanomaterials occur in nature via various geobiophysicochemical processes , such as volcanic eruptions , bacterial metabolism , and other erosion and reduction mechanisms . in recent decades , nanoparticles have been increasingly manufactured and used in daily consumer products , such as textiles , paints , pharmaceuticals , and cosmetic products , as well as in pollution treatment and remediation processes [ 1 , 2 ] . however , it is widely known that nanoparticles may cause potential adverse effects in organisms , including human and mammals [ 37 ] . consequently , the adverse effects of incompletely retrieved nanoparticles pose an increasing threat to the public [ 79 ] . therefore , it is important to develop an understanding of the effects and active mechanisms of nanoparticles . silver ( ag ) is among the most used metals in human life and is well known for its antimicrobial effect . during the recent rapid development of nanotechnology , silver nanoparticles ( ag - nps ) have been used for various types of consumer products , such as cosmetics , heath care products , and textiles . consequently , information about the toxicity and carcinogenic effects of ag is available , with it being considered nontoxic and noncarcinogenic when used in appropriate amounts . however , at relatively high concentrations , ag - nps cause cellular toxicity and other adverse impacts , such as the inhibition of mitochondrial function in rat liver cells , mouse germline stem cells , and human fibroblasts [ 1113 ] . furthermore , an increasing number of recent reports have provided evidence of the cytotoxicity of ag - nps at doses of low exposure [ 14 , 15 ] . ag - nps also have an adverse impact on aquatic organisms , such as zebrafish and medaka fish , and cause oxidative stress , cellular apoptosis , chromosomal aberrations , and other developmental toxicity effects during early life stages and adulthood . this is because changes in the functional groups on the nanoparticle surface may facilitate substances to penetrate deep into the cells of the body , causing unexpected impacts on organisms . however , it is unclear whether the effects of coating materials increase the adverse effects of nanomaterials . in this study , we investigated the effects of ag - nps in the presence of citric acid as a capping material . citric acid is an organic chemical commonly found in the natural environment and human body . therefore , the interaction of citric acid with exposed materials is a common mechanism in the natural environment . common carp ( cyprinus carpio ) is an abundant species in the freshwater environment . due to their large size , carp have a better capacity for resistance to pollutants than other laboratory fish , such as zebrafish and japanese medaka . hence , carp are not usually used in experiments to examine the fatal effects of exposure to low doses of pollutants . however , because of the dominance of this species in the natural environment , carp is considered as one of the most suitable models to assess the non - fatal effects of pollutants by evaluating fluctuations in antioxidant enzyme levels or other changes in fish histology and physiology . the objectives of this study were to investigate the adverse effects of ag - nps capped with citric acid ( ag - nps ) by measuring antioxidant enzymatic activities , including those of catalase ( cat ) , superoxide dismutase ( sod ) , and glutathione - s - transferase ( gst ) . target organs included the brain , liver , and gills of the common carp ( c. carpio ) . in addition , other biochemical parameters in the blood and histological changes in the skin , liver , and gills of the fish were examined . for this reason , we used the same biological samples as those used in the previous study to present detailed data about the toxicity of nanoparticles . all chemicals were purchased from sigma - aldrich and were of analytical grade or better . citrate - capped ag - nps were purchased from abcnanotech ( daejeon , korea ) in a suspension form . the average ph of the original solution was 6.5 , with a particle content of 20% by weight . the dispersion of the particles was confirmed using a transmission electron microscope ( tem ; jeol , japan ) and the dynamic light scattering method ( dls ; els-8000 , otsuka , japan ) . a stock solution of ag - nps was prepared at 10 mg / l . working solutions ( 25 , 50 , 100 , and 200 g / l ) were obtained by diluting the stock solution with respective amounts of di water . the concentration of elemental ag was measured using an inductive coupled plasma mass spectrometer ( icp - ms , perkin elmer , usa ) . mean concentrations were obtained by using the data to plot a standard curve with ag standard solution ( scp science , qc , canada ) and 5% hno3 solution ( electronic grade , scp science , qc , canada ) . the ranges of the standard were 0.0 , 1.0 , 5.0 , 10.0 , 20.0 , 40.0 , 60.0 , 80.0 , and 100.0 juvenile carp were obtained from the national academy of agricultural science , korea . as shown in table 1 , the body weight and length of the fish were 18.6~33.8 g and 10.9~13.5 cm , respectively . water temperature was kept at 24 1c , hardness was maintained at 40~60 mg caco3/l , ph of the water was 7.2 , and oxygen saturation was more than 85% . fish were exposed to ag - nps for a period of 48 h and 96 h , according to the oecd test guideline 203 : fish acute toxicity test . the carp were exposed to ag - np concentrations of 25 , 50 , 100 , and 200 g / l . a blank control test ( without ag - nps ) was performed at the same time and under the same exposure conditions . all the experiments were conducted in an 80-l glass tank containing 60 l of water , with 10 fish in each exposure tank . the water was not changed during the test . after exposure , the gills , liver , and brain of the fish were collected , frozen , and stored separately at 20c for further treatment . the samples were rinsed with 0.15 mm kcl solution and homogenized on ice with 50 mm phosphate buffer ( ph 7.0 ) . the suspension was sonicated in an ultrasonic bath for 30 min and then centrifuged at 10,000 g at 4c for 10 min . gst activity was measured using a gst tag assay kit ( novagen , germany ) , in which a sample was combined with 1-chloro-2,4-dinitrobenzene substrate in the reaction buffer . the absorbance of the reaction was monitored at 340 nm by using a uv spectrophotometer ( tecan infinite f200 , austria ) . cat activity was measured using the abei method , in which 50 mm h2o2 was used as a substrate . a solution of 50 mm peroxide was prepared in 50 mm potassium phosphate buffer . the decomposition of h2o2 catalyzed by catalase can be followed using uv spectroscopy on the basis of the absorbance of h2o2 at a wavelength of 240 nm . sod activity was measured by using an sod assay kit ( dojindo laboratories , kumamoto , japan ) . total protein concentration was measured using the bradford method , with bovine serum albumin as the standard protein . other biochemical parameters were examined in this study , namely , ammonia ( nh3 ) , glucose ( glu ) , total cholesterol ( tcho ) , alkaline phosphates ( alp ) , glutamic oxaloacetic transaminase / aspartate aminotransferase ( got / ast ) , alanine aminotransferase ( gpt / alt ) , -glutamyltransferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) . after exposure , the skin , gills , and liver of the carp were dissected and fixed in bouin 's solution for 12 h or overnight . subsequently , these samples were washed with water and then dehydrated using a graded ethanol series ( 70% , 75% , 80% , 90% , and 100% ) . the samples were embedded in paraffin at 62c and sectioned at a thickness of 5 m by using a precision microtome ( mt-990 ; rmc products , usa ) . the sections were stained using the hematoxylin and eosin ( h&e ) staining method with alcian blue and the periodic acid - schiff ( ab - pas ) reaction . an olympus bx-51 light microscope ( olympus corp . , japan ) coupled with an artcam-150 piii digital camera was used to examine any abnormalities in the samples . data were calculated and analyzed using excel ( microsoft corporation , wa , usa ) software and plotted using sigma plot ( spss inc . the differences between the samples and blank controls were evaluated using one - way analysis of variance and the student 's t - test . rna extraction and dna microarray analysis were performed according to the protocol previously described by lee et al . . the sequences of each clone i d were aligned on the basis of sequence homology by using the basic local alignment search tool ( blast ) in the national center for biotechnology information database . the translated nucleotide sequences were compared with the protein databases by using the blastx analysis software . the size distribution and morphology of ag - nps were examined using tem and dls . as shown in figure 1 , ag - nps became agglomerated in the solution , and the nominal size measured using tem was ~12 nm . however , the mean size of the agglomerated particles varied as much as several hundred nanometers . the dls results showed that the mean diameter of ag - nps at 5 mg / l and 10 mg / l was ~70 nm and ~90 nm , respectively ( table 2 ) . however , particles with a diameter of less than 20 nm were frequently observed in the solution . elemental ag was measured using icp - ms , and the results are shown in table 3 , with relative standard deviations ranging around 10% . in general , ag - nps were well dispersed in di water solution , with some agglomeration and aggregation . the antioxidant enzymes , including those of gst , cat , and sod , were measured , and the results are shown in figures 2 , 3 , and 4 . generally , enzymatic activities changed with different ag - np concentrations . after 48 h of exposure , gst activities in the liver , gills , and brain were not significantly different than that observed when the fish were exposed to 25 g / l and 50 g / l of ag - nps ( figure 2(a ) ) . when the concentration of ag - nps increased to 100 g / l and 200 g / l , gst activity was more significantly reduced in the gills than in the liver and brain . after 96 h of exposure , gst activity was elevated in the liver ; this was correlated with an increase in ag - np concentration in the media ( figure 2(b ) ) . gst activity in liver samples exposed to 100 g / l and 200 g / l was about 190% higher than that observed in the control fish . in comparison , gst activity in the liver of fish after 96 h of exposure was also higher than that in fish subjected to 48 h of exposure , especially at 100 g / l of ag - nps . after 96 h of exposure , gst activity was also higher in the liver than in the brain and gills . as shown in figure 3 , higher levels of cat activity were detected in the liver than in the brain and gills of the experimental fish . similar to gst activity , cat activity in the liver changed at different ag - np concentrations , but it remained relatively stable in the brain and gills . cat concentrations in the liver of the fish after 48 h of exposure showed no significant difference to that in the control group ( figure 3(a ) ) . however , cat activity declined at lower ag - np concentrations ( i.e. , 25 g / l and 50 g / l ) and increased at higher ag - np concentrations ( i.e. , 100 g / l and 200 g / l ) . however , after 96 h of exposure , cat activity was significantly reduced in the liver at 25 g / l and 50 g / l , recovered at 100 g / l , and then decreased again at 200 sod activity differed from that of gst and cat in that it was relatively stable in all examined tissues ( figure 4 ) . however , similar to gst and cat , higher levels of sod activity were detected in the liver than in the brain and gills . there was no significant difference in sod levels among the different exposure groups . in this study , the biochemical parameters in the blood of the exposed fish were also analyzed to understand the changes in metabolism and fish response to the stressor ( i.e. , ag - nps ) . detailed information was published in our previous study , where the concentration of biochemical parameters such as ammonia ( nh3 ) , glutamic oxaloacetic transaminase ( got ) , and total cholesterol ( tcho ) declined at the beginning of exposure and gradually recovered with increase in ag - np concentration . other parameters such as alanine aminotransferase ( gpt ) , -glutamyl transferase ( ggt ) , albumin ( alb ) , blood urea nitrogen ( bun ) , creatinine ( cre ) , and total bilirubin ( tbil ) remained stable or fluctuated around the levels of the control groups , without any significant difference . in comparison , alkaline phosphatase ( alp ) levels increased with increase in ag - nps concentration . after reaching the highest concentration when exposed to 50 g / l of ag - nps , alp concentration began to decline when the fish were exposed to 100 /l and 200 the histological structure of the skin ( figure 5 ) is composed of an epidermal layer and a dermal layer . the epidermis is the outermost layer of the skin , where the epithelial and secretory cells are distributed . when dyed with h&e stain , the vacuolated mucous cells appeared as blank circles or elliptical in shape and were marked with a blue color ( color index 313c ) as a result of the ab - pas ( ph 2.5 ) reaction . vacuolated club cells also appeared elliptical in shape when using the ab - pas ( ph 2.5 ) reaction . as illustrated in figure 5(a ) , the skin histology of the control fish showed that normal - sized mucous cells ( mcs ) were present , with appropriate amounts of club cells ( ccs ) in the epidermal layer . exposure to 50 g / l of ag - nps ( figure 5(b ) ) increased the size and number of mucous cells marked with alcian blue by using the ab - pas ( ph 2.5 ) positive reaction . on increasing the level of exposure to 100 g / l of ag - nps , the amount of club cells in the epidermal layer significantly increased . the size of the mucous cells was also larger than those of the control fish ( figure 5(c ) ) . at 200 g / l of ag - nps , hyperplasia ( ) was frequently recorded in the epidermal layer of the exposed samples ( figure 5(d ) ) . carp have one pair of gills on each side of the body , like other teleosts . the gill raker is lined up at the front of the gill arch . as shown in figure 6(a ) , numerous gill lamellae are lined up along either side of the gill filament . the gill filament consists of a cartilaginous support , which is a multilayered epithelium . in comparison , after staining with h&e , vacuolated mucous cells appeared as blank circles or elliptical in shape but were marked with a blue color ( color index 313c ) as a result of the ab - pas ( ph 2.5 ) reaction . as shown in figure 6(b ) , exposure to 50 g / l of ag - nps caused the bifurcation of the filament ( black arrow ) . figure 6(c ) shows an increased number of mucosa cells ( black arrow ) in the gills of the fish exposed to 100 g / l of ag - nps . the exposure of the fish to 200 g / l of ag - nps caused hyperplasia ( ) of the epithelium in the lamella ; this finding was not observed in the control group ( figure 6(d ) ) . this phenomenon was also recorded at other concentrations of ag - nps , but it was most clearly observed at a concentration of 200 the liver of carp consists of numerous lobules , along with various bile ducts , capillaries , and the pancreas . hepatocytes from the control group were clearly observed to have a round polygon shape with a nucleus ( figure 7(a ) ) . exposure to 100 g / l of ag - nps caused extreme atrophy of the hepatocyte nucleus in the liver of the fish ( figure 7(b ) ) . fish exposed to 200 g / l of ag - nps showed atrophy of the hepatocyte nucleus and an accumulation of eosinophilic granules ( color index 2415c , black arrowhead ) in the liver ( figure 7(c ) ) . as shown in figure 8 , ag - nps penetrated the skin of common carp after 96 h of exposure , regardless of particle concentrations . ag - nps were present in every layer of the skin , including the epithelium ( figure 8(b ) ) , epidermis ( figure 8(a ) ) , and mitochondrion ( figure 8(c ) ) . the particles stayed either in the junctions of the cells , on the cell membrane , or inside the cell . penetration by a large number of ag - nps in the skin would result in damaging the cell junction , membrane , or other cell structures , depending on the size and concentration of the particles . the penetration of large agglomerations of particles into the skin ( as shown in figure 8(b ) ) may destroy the structure and connection between the skin layers , while small amounts of single particles ( as shown in figure 8(c ) ) may stick on the membrane of cells , causing them to malfunction . in this study , the penetration of ag - nps in the gills of common carp after exposure was evaluated . figure 9 shows the presence of nanoparticles in the gill structures , including the gill lamella , the nucleus of gill cells , and blood corpuscles . if the skin is the first defensive structure of the fish body , the gill is an important organ that allows gaseous transfer and filtration of foreign materials . ag - nps were detected in every part of the gill at varying levels of penetration , such as between blood corpuscles and inside cell nuclei . the effects of nanoparticles on cell function might be more severe because of deep penetration . the presence of nanoparticles in different parts of the gills indicated that common carp is significantly affected by exposure to ag - nps . the effects may be directly expressed by damage to the tissue structure or indirectly expressed through abnormal levels of other biochemical markers . in this study , we also investigated the penetration of ag - nps into other important organs of the fish , including the brain and liver . the results are shown in figure 10 , indicating that particles entered the brain and liver of common carp exposed to 200 g / l of ag - nps for 96 h. as shown in figure 10 , the particles were able to penetrate deep into the brain cells . this result indicated that the particles were carried through the body of the fish by the circulation of blood , subsequently accumulating in different important organs . liver is an important organ of the body that facilitates the detoxification of substances , whereas the brain controls the activity of the whole body . damage to these organs may result in the malfunction of fish activities and , more seriously , may cause fish mortality . the findings of this study are comparable to those of a previous report by park et al . [ 23 , 24 ] , who observed that titanium dioxide nanoparticles caused chronic inflammatory disease and oxidative stress by intratracheal instillation in mice . alteration of the level of genomic expression towards 100 g / l of ag - nps exposure for 96 h in the liver of the common carp was analyzed using the dna microarray method . in response to ag - np exposure , there was a 2-fold upregulation and downregulation of 502 genes and 1,850 genes , respectively . differentially expressed genes ( > 2-fold , p < 0.05 ) with defined functions are summarized in table 4 . among the upregulated genes , 12 genes , including that for the myc associate protein x ( max ) , were shown to be involved in cell apoptosis , proliferation , protein synthesis , and energy production ( table 4 ) . generally , max has been known to participate in cell death and cell proliferation , with the interaction of myc . among the downregulated genes , 11 genes , including that for glutathione peroxidase 4a ( gpx ) , gst , and the retinol binding protein ( rbp ) , were shown to play a role in oxidative stress response , cellular defense , cell migration , detoxification , and fibrinolysis ( table 4 ) . both gpx and gst have protective effects against oxidative stress ( caused by the reactive oxygen species ros ) , while rbp has a major function in retinol metabolism [ 26 , 27 ] . our findings indicate that ag - nps might cause biological malfunctions or dysregulation in cellular processes , including cell death , proliferation , and resistance to oxidative stress . ag - nps also contribute towards changing the gene expression of particular sets of genes , which might be considered as plausible biomarkers of ag - nps toxicity . because of its strong antibacterial activity , ag is regularly used in preservative and hygiene products . ag - nps , categorized at a nanoscale , are expected to possess many physical and chemical properties that are different from those of their bulk particles . however , the novel properties of these particles require study , particularly because of their potential effects when exposed to organisms , including human beings . one of the novel properties of nanoparticles in general ( ag - nps in particular ) is their minute diameter , in comparison with that of their bulky counterparts [ 28 , 29 ] . because of the ultrafine particle - size , ag - nps may penetrate deep into the organs and , therefore , alter normal metabolic and bodily functions . fortunately , naturally occurring nanoparticles usually form aggregations , agglomerations , or complexes with other substances , such as natural organic materials . these complexes and/or aggregations / agglomerations reduce the size advantage of nanoparticles , thus reducing their toxicity or other adverse effects on aquatic organisms . to evaluate the impact of other materials on the toxicity of nanoparticles , synthesized particles with coated materials stabilizers of nanoparticles include ligands , surfactants , and polymers with different functional groups , such as cooh and nh2 . however , the toxicity and adverse effects of ag - nps capped with natural organic substances remain unclear . in this study , we used citric acid as a capping agent to stabilize ag - nps in solution . as shown in table 1 , this stabilizer keeps the particles well separated , with a nominal size of 10~20 nm . in addition , citric acid is a common organic substance in the natural environment and our daily life ( e.g. , fruit juice ) . hence , developing an understanding of the mechanisms and effects of citrate - ag - nps would contribute towards evaluating the fate and impacts of ag - nps in environmental matrixes . the antioxidant enzyme system is responsible for the elimination of oxidative stress during the early stage of the body 's defensive mechanism . abnormal changes in enzymes reflect the level of oxidative stress encountered by the body . in the current study , higher levels of gst , cat , and sod activities were detected in the liver compared to the brain and gills . this is because the liver is the major detoxification organ of the body . in this study , after 48 h of exposure , gst and cat activities in the liver were observed to vary , but they generally declined at lower ag - np concentrations and recovered at higher ag - np concentrations . gst is known for its catalysis function in the reduction process of glutathione ( gsh ) , in which endogenous and xenobiotic chemicals are detoxified . the reduction of gst occurs as a result of the overutilization of existing enzymes to overcome oxidative stress caused by ag - nps , which causes an increase in gsh concentration in specific organs . in this study , the carp showed a significant reduction in gst activity in the gills after 48 h of exposure to 200 g / l of ag - nps , but recovered after 96 h of exposure ( figure 2 ) . oxidative stress increased with a reduction in gst concentration ; this may cause the development of toxic effects or even carcinogenic effects . however , higher ag - np concentrations may trigger the production of enzymes to counteract the severe effects of the particles . hence , an increase in gst concentration may be used as an indicator for the depletion of gsh in detoxification processes . the main function of cat is to catalyze the decomposition of h2o2 . in this study , the activity of cat detected in the liver was significantly higher than that in the brain and gills ( figure 3 ) . cat concentration in the liver also declined when the fish were exposed to lower ag - np concentrations and then recovered with an increase in ag - np concentrations . g / l of ag - nps , cat levels in all the examined organs ( i.e. , brain , liver , and gills ) were significantly lower than those in the control group , as well as for fish exposed to 100 g / l of ag - nps ( figure 3(b ) ) . this phenomenon was not observed during the 48 h test under the same concentration . these results indicate that the impact of ag - nps on the fish was more severe under longer periods of exposure . the results of cat and gst activities also indicated that when the fish were exposed to short periods ( 48 h ) of ag - nps at a concentration of < 100 thus , the depletion of this enzyme may indicate that the antioxidant defense system is overwhelmed . in the present study , sod activity in the brain and gills was not significantly different between the control and exposed fish . however , sod activity was significantly lower in the liver of fish exposed to 200 g / l of ag - nps compared to the liver of fish in the control group ( figure 4(a ) ) . it has been suggested that the antioxidant defense system of the liver is severely affected at this concentration , as illustrated by the reduction of other enzymes ( i.e. , cat and gst ) in the liver of fish exposed to lower ag - nps concentrations . however , after 96 h of exposure , sod activity returned to the control level ( figure 4(b ) ) . hence , we hypothesize that gst and cat were produced in sufficient quantities to counteract the oxidative stress caused by ag - nps . however , the correlation and combined effects of gst , cat , and sod , as well as other antioxidant enzymes , require further study . as shown in the results of the biochemical analysis , the concentrations of nh3 and got declined and recovered , similar to that observed for other antioxidant enzymes ( e.g. , cat and gst ) . it is known that nh3 in the blood is associated with hepatic detoxification ( i.e. , the urea cycle ) . therefore , fluctuations in blood nh3 concentrations may be related to the antioxidant defense system . g / l of ag - nps ; hence , changes in the enzyme system may alter the capability of urine transformation . however , other biochemical parameters were not significantly altered . along with slight fluctuations in antioxidant enzymes , this result indicates that < 200 g / l of ag - nps exposure did not have a severe effect on the fish . the histological analysis of the tissues also showed that ag - nps have potential adverse effects on exposed fish . as shown in figure 5 , the histopathology of the skin samples showed an increase in the number and size of mucous and club cells , as well as vacuolation of the epithelium . mucous and club cells located in the epidermal layer of the skin are responsible for the excretion of waste , respiration , ionic and osmotic regulation , disease resistance , communication , and other protection functions . an increase in the number and size of these cells may be a necessary response of the body to counteract the effects of exogenous chemicals , such as nanoparticles . however , if nanoparticle concentrations exceed the resistance level of the skin and excretory systems , they may have a lethal effect on cells . for example , when the fish were exposed to 200 g / l of ag - nps ( as shown in figure 5(d ) ) , vacuolation of the epidermal epithelium was observed . malfunctioning or injured skin may also cause nanoparticles to have a fatal effect on the fish . therefore , in this study , stresses caused by the exposure of common carp to ag - nps may have potentially toxic effects . in this study , however , chronic exposure to ag - nps may cause other types of damage that kill fish . one impact of ag - nps is the inhibition of the oxygen / carbon dioxide exchange process because of injury to the respiratory organ ( gills ) . as shown in figure 6 , the exposure of fish to ag - nps initially caused the bifurcation of the filament ( figure 6(b ) ) , an increase in the number and size of mucous cells ( figure 6(c ) ) , and hyperplasia of the lamellar epithelium ( figure 6(d ) ) . similar to the effects on the skin , these slight changes to the gills did have a severe effect on the fish , but it could result in potentially fatal effects if the carp were exposed to higher concentrations of ag - nps over an extended period . the adverse effects observed in the liver of the exposed carp ( figure 7 ) , such as atrophy of the hepatocyte nucleus and eosinophilic granule formation , may therefore be used as indicators . we used genomic analysis to evaluate changes in gene expression towards ag - np exposure in the liver of common carp , and we subsequently selected several biomarkers of ag - np toxicity . in our microarray data , however , the upregulation of max blocks apoptosis in endothelial cells and is involved in the maintenance of healthy morphology in the retinal ganglion cells of rats , indicating the presence of protective cellular functions . in addition , this observation might indicate that programmed cell death and cell morphology are mediated and orientated by max , leading to cellular defense against ag - np toxicity . in our microarray data , repression of rbp in the epidermis might cause a reduction in the amount of retinol in keratinocytes , indicating cornification of the epidermis . another study documented that the underexpression of rbp is associated with inhibiting differentiation and the formation of large tumors in hepatocellular carcinoma . these observations might indicate that ag - nps cause abnormal retinoid metabolism , resulting in tumorigenesis . in our microarray data , gst has been reported to play a critical role in the detoxification of various types of toxicants ( such as carcinogens ) and has a protective effect on ros - mediated dna damage [ 4145 ] . a recent assessment indicated that gst activity might be useful towards evaluating oxidative stress [ 46 , 47 ] . hence , ag - nps might induce the accumulation of intracellular oxidative stress , giving rise to abnormal biological functions of responsive components , including gst . gene expression level of gst obtained from microarray analysis was found to be downregulated while the gst activity was induced in presence of ag - nps , particularly after 96 h exposure to 100 g / l in common carp liver tissue . possible explanation is that the inhibitory gene expression at mrna level manifests at initial stage due to the ag - nps toxic effect whereas the induction of gst activity would play an important role as defensive mechanism in response to the ag - nps - induced oxidative stress at later stage . at present , while the functions of many common carp genes remain under investigation , the inferred functions of unknown genes might be used as putative bioindicators of ag - np toxicity . the results of the current study indicated that max , gst , and rbp might be viable candidates as ag - np biomarkers . however , detailed study of the toxicity - mediated molecular mechanisms of ag - nps and further functional analysis of common carp genes are necessary to elucidate cellular responses to ag - nps and the detoxification processes . citrate - capped ag nanoparticles dispersed well in freshwater , with an estimated average particle size of 721 nm ( tem ) and 7291 nm ( dls analysis ) . despite the formation of aggregations / agglomerations in the solution , nanosized particles of citrate - ag - nps were dominant . among the examined tissues , liver was the most susceptible to changes in particle concentration . the antioxidant enzyme system was mostly active in the liver . according to the analysis of the antioxidant enzyme system and other biochemical parameters , liver was the most severely affected organ when the carp were exposed to ag - nps . the activities of antioxidant enzymes , such as gst and cat , fluctuated with different ag - np concentrations , whereas sod activity remained stable . the histopathology showed the following : ( 1 ) in the skin : an increase in the number and size of club cells and mucous cells and vacuolation of the epithelium ; ( 2 ) in the gill : bifurcation of the filament , an increase in the number of mucous cells , and hyperplasia of the lamellar epithelium ; ( 3 ) in the liver : atrophy of the hepatocyte nucleus and accumulation of eosinophilic granules . the adverse effects recorded in the current study were not severe enough to cause carp mortality , but they might potentially lead to lethal effects if the fish were exposed to higher concentrations of ag - nps over a longer period .	juvenile common carp ( cyprinus carpio ) were used as a model to investigate acute toxicity and oxidative stress caused by silver nanoparticles ( ag - nps ) . the fish were exposed to different concentrations of ag - nps for 48 h and 96 h. after exposure , antioxidant enzyme levels were measured , including glutathione - s - transferase ( gst ) , superoxidase dismutase , and catalase ( cat ) . other biochemical parameters and histological abnormalities in different tissues ( i.e. , the liver , gills , and brain ) were also examined . the results showed that ag - nps agglomerated in freshwater used during the exposure experiments , with particle size remaining < 100 nm . ag - nps had no lethal effect on fish after 4 days of exposure . biochemical analysis showed that enzymatic activities in the brain of the fish exposed to 200 g / l of ag - nps were significantly reduced . varied antioxidant enzyme activity was recorded in the liver and gills . varied antioxidant enzyme activity was recorded for cat in the liver and gst in the gills of the fish . however , the recovery rate of fish exposed to 200 g / l of ag - nps was slower than when lower particle concentrations were used . other biochemical indices showed no significant difference , except for nh3 and blood urea nitrogen concentrations in fish exposed to 50 g / l of ag - nps . this study provides new evidence about the effects of nanoparticles on aquatic organisms .
You are an expert at summarizing long articles. Proceed to summarize the following text: a 32-year - old married woman presented with complaints of itchy red rash on her forearms and nape of neck for 2 months . she was clinically diagnosed as polymorphic light eruption and started on mild potent topical corticosteroids and antihistaminics , along with instructions for strict photo protection and use of sunscreen . her forearm lesions improved with this treatment , but lesions on the nape of neck had worsened with complaints of significant itching , when the patient came for follow - up 2 weeks later . clinical examination at this point of time revealed presence of annular lichenoid plaques and few lichenoid papules [ figure 1a ] . a skin biopsy was ordered from the lesion on the nape of neck , keeping differentials of annular lichen planus and sarcoidosis . a focal interface infiltrate composed of lymphocytes with a conspicuous presence of the plasma cells as well as eosinophils was seen within the dermis [ figure 2a ] . there was also prominent endothelial inflammation with endothelial cell swelling and red blood cell extravasation [ figure 2b ] . at places , complete obliteration of the vascular lumina ( endarteritis obliterans ) was seen , with presence of plasma cells in the vessel walls [ figure 2b ] . based on these findings , a possibility of syphilis was suggested by the pathologist . about a week later , patient reported to us with new lesions over her genitalia and groin . examination revealed multiple skin coloured to pinkish , flat topped , moist appearing papules in the perianal region , labia majora , pubic region and groin folds , suggestive of condyloma lata [ figure 1c ] . dark ground illumination test performed from the wet lesions of condyloma lata revealed spiral spirochetes with characteristic motility . venereal disease research laboratory assay came out to be positive in the titres of 1:256 for the patient and in 1:8 for her husband . both patient and her husband were treated with a single shot of 2.4 million units of benzathine penicillin after sensitivity testing . all lesions in patient resolved completely within a span of 2 weeks [ figure 1b and d ] . annular lichenoid plaques on the nape of neck ( a ) subsiding completely 2 weeks posttreatment ( b ) . condyloma lata involving groins , labia majora and pubic region ( c ) with complete resolution post injection penicillin ( d ) interface dermatitis with conspicuous presence of plasma cells and eosinophils ( h and e , 100 ) ( a ) ; prominent endarteritis obliterans with complete obliteration of the vascular lumina with plasma cell infiltrate ( arrow ) ( h and e , 400 ) ( b ) syphilis continues to be a public health problem , with various studies showing a rise in its prevalence , in recent years , in india as well as in the west . cutaneous signs are the commonest and most important manifestations of secondary syphilis , with up to 80% of patients developing lesions of the skin or mucous membranes . a dermatologist needs to be aware of the protean mucocutaneous manifestations that secondary syphilis can present with , to avoid misdiagnosis of this infection . however , they were believed to be caused by arsenicals , which formed the mainstay of treatment of syphilis in those days . jeerapaet and ackerman were the first to report lichenoid histological changes in patients of s2 . in 1974 , lochner and pomeranz established the clinical - histopathologic correlation for lichenoid lesions occurring in syphilis . in the penicillin era as well , lichenoid lesions have been reported , albeit occasionally . most of these cases including the present case had associated pruritus , which is otherwise an unusual symptom in s2 per se . annular lichenoid lesions are even rare and have been reported previously on only two occasions . the localized annular lichenoid lesions may present as a diagnostic dilemma , more so when other features of secondary syphilis are absent , as seen on the first visit in the present case . in this situation , a clinician has to entertain other conventional differentials such as lichen planus , annular sarcoidosis and granuloma annulare . in addition , like secondary syphilis , all the aforesaid disorders may also show interface inflammatory infiltrate on histology . however , in the present case , the presence of profuse plasma cell infiltrate and prominent endarteritis strongly suggested a diagnosis of syphilis . further , presence of eosinophils in the infiltrate as in the present case is of rare occurrence . histology mimicking lichenoid drug eruption with the presence of eosinophils and high level civatte bodies has been described by tang et al . the presence of lichenoid lesions in a sexually active individual not responding to conventional therapy should arouse the suspicion of secondary syphilis . a thorough and serial clinical examination and careful histological	syphilis is a disease known for centuries , but still continues to be a diagnostic challenge as the myriad manifestations of secondary syphilis can mimic a lot many dermatological disorders . lichenoid syphilis is an uncommon entity , reported only occasionally in the penicillin era . we present the case of a 32-year - old woman presenting with localized annular lichenoid lesions on the neck .
You are an expert at summarizing long articles. Proceed to summarize the following text: perampanel ( per ) is the first - in - class orally active antagonist of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptors , . perampanel was licensed in europe and the us for the add - on treatment of patients with epilepsy with partial - onset seizures after three phase iii trials that assessed the clinical efficacy and tolerability of per as adjunctive antiepileptic drug ( aed ) in patients with difficult - to - treat partial - onset epileptic seizures , , , . in addition , per was approved for the treatment of patients with primary generalized tonic clonic seizures because superiority over placebo was shown in a randomized controlled trial . in this trial , furthermore , single reports indicated a potential beneficial effect of per when used in an off - label manner in lafora disease , a form of progressive myoclonic epilepsy , . however , our own clinical experience beyond the label and one report on convincing efficacy of per in a case of superrefractory acute posthypoxic myoclonus encouraged us to start add - on per in a chronic case of drug - resistant posthypoxic myoclonus ( lance adams syndrome ) . this 36-year - old male had suffered from drug - resistant posthypoxic myoclonus for one year after a cardiac arrest and reanimation because of cardiologically proven brugada syndrome . on admission , he presented with almost continuous stimulus - sensitive polytopic myoclonic jerks that required the permanent use of a wheelchair . we did not perform magnetic resonance imaging ( mri ) because a pacemaker with defibrillator had been implanted . his medication consisted of 2000 mg of levetiracetam , 1500 mg of valproic acid , 2 mg of clonazepam , and 7600 mg of piracetam . in addition , 100 mg of lacosamide had been added few weeks prior to admission in our hospital . perampanel was initiated at a dose of 2 mg and increased to 4 mg once daily in the evening after three days . since the patient complained about per - related somnolence , he wished to try a withdrawal . the next day , myoclonus was very prominent again and remained over the following five days before we decided to reintroduce per ( 4 mg per day ) . myoclonus was suppressed again and has not been reported for the follow - up of 4 weeks . . they may not only be part of various epilepsy syndromes but also occur as the result of nonepileptic pathogenesis as a cerebellar phenomenon in patients with posthypoxic brain damage which is well - known as so - called lance adams syndrome . although the action myoclonus in lance adams syndrome is usually not of epileptic origin , antiepileptic drugs are recommended for its treatment , , , , . in addition , piracetam has been recommended . because of the nonepileptic origin of the myoclonus , other therapeutic approaches addressed the idea of a movement disorder and suggested the use of l-5-hydroxytryptophan , levodopa , and gabaergic drugs including alcohol , sodium oxybate , baclofen , or deep - brain stimulation , . the prognosis is bad ; the efficacy of aeds is usually limited . in our patient , therefore , it was tempting to speculate if per might be a suitable option since beneficial effects have been reported in single patients with juvenile myoclonic epilepsy and lafora disease , as well as in an acute myoclonic status after severe hypoxia . somnolence and dizziness belong to the most common adverse events under add - on per , . the rather fast up - titration to 4 mg was performed more quickly than generally recommended because we wanted to estimate the potential efficacy of per in this severe case with numerous myoclonic jerks as fast as possible . therefore , somnolence might have been partly caused by this titration regimen . in patients with epilepsy , we discontinued per in our case in spite of the apparent beneficial effect against the myoclonic jerks because of the sedating effects the patient complained about . after discontinuation , myoclonus exaggerated and was suppressed almost completely again when we reintroduced per . interestingly , this time tolerability was better , and so , we assume that a sort of adaptation had happened . because of the reproducible effect of add - on per after reintroduction , we are convinced about its efficacy in our patient and recommend trying it in comparable cases . since patients with lance adams syndrome suffer from very frequent myoclonus , one should be able to judge the efficacy of per rather promptly in spite of the long elimination half - life and the considerable delay until plasma steady state is reached . we hope that our patient will be able to cope with the sedating profile of per . this observation justifies trying add - on per beyond the labeling in difficult - to - treat cases of lance adams syndrome on an individualized basis . bjs has received speaker 's honoraria from desitin , eisai , med update , omnia med , ucb , and viropharma . he was a member of advisory boards of or has consultancy agreements with actelion , b.braun , eisai , shire , and ucb .	perampanel ( per ) is the first - in - class selective , noncompetitive -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor antagonist that has been licensed and marketed as antiepileptic drug ( aed ) indicated for patients with partial - onset and primary generalized tonic clonic seizures . a positive effect was reported in some patients with epileptic myoclonic jerks in idiopathic generalized epilepsy and in progressive myoclonic epilepsy.we treated a male patient with posthypoxic nonepileptic myoclonus ( lance adams syndrome ) with add - on per and achieved an almost complete cessation of jerks . this effect was reproducible and , therefore , we suggest that it might be worth trying per in comparable cases .
You are an expert at summarizing long articles. Proceed to summarize the following text: near - infrared ( nir ) fluorescent probes have been widely used in biological and biomedical research because light in this wavelength region has a large penetration depth in tissue and causes minimal cellular autofluorescence . organic dyes are the most commonly used nir fluorescent probes , despite their non - optimal brightness and photostability . they are extensively used in fluorescence imaging and assays because of their well - established labeling protocols and their molecular dimensions . however , the use of organic nir probes for bioimaging applications encounters several difficulties . for example , organic nir dyes usually consist of large , hydrophobic conjugated systems that may lead to aggregation in physiological environments , which can dramatically reduce their emission quantum yield ( qy ) and broaden their emission spectra as compared with dyes emitting in the visible range . in addition , the stokes shifts for organic nir dyes are generally very small , which is not optimal for imaging applications because cross - talk between the excitation light and fluorescence can make it difficult to capture the pertinent signals . in the past decade , inorganic semiconductor quantum dots ( qdots ) were developed as nir probes because they offer significantly higher photostability and brightness compared to conventional dyes . the potential leakage of heavy metal ions from the qdots and the subsequent cell toxicity , however , are critical concerns for biological applications . semiconducting polymer dots ( pdots ) with small sizes ( < 30 nm ) are a new class of highly fluorescent probes that possess extraordinary brightness , fast emission rates , good photostability , and minimal toxicity to biological cells and tissues . one strategy is based on encapsulation of a hydrophobic nir dye in pdots with visible emission . upon light excitation , the pdots transfer the energy efficiently to the dopant dye , resulting in quenching of visible emission with concomitant nir emission . however , this dye - doping approach can have a dye leakage problem in applications that occur over relatively long periods of time and during storage . another method is to blend a nir - emitting polymer as an acceptor with several semiconducting polymers such as red , green , and blue - emitting polymers as donors that work together via cascade fluorescence resonance energy transfer ( fret ) to generate nir - emitting pdots . the design and synthesis of these polymers for cascade fret are relatively complicated , and much work must be done to optimize the blending ratios of the semiconducting polymers to get high fluorescence qy and efficient energy transfer . many biological applications require that the fluorescent probes possess narrow - band emissions for multiplexed detections . for example , in a wavelength - and - intensity multiplexing scheme for optical encoding , the number of codes increases greatly with increasing number of color and intensity levels . previous studies suggest that it is better to use more colors , rather than more intensity levels , in order to increase the number of usable codes . therefore , development of narrow , bright nir pdots remains an important task for widespread biological applications . this article describes the design and synthesis of squaraine - based pdots that show large stokes shifts and narrow - band emissions in the nir region . the squaraine pdots exhibit an emission bandwidth of 36 nm ( full width at half - maximum , fwhm ) , about 2 times narrower than those of qdots in the same wavelength region . to our knowledge , this is the narrowest emission bandwidth among various pdot species reported so far . the narrow bandwidth , high qy , and large stokes shifts of the squaraine - based pdots are promising for multiplexed biological detections . scheme 1 shows the chemical structures of the squaraine - based polymers ( pfs1 and pfs2 ) and the scheme for forming pdot bioconjugates . the squaraine units were chosen because of their strong absorption and narrow emission band centered in the nir region ( 700720 nm ) . two types of squaraine - based units were synthesized and incorporated into the polyfluorene backbone with different squaraine fluorene ratios ( squaraine monomer for polymer pfs1 and benzene fused squaraine monomer for polymer pfs2 ) . the detailed synthesis procedure of the squaraine monomer and polymer pfs1 is included in schemes s1 and s2 ( supporting information ) . to further shift the emission peak to the nir , a benzene ring fused squaraine monomer was also designed and prepared for polymer synthesis . benzene fused squaraine shows increased conjugation length that generally produces a red - shifted emission . the synthesis procedure for this fused squaraine monomer and polymer pfs2 is shown in scheme s3 and s4 ( supporting information ) . the schematic depicts the process of pdot preparation , bioconjugation , and cellular labeling . we systematically studied the optical properties of the pdots as we varied the monomer ratios , as squaraine concentration greatly affects the emission spectra and qy of the pdots . from the fluorescence spectra ( figure 1 ) , all the copolymers in thf solutions show two emission peaks , around 420 and 700 nm , respectively . the former originates from the polyfluorene backbone and the latter is from the squaraine derivatives . the intensity ratio ( r ) of the nir emission peak to the blue emission peak increases with increasing feeding ratios of squaraine ( table s1 ) . when these polymers were prepared into pdots , the nanoparticles in aqueous solution showed similar trend in spectral evolution with dramatically increased nir emissions relative to the blue emission . however , the qy of the nir emission were also dramatically decreased with the higher feeding ratio of squaraine monomers . ( a ) emission spectrum for squaraine monomer ( sq1 ) in thf ( ex = 600 nm , green ) , and emission spectra of pfs1 polymers in thf solution with different molar ratios of acceptor squaraine ( ex = 405 nm ) : 1.5% ( black ) , 3% ( red ) , 7% ( blue ) , 9.4% ( cyan ) , and 19% ( magenta ) . the inset shows the enlarged region of the fluorescence spectra of 1.5% ( black ) and 3% ( red ) ( b ) emission spectrum for fused squaraine monomer ( sq2 ) in thf ( ex = 620 nm , green ) , and emission spectra of pfs2 polymers in thf solution with different molar ratios of acceptor fused squaraine ( ex = 405 nm ) : 1.5% ( black ) , 5% ( red ) , and 10% ( blue ) . the inset shows the enlarged region of the fluorescence spectra of 1.5% ( black ) and 5% ( red ) pfs2 polymers in the range of 420550 nm . ( c ) emission spectra of different pfs1 pdots made from the above pfs1 polymers in h2o ( ex = 405 nm ) : 1.5% ( black ) , 3% ( red ) , 7% ( green ) , 9.4% ( blue ) , and 19% ( magenta ) of acceptor squaraine . ( d ) emission spectra of different pfs2 pdots made from the above pfs2 polymers in h2o ( ex = 405 nm ) : 1.5% ( black ) , 5% ( red ) , and 10% ( blue ) of fused squaraine acceptors . when the pdots were excited with 405 nm light , we observed strong nir emission , despite the fact that there was almost no spectral overlap ( figure 2 ) between the emission of the energy donor ( polyfluorene ) and the absorption of the acceptor / emitter ( squaraine monomer ) . we believe the mechanism for the efficient energy transfer from polyfluorene to squaraine likely is not based on fret . rather , energy transfer likely occurred first through exciton diffusion along the fluorene polymer backbone followed by through - bond energy transfer to the squaraine monomer . ( a ) absorption spectra of squaraine monomer ( sq1 ) in thf ( red dash ) and pfs1 polymer in thf ( violet dash ) , and emission spectra of pfo polymer in thf ( blue solid ) , pfs1 polymer in thf ( red solid ) , and pfo polymer mixed with 1.5% ( in molar ratio ) sq1 monomer in thf ( cyan dash - dot ) . ( b ) absorption spectra of sq1 monomer doped in amphiphilic ps - peg - cooh ( 20 wt% ) polymer nanoparticle in h2o ( red dash ) , pfs1 pdots in h2o ( violet dash - dot ) , and pfo polymer blended with 1.5% ( in molar ) sq1 monomer pdots in h2o ( black dot ) , and emission spectra of pfo pdots in h2o ( blue solid ) , pfs1 pdots in h2o ( burgundy solid ) , and blended pdots prepared from pfo polymer mixed with 1.5% ( in molar ratio ) sq1 monomer ( red solid ) . ( c ) instrument response function ( irf ) curve ( black ) , lifetime decay curves of the fluorescence from blended sq1/pfo pdots in h2o , which include the lifetime decay for the fluorescence from fluorene groups of pfo polymer in pdots ( blue ) and from squaraine monomer sq1 in pdots ( magenta ) , and lifetime decay curves of the fluorescence from pfs1 pdots in h2o , which include the lifetime decay for the fluorescence from fluorene groups in pfs1 pdots ( cyan ) and from squaraine groups in pfs1 pdots ( red ) . ( d ) lifetime decay curves of the fluorescence from fluorene groups in pfs2 pdots in h2o ( blue ) and from squaraine groups in pfs2 pdots in h2o ( red ) . all fluorescence lifetime measurements were obtained at an excitation from a laser tuned to ex = 375 nm . fluorescence lifetime measurements ( figure 2c , d ) are helpful for clarifying the mechanism of energy transfer processes . we measured the fluorescence lifetimes of different donors and acceptors in blended squaraine sq1/pfo and pfs1 pdots ( figure 2c ) . from the fitted lifetime decay curves , the lifetime values were 0.27 and 0.08 ns for fluorene in blended squaraine sq1/pfo pdots and in pfs1 pdots , respectively ; for squaraine in blended squaraine sq1/pfo pdots and in pfs1 pdots , the values were 2.0 and 2.24 ns , respectively . these values indicate fluorenes in pfs1 pdots have a significantly shorter fluorescence lifetime than those in the blended squaraine sq1/pfo pdots , which is comparable to the published values for pfo pdots . this result suggests energy transfer from the fluorene donor to the squaraine acceptor in pfs1 pdots was much more efficient than that in the blended pdots ; the enhanced intraparticle energy transfer is the dominant factor that made pfs1 pdots strong nir emitters . similarly , in pfs2 pdots , the luminescence lifetime of fluorene donor was also shortened to 0.08 ns , which indicates there was also an enhanced energy transfer in these pdots . in pfs2 pdots , the nir emission from the fused squaraine acceptor also had a longer lifetime of 2.12 ns . table s1 summarizes the photophysical properties of the two sets of pdots with different monomer ratios . for example , the qy of pfs1 pdots decreased from 30% to 2% when the molar ratio of the squaraine chromophore was increased from 1.5% to 10% . similarly , the pfs2 pdots exhibited a decrease in qy from 17% to 1% as the molar content of benzene - fused squaraine increased from 1.5% to 10% . we attribute this phenomenon to aggregation of the squaraine chromophores in pdots , consistent with the slight red - shift observed in the pdot fluorescence spectra . from these experiments , we determined that pdots consisting of 1.5% squaraine offered the best overall performance in terms of the emission bandwidth and brightness . figure 3 shows the spectroscopic and colloidal properties for the final optimized form of the two types of pdots . an average diameter of 18 nm was determined for both pdots by dynamic light scattering ( dls , figure 3e , f ) , and further confirmed by transmission electron microscopy ( tem , figure 3c , d ) . from the absorption spectra , both pdots showed strong absorption peaks around 375 nm due to the polyfluorene backbone . the fluorescence spectra show strong emission centers at 693 nm for pfs1 pdots , and 711 nm for pfs2 pdots . both pdots show large stokes shifts of over 320 nm and exhibit narrow emission bandwidths ( fwhm 36 nm ) , which is comparable to or narrower than most nir organic dyes , and about 2 time narrower than that of qdots emitting in similar wavelength region ( 66 nm for commercially available qdot705 ) . because the emission spectra of pdots show a tail at longer wavelengths , we also compared the full width at 10% of maximal intensity between pfs1 pdot and qdot705 ( figure s1 ) ; here , pdot is still 37 nm narrower than qdot . the narrow emission bandwidth is important for preventing emission cross - talk among probes , particularly for multicolor labeling and multiplexed biological detections . absorption and emission spectra ( ex = 380 nm ) of ( a ) pfs1 and ( b ) pfs2 pdots in water . tem images for ( c ) pfs1 pdots and ( d ) pfs2 pdots ; scale bar is 50 nm . histograms of size distribution measured by dls for ( e ) pfs1 pdots and ( f ) pfs2 pdots ; both show an average diameter of 18 nm . for using pdots as fluorescent labels , the final brightness of pdots can be estimated by the product of the peak absorption cross - section and the fluorescence qy of the nir emission of the pdots . the absorption cross - section is the light - harvesting capability of a fluorescent probe at a given wavelength , which can be determined from the absorption spectra . an analysis of the uv vis spectra at known pfs1 and pfs2 pdot concentrations shows the per - particle absorption cross sections of the pdots ( 18 nm in diameter ) at 405 nm were 2.4 10 cm for pfs1 pdots and 1.9 10 cm for pfs2 pdots , which are close to the reported values of other types of pdots . we performed single - particle imaging to experimentally compare the single - particle brightness between qdot705 and the pdots under identical excitation and collection conditions . the pfs1 pdots , pfs2 pdots , and qdot705 were excited with a 405 nm laser , and a long - pass filter of 685 nm was used . figure 4 shows the single - particle images of the three nanoparticles ; intensity histograms were obtained by analyzing hundreds of nanoparticles from each sample . we found pfs1 pdots were about six times brighter than qdot705 and pfs2 pdots were about three times brighter than qdot705 , consistent with their measured photophyiscal parameters . single - particle fluorescence images of ( a ) qdot705 , ( b ) pfs1 pdots , and ( c ) pfs2 pdots obtained with a 405 nm laser using the same excitation power and under identical detection conditions . histograms of the distributions of single - particle brightness of ( d ) qdots705 , ( e ) pfs1 pdots , and ( f ) pfs2 pdots . the black curves were obtained by fitting a log - normal function to the histograms , which gave mean brightness of 4500 , 25 300 , and 13 300 ccd counts for qdot705 and pfs1 and pfs2 pdots , respectively . to use the nir - emitting pdots for biological labeling and targeting , we functionalized the pdot surface with carboxyl groups and carried out bioconjugation with streptavidin using edc - catalyzed coupling as described in our previous reports ( scheme 1 ) . pdot - streptavidin ( pdot - sa ) was used to label cell - surface markers on mcf-7 breast cancer cells . the cells were sequentially incubated with biotinylated primary anti - epcam antibody and pdot - sa probes . flow cytometry was performed to examine the specificity and brightness of labeling by the pdot - streptavidin probes ( figure 5 ) . the labeled cells exhibited stronger fluorescence compared with the control samples , indicating the two pdots effectively and specifically labeled epcam receptors on the cell surface with excellent brightness . the negative control samples were prepared under identical conditions as the positive ones but without primary biotinylated antibodies . we compared the pfs1 and pfs2 pdots - sa with commercial qdot705-sa under the same labeling concentrations and identical experimental conditions ( figure 5 ) . the results indicate pfs1-sa and pfs2-sa pdots were about six and three times brighter than that of qdot705-sa , respectively , which are consistent with the single - particle brightness measurements shown in figure 4 . from the negative control samples , it can be seen that both pdot probes also exhibited less non - specific binding to cells than the qdot probe . flow cytometry measurements of the labeling intensity of mcf-7 breast - cancer cells labeled with qdot705-streptavidin , pfs1 pdot - streptavidin , and pfs2 pdot - streptavidin in the presence ( positive labeling , p ) and absence ( negative control , n ) of biotinylated primary antibody against the cell - surface protein epcam . ( a ) comparison between qdot705 and pfs1 pdots ; fluorescence of labeled cells was collected with a 695/40 nm band - pass filter ; excitation at 405 nm . ( b ) comparison between qdot705 and pfs2 pdots ; fluorescence of labeled cells was collected with a 710/20 nm band - pass filter ; excitation at 405 nm . we further confirmed specific cellular labeling with pfs1 pdot - sa and pfs2 pdot - sa probes by confocal fluorescence imaging ( figure 6 ) . these results also show pfs1 ( figure 6a ) and pfs2 pdot - sa ( figure 6b ) probes effectively and specifically labeled epcam receptors on the mcf-7 cell surface ; in the equivalent negative control experiments ( data not shown ) where the primary biotinylated antibody was absent , no fluorescence was detected , again indicating little or no non - specific binding of the pdots to the cells . confocal fluorescence microscopy images of mcf-7 cells labeled with ( a ) pfs1 pdot - streptavidin via binding to biotinylated anti - epcam primary antibody and ( b ) pfs2 pdot - streptavidin via binding to biotinylated anti - epcam primary antibody . from left to right : fluorescence images , nomarski images , and merged fluorescence and nomarski images ; scale bars are 50 m . in summary , we successfully developed two types of near - infrared - emitting polymer dots with large stokes shift based on narrow - band emissive squaraine derivatives . rather than fret , we believe that efficient exciton diffusion along the fluorene polymer followed by through - bond energy transfer occurred in the pfs pdots . this mechanism enabled us to develop nir - emitting pdots with large stokes shift , high quantum yield , and narrow emission bandwidth , even in the absence of significant spectral overlap between energy donor and acceptor . single - particle brightness measurements and flow - cytometry experiments both indicate that the pfs pdots can be up to 6 times brighter when compared with qdot705 , while the emission bandwidths of the pfs pdots are about 2 times narrower than that of qdot705 . these superior performance features should make pfs pdots a valuable new addition in the near - ir region for multiplex biological imaging and analysis .	this article describes the design and development of squaraine - based semiconducting polymer dots ( pdots ) that show large stokes shifts and narrow - band emissions in the near - infrared ( nir ) region . fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the pdots , where exciton diffusion and likely through - bond energy transfer led to highly bright and narrow - band nir emissions . the resulting pdots exhibit the emission full width at half - maximum of 36 nm , which is 2 times narrower than those of inorganic quantum dots in the same wavelength region ( 66 nm for qdot705 ) . the squaraine - based pdots show a high fluorescence quantum yield ( qy ) of 0.30 and a large stokes shift of 340 nm . single - particle analysis indicates that the average per - particle brightness of the pdots is 6 times higher than that of qdot705 . we demonstrate bioconjugation of the squaraine pdots and employ the pdot bioconjugates in flow cytometry and cellular imaging applications . our results suggest that the narrow bandwidth , high qy , and large stokes shift are promising for multiplexed biological detections .
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Proceed to summarize the following text: neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections . invading microorganisms there , neutrophils destroy the microorganism by a series of mechanisms , mainly phagocytosis , release of antimicrobial substances , and the formation of neutrophil extracellular traps ( nets ) . activated neutrophils also release proteinases into the surrounding tissue , causing damage to the host . in addition , neutrophils are capable of producing many cytokines and chemokines , which can influence the inflammatory response , as well as the immune response [ 4 , 5 ] . besides this classical role in antimicrobial functions , neutrophils early studies suggested that these tumor - associated neutrophils ( tans ) were mere bystanders because it was hard to imagine that neutrophils , being short - lived cells , could have an effect on chronic and progressive diseases such as cancer . however , more recently it is becoming clear that tans have relevant roles in malignant disease . this renewed interest comes in part from the recognition that cancer - related inflammation is an important feature for the development of many tumors and it is a hallmark of cancer . the various antimicrobial and cytotoxic compounds contained in granules can destroy malignant cells , and cytokines and chemokines secreted by neutrophils can also recruit other cells with antitumor activity [ 5 , 9 ] . however , an increasing number of clinical observations and laboratory studies have shown that presence of neutrophils in tumors correlates with poor prognosis . this has been well documented for bronchoalveolar carcinoma , melanoma , renal carcinoma , and head and neck squamous cell carcinoma ( hnscc ) . in all these cases , tans are different from circulating neutrophils ( as discussed later ) , and , in untreated tumors of murine models , they can display a protumorigenic phenotype . the mechanisms for this phenotype are just beginning to be elucidated , but some of them involve genotoxicity , angiogenesis , and immunosuppression . these two types of tans described in mice have been named n1 and n2 in a similar manner as antitumor and protumor macrophages ( tams ) . it is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor , for neutrophils support to the tumor , and for neutrophil activation to enhance their antitumor functions and in the future improve cancer immunotherapy . our knowledge on the role of neutrophils in human cancers is relatively small . from an initial interest in the 1980s , the number of publications on neutrophils in cancer - related studies has been steadily going down . however , this trend is now beginning to change with the realization that neutrophils are indeed important players in cancer development , as reflected by several recent reviews [ 1618 ] , and as we will see next . in many patients with advanced cancer , elevated counts of neutrophils in blood are found . how tumors induce neutrophilia is uncertain , but production of granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a possible mechanism in several types of cancer . in addition , other cytokines such as granulocyte colony - stimulating factor ( g - csf ) , interleukin- ( il- ) 1 , and il-6 produced by tumors seem to contribute to elevated neutrophil numbers in blood . this neutrophilia is associated with poor prognosis in several types of cancers , such as lung , melanoma , and renal carcinomas [ 11 , 21 , 22 ] . in agreement with this , the presence of neutrophils within certain tumors seems also to be an indicator of poor prognosis . reduced recurrence - free time and overall survival were reported for neutrophil - infiltrated tumors in renal carcinomas , hnscc , pancreatic adenocarcinomas , and liver carcinoma . because neutrophilia is frequently associated with inflammatory responses to infections and tissue damage , neutrophilia represents evidence for the concept of cancer - related inflammation inducing tumor progression . the relation of neutrophil numbers in blood to other leukocyte counts has been suggested to serve as a prognostic factor for cancer . thus , the neutrophil - to - lymphocyte ratio ( nlr ) was introduced as prognostic factor for colorectal cancer . due to its simplicity , nlr has shown to be a readily available and inexpensive biomarker for many types of tumors including non - small - cell lung cancer , hepatocellular carcinoma , nasopharyngeal carcinoma , colorectal cancer , melanoma , and breast cancer [ 29 , 30 ] . in general , the blood nlr is elevated in patients with more advanced or aggressive disease , as indicated by increased tumor size , nodal stage , and number of metastatic lesions . also , a high nlr correlates with adverse overall survival in many solid tumors [ 32 , 33 ] . despite the clinical evidence from the many studies mentioned above , neutrophilia ( larger numbers of neutrophils in blood as a consequence of elevated egress of cells from the bone marrow ) is not always a bad indicator for cancer progression . in some types of tumors , for example in fact , the capacity of neutrophils to directly kill tumor cells both in vitro and in vivo was reported long time ago [ 3537 ] . also , neutrophils from tumor - bearing animals were reported to have enhanced cytotoxic activity [ 38 , 39 ] . and recently , neutrophils isolated from blood of some healthy individuals presented direct cytotoxicity against several tumor cell lines . therefore , the exact role of neutrophils within the tumor is a controversial matter [ 14 , 41 ] . in addition to the elevated number of neutrophils in blood , an increase in the frequency of immature myeloid cells at earlier stages of differentiation has also been detected in several types of tumors , including terminal patients with lung , breast , and gastrointestinal cancer . these immature cells consist of a heterogeneous population of immunosuppressive cells defined as myeloid - derived suppressor cells ( mdscs ) . these mdscs can be divided phenotypically into granulocytic ( g - mdsc ) and monocytic ( mo - mdsc ) subgroups [ 45 , 46 ] and are found in great numbers in the spleens of tumor - bearing animals , where they display an immunosuppressive phenotype helping tumor progression [ 47 , 48 ] . the g - mdscs have immature neutrophil morphology and the consensus phenotype cd33/cd11b / hla - dr / cd15 in humans . they have been found in peripheral blood of patients with glioblastoma , multiple myeloma , hodgkin lymphoma , or head and neck cancer . the main mechanism involves production of reactive oxygen species ( ros ) by the respiratory burst of these cells . in advanced cancer patients , the hydrogen peroxide ( h2o2 ) produced by activated granulocytes reduced expression of the t cell receptor ( tcr ) cd3 chain and decreased cytokine production by patients ' t cells . these oxidized human t cells had defective chemotaxis and presented impaired f - actin remodeling . the effect was found to be mediated by oxidation of the actin - remodeling protein cofilin . cofilin is activated through dephosphorylation at ser3 , and then it mediates severing and depolymerization of f - actin for formation of the immune synapse and t cell activation . cofilin oxidation induced formation of an intramolecular disulfide bridge that prevents its activation , thus leading to impaired t cell activation . also , long - term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic - like programmed cell death takes place in human t cells . in addition , exposure of ros to memory / effector cd45ro t cells results in inhibition of nf-b activation and reduction in th1 cytokines production . furthermore , mdsc - produced ros can lead to cd8 t cell tolerance by another mechanism involving peroxynitrite . ros can combine with nitric oxide and form peroxynitrite , which is highly reactive at short distances . during mdsc - t cell contact , this process makes cd8-expressing t cells unable to bind peptide - mhc complexes and to respond to the specific peptide . another mechanism for t cell suppression is production of arginase 1 ( arg1 ) by mdscs . arg1 inhibits t cell proliferation by degrading extracellular arginine , which results in decreased responsiveness of t cells to cd3/tcr stimulation . for example , in patients with non - small cell lung cancer , tans had reduced intracellular arg1 and tumor - infiltrating lymphocytes showed reduced proliferation in response to cd3/tcr stimulation . all non - small cell lung cancer cell lines secreted il-8 , and il-8 was effective in triggering arg1 release . also , in patients with glioblastoma , degranulated neutrophils associated with elevated levels of serum arg1 correlated with decreased t cell cd3 zeta chain expression in peripheral blood t cells , resulting in immunosuppression . together , these mechanisms explain how mdsc - produced ros and arg1 mediate t cell suppression in cancer settings . because , g - mdscs share many properties with neutrophils but seem to be functionally different from mature neutrophils [ 13 , 62 ] , a transcriptomic analysis was conducted to compare in tumor - bearing animals circulating neutrophils with tans and with mdscs . however , at present it is not clear whether tans are mature neutrophils or represent a special category of cells such as immature neutrophils with protumor properties . depending on the phenotype displayed by tans , they have been classified in tumor - bearing mice as n1 or n2 . similarly to antitumor tumor - infiltrating macrophages ( m1 ) , n1 cells display proinflammatory and antitumorigenic functions . tans seem to be different from circulating neutrophils and also from g - mdsc in the bone marrow and spleen [ 44 , 63 ] . upon transforming growth factor - beta ( tgf- ) blockade , murine cd11b / ly6 g neutrophils recruited to tumors were hypersegmented and more cytotoxic to tumor cells and expressed higher levels of proinflammatory cytokines . in contrast , depletion of these neutrophils decreased tumor growth and resulted in more activated cd8 t cells intratumorally . thus , it seems that tgf- within the tumor microenvironment induces a population of tans with a protumor phenotype . in support of this idea , in two models of murine tumor cancer cell lines ( lewis lung carcinoma and ab12 mesothelioma ) , neutrophils were found primarily at the periphery of the tumor at early stages of tumor development . these tans were more cytotoxic toward tumor cells and produced higher levels of tumor necrosis factor - alpha ( tnf- ) , no , and h2o2 . in contrast , tans in established tumors had these functions downregulated and presented a more protumorigenic phenotype . these results showed that neutrophils enter the tumor and become more protumor with tumor progression . therefore , murine tans can have an antitumorigenic ( n1 ) phenotype but also a protumorigenic ( n2 ) phenotype capable of supporting tumor growth and suppressing the antitumor immune responses [ 14 , 41 ] , depending on the tumor microenvironment . despite this classification , the nature and function of tans in the cancer microenvironment remain largely unknown , particularly with human tumors . however , two recent publications describe the phenotype of neutrophils infiltrated into human tumors . in one study of surgically resected lung cancer patients , tans were isolated from digested human lung tumors and constituted 5%25% of the cells in the tumor . these tans presented an activated phenotype ( cd62l / cd54 ) with expression of a distinct repertoire of chemokine receptors that included ccr5 , ccr7 , cxcr3 , and cxcr4 . in addition , tans produced larger quantities of the proinflammatory factors mcp-1 , il-8 , mip-1 , and il-6 than blood neutrophils did . these results indicate that , in the earliest stages of lung cancer , tans are not immunosuppressive but rather stimulate t cell responses . in the second study , the role of chronic inflammation , particularly via il-23 and il-17 , in developing human colorectal cancer authors found that innate t ( t17 ) cells were the major cellular source of il-17 in colorectal cancer . tumor growth led to epithelial barrier disruption allowing microbial products to induce inflammatory dendritic cell accumulation and t17 polarization in human tumors . these activated dendritic cells induced t17 cells to secrete il-8 , tnf- , and gm - csf , thus leading to accumulation of neutrophils in the tumor . these tans were characterized by cd45/lin / hladr / cd11b / cd33/cd66b and displayed typical polymorphonuclear morphology . thus , they were described as g - mdsc . these tans ( g - mdsc ) produced much more arg1 and ros than autologous neutrophils and inhibited proliferation of activated autologous t cells and ifn- production . the tans described in these reports show that in human tumors the dual role of neutrophils is also observed . in early tumors , tans seem to be able to stimulate t cell responses , but later in established tumors tans are immunosuppressive . these important reports are just the tip of the iceberg in our understanding of the origin and function of tans . many questions remain for example , are tans in early tumors mature neutrophils with antitumor properties and tans in established tumors immature cells ( g - mdscs ) with immunosuppressive properties directly recruited from the circulation ? or are tans mature neutrophils that develop a more protumor phenotype with tumor progression ? as suggested by several tumor animal models and cancer patients [ 17 , 64 ] . a very recent publication identifies several subpopulations of neutrophils in the blood of tumor - bearing mice and in human cancer patients and describes several relationships of these cells in connection to cancer progression . in this study , the other subpopulation has lower density neutrophils ( ldns ) that copurify with the low - density mononuclear cells layer formed when separating leukocytes by density gradient centrifugation . in tumor - free mice , most neutrophils were hdns , but in tumor - bearing animals ldns increased progressively and often became the dominant neutrophil type in circulation . the hdns from cancer animals , which were previously reported as tumor - entrained neutrophils ( tens ) , displayed high cytotoxicity toward tumor cells in culture , whereas ldns were not cytotoxic . also , the ldns had reduced expression of various chemokines ( cxcl1 , cxcl2 , cxcl10 , ccl2 , and ccl3 ) and chemokine receptors ( cxcr2 and ccr5 ) , consistent with a reduced inflammatory state . the ldn subpopulation consists of large mature ( fully formed lobulated nucleus ) neutrophils and also of immature neutrophils , similar to g - mdsc . the authors then showed by brdu labeling that ldns rapidly accumulate in the circulation , whereas hdns appear in the circulation much later . this is consistent with the idea that some of the ldns are indeed immature neutrophils . in addition , authors showed that hdns are capable of becoming ldns upon treatment with tgf- . it is interesting to note that tgf- was able to induce the change of hdns from tumor - bearing mice into ldns , but it had no effect on hdns from tumor - free mice . this indicates that other stimuli are also needed for this change in animals with cancer . for example , treatment of nave healthy mice with recombinant g - csf protein elicited g - mdsc similar to those induced in tumor - bearing animals . together , all these results support a model proposed by the authors , in which neutrophils are present in three subpopulations in cancer : normal high - density neutrophils , immature low - density neutrophils ( g - mdsc ) , and large mature low - density neutrophils . while the hdns are antitumor and the ldns are protumor , they can change under the influence of the different chemokines and cytokines in the tumor microenvironment . solid tumors are composed of several cell types , including tumor cells and stromal cells . the tumor stroma contains fibroblasts , endothelial cells of blood vessels , and in many cases immune cells . these tumor - infiltrating immune cells highlight the inflammatory microenvironment that is commonly associated with tumor progression . in addition to lymphocytes and macrophages , neutrophils are found in great numbers in a wide variety of tumors [ 12 , 13 , 23 , 24 ] . clearly , neutrophils are recruited to the tumor by the action of neutrophil - attracting chemokines that can be produced not only by other immune cells but also directly by several tumor cells ( figure 1 ) . it was found that oncogenic ras induced il-8 expression , and ras - expressing mouse adenomas produced kc / cxcl1 and mip-2/cxcl2 , the murine equivalents of il-8 , to attract tans . accordingly , increased il-8 levels were found in hnscc patients , and elimination of neutrophils in cancer murine models reduced tumor burden and metastasis . deleting il-8 receptors also reduced tumor growth . these findings support the notion that tumor - produced il-8 is important for neutrophil recruitment to help tumor progression . however , the il-8 receptors cxcr1 and cxcr2 are also expressed on other cell types including endothelial cells and tumor cells . thus , determining the extent of neutrophil involvement in il-8-mediated tumor progression will require future studies . using the same cxcr1 and cxcr2 receptors , neutrophils can also respond to other chemokines such as cxcl1 , cxcl2 , cxcl5 , cxcl6 , and cxcl7 ( figure 1 ) . cxcl2 can induce neutrophil infiltration in tumors , and it was suggested that this is an autocrine effect . supporting this idea , it was also found that in tans the expression of cxcl2 , and also cxcl1 , was upregulated more than 150-fold . therefore , it seems that neutrophils activate a positive feedback mechanism by releasing neutrophil chemokines that attract more neutrophils into the tumor , similarly to neutrophil recruitment into sites of infection . the role of ena-78/cxcl5 in appearance of tans in carcinoma of the liver was investigated in 919 patients with hepatocellular carcinoma . cxcl5 was found to be overexpressed in patients with recurrent tumors , and the levels of cxcl5 correlated with greater appearance of tans and with shorter overall survival . another chemokine that also participates in neutrophil recruitment to tumors is gcp-2/cxcl6 . in a melanoma mouse model , specific anti - cxcl6 monoclonal antibodies reduced the number of tans and also tumor size . in addition , migration inhibitory factor ( mif ) , another tumor - derived chemokine for neutrophils , was identified in hnscc tumors . mif was described as an inhibitor of macrophage migration in vitro , but it is now known that it also binds cxcr2 ( figure 1 ) . tumor - derived mif levels correlated with higher tans levels and poor survival of these patients . many tumor cells can directly produce chemokines for neutrophils , but various other cells within the tumor may also be the source for these chemokines and other cytokines . in particular , activated t cells are known to produce gm - csf , cxcl1 , cxcl2 , tnf- , and ifn- . although the influence of activated t cells in neutrophil recruitment to tumors is not known , regulatory t cells ( treg ) seem to be important for neutrophil infiltrating tumors . in one study , treg were found to inhibit neutrophil recruitment to a tumor site by reducing the expression of cxcl1 and cxcl2 . in contrast , in another study , treg promoted neutrophil infiltration to tumors by producing il-8 . thus , the influence of t cell function on the appearance of tans needs to be further explored . murine tans secrete ccl17 , a potent chemokine for treg , at higher levels than circulating or splenic neutrophils ( figure 1 ) . moreover , the amounts of ccl17 increased progressively during tumor progression . a large body of clinical evidence indicates that neutrophils are involved in cancer development and tumor progression . in most cases , large numbers of tans are associated with advanced disease and poor prognosis for cancer patients . this negative association has been reported for several solid tumors , such as melanoma , hepatocellular carcinoma , non - small cell lung carcinoma , glioma , hnscc , adenocarcinoma , and colon cancer [ 41 , 88 ] . these functions involve the same molecules neutrophils use to destroy microorganisms and to modulate inflammation . important molecules that can modify growth and invasiveness of tumors involve granule proteins , matrix - degrading proteinases , reactive oxygen species ( ros ) , chemokines , and cytokines . recent reports describe how tans use these molecules to affect cell proliferation , angiogenesis , metastasis , and immune surveillance ( figure 2 ) . neutrophil elastase ( ne ) is a major protein of azurophilic granules that is released upon cell degranulation . the main physiologic function of ne seems to be elimination of invading microorganisms , but it also has important inflammatory effects . ne is a serine protease with a broad range of substrates ; among them are neutrophil - derived antibacterial proteins , extracellular matrix proteins , integrins , cytokines , and cytokine receptors . in addition to its roles in inflammation and bacteria destruction , ne has presented various protumor effects both in vivo and in vitro . ne was found to directly promote a459 tumor cell proliferation when murine neutrophils were cocultured with this lung carcinoma cell line . this effect was markedly reduced when tumor cells were cocultured with ne neutrophils , or in the presence of an ne inhibitor . the effect of ne on tumor growth was dependent on phosphatidylinositol 3-kinase ( pi-3k ) , since it was also reduced in the presence of a pi-3k inhibitor . staining experiments showed that ne got inside the tumor cells via clathrin - coated pits and localized at early endosomes . once inside the cell , ne acted on insulin receptor substrate-1 ( irs-1 ) . because irs-1 binds to the regulatory unit of pi-3k , its degradation by ne led to more pi-3k available to enhance the proliferation pathway . similar results have been found with other types of tumor cells , including esophageal cancer , gastric cancer , and breast cancer . in these cases , ne mediated release of transforming growth factor - alpha ( tgf- ) from the cell surface . coculture of human neutrophils with pancreatic ductal adenocarcinoma cells ( pdac ) resulted in dyshesion of cells from the monolayer . the same effect was observed by adding ne to pdac cultures and correlated with loss of surface expression of e - cadherin . cathepsin g is a peptidase from azurophilic granules that participates in degradation of phagocytosed microorganisms and in remodeling of extracellular matrix ( ecm ) proteins . first cathepsin g binds to the tumor cell surface , independently of its catalytic site , and then induces cell aggregation , which is dependent on its enzymatic activity ( figure 2 ) . cathepsin g degrades ecm molecules such as fibronectin and attenuates binding between integrins and fibronectin . this leads to e - cadherin - mediated homotypic cell - cell adhesion , which is protease - resistant . the formation of these tumor cell aggregates would allow tumor cells to disseminate via the circulation to distant sites and establish new metastases . once at the new site , tumor cells would need new vasculature . in a model of breast cancer metastasis to the bone , it was also found that cathepsin g enhanced tgf- signaling and upregulated vascular endothelial growth factor ( vegf ) to promote angiogenesis . together , these reports indicate that tans - derived cathepsin g may induce ecm remodeling and promote tumor progression and metastasis [ 102 , 103 ] . matrix metalloproteinase-9 ( mmp-9/gelatinase b ) is released from secondary ( specific ) granules and is believed to help neutrophils in the process of extravasation via degradation of ecm proteins . mmp-9 was found to promote tumor proliferation in a human papilloma virus- ( hpv- ) 16 skin carcinogenesis model . mmp-9 mice showed reduced keratinocyte proliferation , but this phenotype was reversed when bone marrow - derived leukocytes were transplanted into irradiated mice . also , immunostaining of mmp-9 in squamous cell carcinoma tumors showed that mmp-9 was found only in tumor infiltrating leukocytes and not in tumor cells . in addition , mmp-9 has been shown to inhibit apoptosis of tumor cells in the lung . thus , mmp-9 supplied by bone marrow - derived cells is responsible for enhancing tumor proliferation via both increased proliferation and reduced apoptosis of tumor cells . the vascular endothelial growth factor ( vegf ) is sequestered in the ecm after it is produced by cells ( figure 2 ) . the proteolytic release of vegf from tissue ecm via mmps is regarded as a prerequisite for in vivo induced angiogenesis [ 106 , 107 ] . melanoma cells were transfected to overexpress the gcp-2/cxcl6 chemokine and then implanted into nude mice . the new cxcl6-melanoma tumors grew larger and with a well - developed vasculature than wild type ( wt ) melanomas . these larger tumors also presented higher levels of mmp-9 and induced a strong influx of tans . similarly , in a model of pancreatic adenocarcinoma , new dysplastic lesions that develop into carcinomas are formed with enhanced angiogenesis . this process has been named the angiogenic switch . in these new lesions , mmp-2 and mmp-9 were upregulated . mmp inhibitors and genetic ablation of mmp-9 reduced the angiogenic switching , tumor number , and tumor growth , indicating that mmp-9 can render normal islets angiogenic . in addition , malignant keratinocyte transplantation resulted in tumors with neutrophils expressing predominantly mmp-9 and stromal cells expressing mainly mmp-2 and mmp-3 . these reports suggested a direct role for mmp-9 in tumor angiogenesis , but they did not identify the cell type producing this protease . reconstitution of tumor - bearing mmp-9 mice with wild type , mmp-9-competent hematopoietic cells demonstrated that tumor - infiltrating myeloid cells were the source for mmp-9 [ 111 , 112 ] . in a murine model of pancreatic islet carcinogenesis , mmp-9-expressing neutrophils were predominantly found inside angiogenic islet dysplasias and tumors , whereas mmp-9-expressing macrophages were localized along the periphery of such lesions . transient depletion of neutrophils significantly reduced the frequency of initial angiogenic switching in dysplasias . also tans in melanoma or fibrosarcoma tumors expressed high levels of mmp-9 and vegf , and elimination of these tans resulted in reduced tumor growth . also , reducing tans in prostate carcinoma tumors reduced angiogenesis and tumor cell intravasation . moreover , in cancer patients , neutrophils expressing high levels of mmp-9 have also been found . in hnscc , expression of mmp-9 was larger by tans than by any other cell type in the tumor , and in hepatocellular carcinoma larger numbers of tans correlated with more angiogenesis [ 117 , 118 ] . direct proof for neutrophils being the major tumor - associated leukocyte type expressing mmp-9 was recently provided in a study employing human xenografts and syngeneic murine tumors . when tumors or isolated tams and tans were double - stained for mmp-9 and for respective macrophage- or neutrophil - specific antigens , only tans gave a strong signal for mmp-9 [ 119 , 120 ] . in addition , it was calculated that 1 10 neutrophils or tans could release approximately 100200 ng prommp-9 within 1 - 2 h of incubation . in contrast , 1 10 macrophages or tams would require several weeks to produce the same amount of prommp-9 [ 119 , 120 ] . hence , neutrophil - derived mmp-9 is responsible for enhancing angiogenesis via release of vegf from the ecm in many types of tumors ( figure 2 ) . the unusual angiogenic potency of neutrophil mmp-9 is related to its unique way of production . in other cell types , the zymogen prommp-9 is released together with the inhibitor of metalloprotease 1 ( timp-1 ) , which slows the activation of mmp-9 and can also inhibit the proteolytic activity of the once activated enzyme . therefore , the timp-1-free prommp-9 from neutrophils can be activated easier and function much longer than mmp-9 from other cell types [ 122 , 123 ] . neutrophils are efficient producers of reactive oxygen species ( ros ) for destruction of microorganisms . first , neutrophils generate hydrogen peroxide ( h2o2 ) , which is next converted to hypochlorous acid ( hocl ) by myeloperoxidase ( mpo ) . hocl can then activate several ecm - degrading mmps , including mmp-2 , mmp-7 , mmp-8 , and mmp-9 . also , hocl can block timp-1 and in this manner potentiate the proteolytic activity of mmps [ 124 , 125 ] . finally , as indicated above , mmp activity leads to enhanced tumor progression by inducing proliferation and angiogenesis . nevertheless , a more potent and direct effect of ros on tumor cells is genotoxicity , which might lead to carcinogenesis ( figure 2 ) . although neutrophil - derived ros and hocl can directly damage and destroy tumor cells , they can also cause genotoxicity in circumstances when they do not kill cells . ros - mediated genotoxicity is induced by two major pathways : oxidative dna damage and mpo catalyzed activation of chemical carcinogens . point mutations and dna strand breaks are induced in many different cell types when cocultured with neutrophils , and hocl has been reported to be mutagenic in lung epithelial a549 cells . upon release from neutrophil granules , arg1 gets activated to degrade extracellular arginine , an essential amino acid for proper activation of t cells . thus , degranulation of neutrophils may exert an immunosuppressive effect in tumors by inhibiting t cells in a similar manner to the one described for g - mdsc . in fact , depletion of tans in tumor - bearing animals increased the numbers of activated cd8 t cells and promoted smaller tumors . similarly , non - small cell lung cancer cells stimulated neutrophils through il-8 to release arg1 , and in tumors tans had reduced levels of arg1 . more recently , the same group found that arg1 released from gelatinase granules was inactive at physiological ph unless activated by factor(s ) stored in azurophil granules . thus , tans can induce arg1-dependent immunosuppression through concomitant exocytosis of gelatinase and azurophil granules ( figure 2 ) . neutrophils can also produce cytokines or growth factors , which increase the tumorigenic potential of cancer cells . two clear examples have been described for oncostatin - m [ 128130 ] and for hepatocyte growth factor [ 10 , 131 , 132 ] . breast cancer cells can stimulate neutrophils to release oncostatin - m , an il-6-like cytokine . oncostatin - m in turn stimulated breast cancer cells to secrete vegf ( figure 2 ) . similarly , hepatocellular carcinoma cells stimulated neutrophils to release hepatocyte growth factor ( hgf ) . in turn , hgf stimulated tumor cells to become more invasive ( figure 2 ) . neutrophils can also influence the migration potential of cancer cells . in several types of cancer these tumors include skin squamous cell carcinoma , melanoma , adenocarcinomas , hnscc , and breast cancer . the way neutrophils augment the migratory capacity of tumor cells involves many different mechanisms that are just beginning to be elucidated . in hnscc , tumor - derived mif not only recruits tans but also induced these cells to display promigratory effects on the tumor cells . similar responses have been documented for different cancer cell lines but through a different mediator . various tumor cells release hyaluronan , which can then activate neutrophils via tlr4 and the pi-3k / akt signaling pathway . in turn , neutrophils induce enhanced migration of the tumor cells . very early reports suggested that tans release enzymes that degrade the basement membrane and promote tumor cell invasion through the basement membrane ( figure 3 ) . in vitro studies showed that human neutrophils assist the human breast tumor cell line mda - mb-231 to cross a monolayer of endothelial cells . tumor cell - conditioned medium downregulated neutrophil cytotoxicity and upregulated expression of adhesion molecules , facilitating tumor cell migration . also , in the presence of neutrophils , melanoma cell adhesion and transmigration through an endothelial cell monolayer were increased [ 141 , 142 ] ( figure 3 ) . this process seems to involve at least in part the protease ne , which can induce severe tissue damage , and as mentioned before correlates with poor prognosis . elevated amounts of ne in various types of cancer can induce tumor invasion and metastasis by degrading ecm proteins . in support of this once in circulation , neutrophils can also help tumor cells to survive by inducing tumor cell aggregation ( figure 3 ) . in patients with breast and prostate cancers , tumor cell clusters in blood have been associated with poor survival , and in animal models , injection of tumor cell clusters resulted in more metastases than injection of dispersed tumor cells . at least , for breast cancer mcf-7 cells , neutrophils can promote aggregation in vitro [ 99 , 101 ] . however , metastasis induced by neutrophil - mediated aggregation of tumor cells has not yet been directly demonstrated in vivo . circulating tumor cells directly adhere to the vascular endothelium promoting extravasation for establishing new metastases . at the site of exit , lung cancer tumor cells have been observed in close association with neutrophils . in this process , neutrophils enhance tumor cell retention and in consequence induce more metastasis ( figure 3 ) . direct cell - cell interaction of neutrophils with breast carcinoma cells has been shown to involve the adhesion molecule icam-1 on the tumor cells and 2 integrins on neutrophils . neutrophils bound tumor cells engaging integrins and inducing icam-1 clustering on the tumor cell ( figure 3 ) . this activated in the tumor cell a signaling pathway involving focal adhesion kinase ( fak ) and p38-mapk that resulted in enhanced migration . in addition , this enhanced migration was shown in vivo to result in increased metastasis to the liver . here , the cancer cells adhered directly on top of arrested neutrophils , which acted as a bridge to facilitate interactions between the tumor cells and the liver parenchyma . moreover , neutrophils seem to participate in facilitating metastasis even before the tumor cells arrive to the new site , the metastatic niche . this is a potential metastatic site where leukocytes create a permissive growth environment prior to the arrival of tumor cells [ 150 , 151 ] . vegfr1-positive bone marrow - derived cells are found in premetastatic niches of organs involved in metastasis of particular tumor types . once at the metastatic niche , these bone marrow - derived cells secrete factors that promote tumor cell growth [ 152 , 153 ] ( figure 3 ) . in lungs of mice bearing mammary adenocarcinomas , these granulocytic cells had decreased ifn- production and increased mmp-9 production , thus promoting angiogenesis . in addition , coinjection of with 4t1 tumor cells with these gr-1cd11b cells , isolated from tumors and spleens of 4t1 mammary tumor - bearing mice , resulted in increased metastases to lungs . but because these gr-1cd11b cells are a heterogeneous population of cells , including neutrophils , macrophages , dendritic cells , and other immature myeloid cells , the particular cell type(s ) needed to promote metastasis remains unclear . however , neutrophils are a good candidate because it has been reported that circulating neutrophils augment in number with increasing metastatic potential of various rat mammary adenocarcinomas , and tumors secreting il-8 also have an increased metastatic potential . clearly , the mechanisms tans use to promote tumor cell migration and metastasis are diverse and complex ( figure 3 ) . despite the large amount of evidence for a negative role of neutrophils during tumor progression , there is also clear evidence for a positive role of neutrophils in carcinogenesis . early murine neutrophils infiltrating tumors have been named n1 since they clearly display an active proinflammatory and an antitumor phenotype . in fact , the antitumor capacity of neutrophils has been recognized for more than three decades . for example , a colon adenocarcinoma cell line transfected to express g - csf lost tumorigenic activity after considerable concentration of neutrophils at the tumor site . interestingly , neutrophils could discriminate between g - csf - producing and g - csf - nonproducing cells and directly inhibited only g - csf - producing tumor cells . this antitumor effect of activated neutrophils can also be transferred to other animals , as demonstrated with spontaneous regression / complete resistance ( sr / cr ) mice . sr / cr mice resist very high doses of cancer cells that are lethal to wt mice even at low doses . the genetic , cellular , and molecular effector mechanisms in this model are largely unknown . however , purified neutrophils from the sr / cr mice independently killed cancer cells in vitro and completely transferred resistance to wt recipient mice . also , the cancer disappeared gradually following infiltration of a large number of neutrophils and few lymphocytes into the remaining tumor tissues . the importance of n1 type tans in antitumor responses is also highlighted by reports showing that depletion of murine neutrophils results in enhanced tumor growth [ 15 , 160 , 161 ] . despite the evidence presented before on neutrophils helping metastasis by preparing the metastatic niche , a complete opposite effect has also been demonstrated for metastatic breast cancer and renal carcinoma . in both models , neutrophils prevented metastasis to the lung . in the breast cancer model , the tumor cells produced ccl2 that induced neutrophil ros production , while , in the renal carcinoma model , tumor - derived il-8 recruited tumor cytotoxic neutrophils . this goes against the majority of reports implicating il-8 in protumor functions of neutrophils . nevertheless , these findings underline the dual antitumor and protumor potential of neutrophils and suggest that neutrophils could be induced to enhance their antitumor responses . the mechanisms by which neutrophils accomplish this function are numerous and not completely understood , but they involve many of the same antimicrobial and immune regulatory functions of neutrophils ( figure 4 ) . early reports indicated that neutrophils from tumor - bearing animals displayed enhanced superoxide anion generation and phagocytosis . this led to reduced tumors and less metastatic foci in lungs [ 38 , 39 ] . also , it has been shown that indeed ros produced by neutrophils can induce tumor cell lysis , through hocl delivered directly at the cell membrane . although ros could be genotoxic for tumor cells , it is clear that , in the case of rapidly growing tumors , activated neutrophils producing sufficient singlet oxygen can eliminate tumor cells at the early phase of tumor development ( figure 4 ) . because neutrophils require close contact mediated by integrins to induce killing , it is also possible that neutrophils may induce direct lysis of tumor cells by a mechanism similar to the one used by nk cells via the enzymes perforin and granzyme . neutrophils induced apoptosis of human breast cancer cells , when stimulated by antibodies targeted to her-2 . neutrophils have another way of eliminating tumor cells by inducing apoptosis of the malignant cell . this effect is mediated by the tumor necrosis factor - related apoptosis inducing ligand ( trail ) ( figure 4 ) . for a long time , carcinoma in situ of the bladder has been treated with intravesical administration of mycobacterium bovis bacillus calmette - gurin ( bcg ) . this kind of immunotherapy is very effective for treatment of this type of cancer , but the mechanism is only partially known . it was then found that neutrophils in urine of patients with carcinoma of the bladder and under bcg immunotherapy expressed high levels of trail . trail is expressed on these neutrophils at high levels both as a type ii membrane protein ( intracellular amino terminal portion and carboxyl terminus outside the cell ) and as a biologically active soluble form , which is released from intracellular stores after interaction with components of the bcg cell wall . trail is a member of the tnf family of molecules , known to have apoptosis - inducing functions . trail binds to target cells through two death receptors ( drs ) ( dr4/trail - r1 and dr5/trail - r2 ) and three decoy receptors ( dcrs ) [ dcr1/trail - r3 , dcr2/trail - r4 , and osteoprotegerin ] . drs activate the formation of a death - inducing signaling complex for caspase activation and initiation of apoptosis . an important feature of neutrophil trail - induced apoptosis is that it can kill tumorigenic and transformed cells but not normal cells and tissues [ 170 , 178 ] . for this reason , trail is becoming a major physiologic weapon against cancer , and several research laboratories and pharmaceutical companies are developing recombinant forms of trail or trail receptor agonists for therapeutic purposes . in addition , the importance of trail in other clinical conditions , such as infectious diseases , autoimmunity , and cardiovascular diseases , is becoming more apparent . therefore , understanding the regulatory mechanisms of trail signaling will help in the future to control these health problems . neutrophils can protect against some tumors by secreting mmp-8 . in mice deficient in mmp-8 , an increase in skin tumors with an increase in neutrophil infiltrates to the tumors was reported . this protective effect is not clearly defined , but it involves the inhibition of neutrophil migration into the tumor site . antibodies directed to tumor cells can also bind to fc receptors on the membrane of immune cells . in many cases this antibody - dependent cell - mediated cytotoxicity ( adcc ) is capable of eliminating efficiently various types of tumors . nk cells are particularly efficient in this response via the fc receptors ( figure 4 ) . however , the mechanism of killing is not completely described , but it seems to be different from the classic adcc mechanism used by nk cells . it is worth noting that an important difference exists between murine and human neutrophils regarding fcr expression . in addition to fcriii , the only fc receptor on murine nk cells , murine neutrophils also express fcriv . in contrast , human neutrophils express two unique fc receptors not present in other species : fcriia ( cd32a ) ( homolog to murine fcriii ) and fcriiib ( cd16b ) ( a glycosylphosphatidylinositol- ( gpi- ) linked receptor ) . therefore special attention should be paid when interpreting data from murine models on adcc against tumors . human neutrophils present a more efficient adcc when they engage fcriia [ 184 , 185 ] . under stimulated conditions mainly with ifn- but also with g - csf , neutrophils can upregulate expression of fcri ( cd64 ) . this receptor seems also capable of promoting neutrophil adcc against tumors and in particular with squamous head and neck cancer . however , in other studies , it was shown that immature neutrophils with high expression of fcri had reduced adcc activity via this receptor . in fact , ample reports have demonstrated that the high affinity receptor for iga , fcri ( cd89 ) , is a more potent inducer of adcc by neutrophils [ 188 , 189 ] . the mechanism for tumor cytotoxicity from neutrophils is not completely known , and it seems to be multifactorial . both ros - dependent and ros - independent mechanisms have been suggested for neutrophil adcc . for the oxidative mechanism , close cell contact mediated by integrins however , studies with neutrophils from chronic granulomatous disease ( cgd ) patients and with ros scavengers suggest that ros are not as important for adcc as they are for antimicrobial functions . however , as mentioned before , expression of these enzymes in neutrophils remains controversial [ 166 , 167 ] . neutrophils invading tumors can modify t cell effector functions and in this way instruct t cells to reject tumors . cytotoxic cd8 t cells are key contributors in any immune response towards tumors . as mentioned before n2 neutrophils can be inhibitors of t cell functions [ 15 , 192 ] . however , the proinflammatory n1 neutrophils can recruit and activate cd8 t cells [ 15 , 193 ] ( figure 4 ) . also , after photodynamic therapy , there was a rapid neutrophil infiltration into the treated tumor bed . neutrophils were necessary for generation of tumor - specific primary and memory cd8 t cell responses . together , these reports indicate that neutrophils can influence the outcome of t cell functions depending on the type of cytokines they produce [ 4 , 194 ] . neutrophil extracellular traps ( nets ) constitute a recently described form of the antimicrobial arsenal of neutrophils . nets are fibers of chromatin released from neutrophils in an active process named netosis . in this process , neutrophils undergo dramatic changes starting with flattening of the cells . citrullination of histone h3 by peptidylarginine deiminase 4 ( pad4 ) is a major modification during netosis . dna fibers in nets are also decorated with various antimicrobial proteins from the neutrophil granules , including neutrophil elastase , mpo , cathepsin g , proteinase 3 , mmp-9 , and bactericidal / permeability increasing protein ( bpi ) . nets form a mesh - like structure where microorganisms get trapped and are either directly killed on some cases or more often subsequently phagocytosed by other neutrophils [ 198 , 199 ] . many microorganisms and various stimuli bacterial products such as lipopolysaccharide ( lps ) , formyl - methionyl - leucyl - phenylalanine ( fmlf ) , and also phorbol esters such as phorbol myristate acetate ( pma ) are efficient net inducers . recent reports also indicated that antigen - antibody complexes are capable of inducing net formation , thus suggesting a direct role for fc receptors in this function . in fact both fcri and fcriiib have been shown to induce net formation . this is interesting because various tumors are known to produce these cytokines and thus it is possible that tumors can enhance net formation . very little is known about the presence and effect of nets in different types of tumors . it is also not clear if distinct tans can make nets with different efficiency . in an initial study , tumor samples from eight patients with ewing sarcoma were evaluated for the presence of tans and nets , defined as extracellular staining for mop . in two ( 25% ) patients , intratumoral nets were found . thus , it was proposed that at least this type of tumor could induce tans to release nets . however , in cancer models of chronic myelogenous leukemia and mammary and lung carcinoma , peripheral neutrophils were more prone to net formation . neutrophils from tumor - bearing animals responded to platelet - activating factor ( paf ) forming more nets than neutrophils from tumor - free animals . in addition , higher amounts of circulating neutrophils and plasma cell - free dna were found in tumor - bearing animals . this free dna is probably in the form of nets , since a concomitant increase in neutrophils with hypercitrullinated histone h3 was also found . it seems then that some cancers may present a systemic effect on the host that predisposes neutrophils to form nets . as discussed earlier , many tumors presenting tans are associated with poor prognosis . in many of these tumors , thus the presence of nets in these tumors most certainly would be associated with tumor progression . supporting this idea , there are studies looking at the phenotype of tans during tumor development . in one study , neutrophil depletion at 14 days after implantation of lewis lung carcinoma ( llc ) and ab12 mesothelioma tumors resulted in reduced tumor growth . in contrast , neutrophil depletion at 7 days after implantation had no effect on tumor growth . tans from early tumors were more cytotoxic toward tumor cells , while tans from established tumors acquired a more protumorigenic phenotype . moreover , in initial tumors , tans were found in the periphery of the tumor , but in mature tumors tans and free dna were within the tumor . in another study , this defect caused an uneven compartmentalization of lymphoid and myeloid populations that led to aberrant interactions between nets and b cells . under these conditions , nets induced b cell proliferation and inhibition of apoptosis , resulting in malignant transformation . neutrophils would migrate to the new tumor and there tans would produce nets , which would promote tumor growth ( figure 5 ) . although evidence strongly indicates that nets within primary tumors can promote tumor progression , no mechanism for this effect has been revealed yet . however , because nets are made of chromatin fibers decorated with antimicrobial proteins such as neutrophil elastase , cathepsin g , and mpo , it is very likely that nets concentrate these factors to high local concentrations within the tumor microenvironment . as discussed above , these factors have all been implicated in tumor promotion . therefore , nets may be a way to enhance exposure of tumor cells to these bioactive proteins and in turn increase proliferation , inhibit apoptosis , and induce migration ( figure 5 ) . although in many instances the presence of neutrophils in tumors has a negative effect in cancer disease , these cells clearly have the capacity to destroy tumor cells . several novel therapeutic approaches are being considered to enhance the antitumor potential of neutrophils or to block the access of tans into growing tumors . n1 type murine neutrophils display an activated phenotype that leads to tumor control . in consequence , tumor cells modified to express activation of neutrophils with g - csf and ifn- can generate cells with an antitumor phenotype . due to the important role of neutrophils in antimicrobial responses , general activation of these cells is not good therapeutic approach since highly activated neutrophils without targeting specificity could cause excessive tissue damage . the two types of tans , n1 and n2 , suggest that the tumor microenvironment could be manipulated to generate more antitumor tans . this idea is supported by studies in murine cancer models where inhibition of tgf- induced the appearance of antitumor neutrophils . another therapeutic approach aims to block infiltration of neutrophils into tumors . as indicated before , the use of il-8 antagonists ( such as the fully humanized neutralizing monoclonal antibody abx - il8 ) to il-8 was shown to reduce tumor growth , metastasis , and angiogenesis of melanoma and lung cancer . because other chemokines also interact with the receptors cxcr1 and cxcr2 , a more effective way to block neutrophil migration may be the inhibition of these receptors . specific inhibitors for these receptors are now being developed with the idea of preventing neutrophil infiltration and retarding tumor progression . for example , the cxcr2 receptor antagonist , gsk135756 , is being considered to be used as an anti - inflammatory drug for chronic obstructive pulmonary disease . this inhibitor has shown to efficiently block neutrophil recruitment into tissues and to selectively target human breast cancer stem cells in xenograft models in mice . in addition to blocking neutrophil infiltration , inhibition of particular neutrophil - specific enzymes known to promote tumor progression is another therapeutic avenue being explored . for example , inhibition of ne was able to reduce significantly the growth of lung adenocarcinomas in a mouse model . also inhibition of mmp has been tried to prevent tumor angiogenesis . the bisphosphonate zoledronic acid , a strong mmp inhibitor , blocked mmp-9 expression and metalloprotease activity reducing angiogenesis and cervical cancer burden . however , in other models and clinical trials , inhibition of mmp-9 was not effective at reducing tumor growth [ 214 , 215 ] . a more promising approach is the use of antitumor monoclonal antibodies ( mabs ) to activate the adcc potential of neutrophils . upon fc receptor activation , today most mabs used in immunotherapy belong to the igg1 class , and they are effective at activating nk cells via fcriiia ( cd16a ) . in contrast , neutrophils activate adcc via fcriia ( cd32 ) by preferentially engaging igg2 class antibodies . this igg2-mediated adcc was influenced by the functional fcriia - r131h polymorphism and was induced more effectively by neutrophils from fcriia-131h homozygous donors than from fcriia-131r individuals . based on these findings , it has been proposed that fc receptor polymorphisms could be biomarkers for egfr antibodies such as panitumumab , the only human igg2 antibody approved for immunotherapy and inhibition of egfr . therefore , there is a big interest in developing new improved antibodies through fc engineering technologies in order to potentiate fcr - mediated functions . based on this methodology , it was possible to change the ability of an fcriii - optimized ( for nk cell ) anti - egfr antibody to efficiently activate neutrophil adcc against egfr - expressing tumors . however , it seems that fcri ( cd89 ) is a more potent inducer of adcc by neutrophils [ 188 , 189 ] . thus , it has been proposed that a new generation of cancer therapeutic mab should include iga class antibodies to fully take advantage of the cytotoxic potential of neutrophils . indeed , this idea is supported by a new iga2 anti - egfr antibody derived from the igg anti - egfr mab cetuximab . iga2 egfr was more effective than cetuximab in vivo against egfr - transfected ba / f3 target cells . very recently , it was also shown that the combination of igg and iga mabs to two different tumor targets ( egfr and her2 ) led to enhanced cytotoxicity compared with each isotype alone . the exact role for these tumor - associated neutrophils ( tans ) has yet to be completely elucidated . however , a tremendous body of clinical evidence has shown that neutrophils promote tumor progression in various ways . neutrophils can induce tumor proliferation and angiogenesis and can enhance tumor cell migration and metastasis . yet , a type of tans , named n1 , can indeed display antitumor functions . new therapeutic ways to recruit and activate these n1 type neutrophils are being investigated in order to turn protumorigenic neutrophils into antitumor effector cells . blocking neutrophil - derived components known to help tumor growth is a field of active research . also , very promising results have been found with the use of therapeutic antibodies , which induce neutrophils to perform adcc and to release cytokines that modulate the immune response against tumors . new antibodies are being designed so that they have better affinity for particular fc receptors and induce stronger antitumor responses . learning how to flip the neutrophil coin to the winning side , namely , functioning as antitumor effector cells , is a challenge for future research that will certainly provide us with new therapeutic options for cancer treatment .	neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections . in addition , neutrophils are also found infiltrating many types of tumors . tumor - associated neutrophils ( tans ) have relevant roles in malignant disease . indeed neutrophils may be potent antitumor effector cells . however , increasing clinical evidence shows tans correlate with poor prognosis . the tumor microenvironment controls neutrophil recruitment and in turn tans help tumor progression . hence , tans can be beneficial or detrimental to the host . it is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor , for neutrophils supporting tumor progression , and for neutrophil activation to enhance their antitumor functions .
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Proceed to summarize the following text: primary malignant pericardial mesothelioma is an extremely rare disease with a reported incidence of 0.0022%.1 ) initial presenting symptoms of this disease are dyspnea , fever and chest pain . patients may also suffer from acute myocardial infarction or embolic stroke due to extension of tumor into myocardium or cardiac chambers . chest x - ray may shows cardiomegaly and echocardiographic examination frequently reveals pericardial effusion . because presenting signs and symptoms are non - specific , diagnosis of the disease has occurred predominantly in men , with the majority of cases occurring in the fifth to seventh decades of life.2 ) the prognosis is dismal , even with radio- and chemotherapy . a 21-year - old man was transferred to our hospital because of cough with sputum , and dyspnea beginning 14 days prior to admission . the cough was persistent and associated with intermittent fever up to 38.3. the patient had been well until 2 weeks earlier , when he inoculated with influenza vaccine ( h1n1 ) . five days before admission , he visited another hospital because of chest pain and aggravating dyspnea . on arrival in the emergency department , the patient reported fever , chills , pleuritic chest pain and orthopnea . on examination , the blood pressure was 105/78 mmhg , the pulse 97 beats per minute , and the temperature was 37.4. the heart rhythm was regular without murmur . initial white blood cell count showed 11900 per microliter of which 71.6% were segmented neutrophils . c - reactive protein ( crp ) was elevated up to 15 mg / dl . a 12-lead electrocardiogram demonstrated regular sinus tachycardia with anterior , inferior lead st - segment elevation . pericardial fluid analysis showed 900 white blood cells per microliter of which 78% were segmented neutrophils . cytological examinations were negative for malignant cells , and cultures and smears for bacteria , acid - fast bacilli , and fungi were negative . the patient was tentatively diagnosed with viral pericarditis and given nonsteroidal anti - inflammatory drugs ( nsaids ) and colchicine . after 1 week of treatment , fever and dyspnea were subsided and an echocardiography showed minimal pericardial effusion . the patient was discharged on colchicine and nsaids , and followed in the outpatient department . one month after discharge , the patient was rehospitalized because of the recurrence of chest pain and dyspnea . an echocardiography revealed increased pericardial thickness with a moderate amount of pericardial effusion with adhesion ( fig . 2 ) . because of increased pericardial thickness and recurrent effusion , pericardial biopsy was performed . histopathological examination of pericardial tissue revealed chronic active inflammation and a few proliferating atypical mesothelial cells in inflamed granulation tissue . the patient was treated with high dose prednisolone ( 1 mg / kg / day ) on the top of nsaid and colchicine . chest computed tomography ( ct ) after 4 days of systemic steroid treatment revealed improved pericardial effusion with normal pericardial thickness ( fig . the subjective symptoms were rapidly improved and the patient was discharged on steroids and additional nsaids . during the regular follow - up at outpatient department , the patient was in well being state . the prednisolone was gradually decreased to 5 mg / day with guide of hscrp level . after 7 months of treatment , the patient was readmitted after complaining of general weakness , chest pain , dyspnea , and lower leg edema . a diagnostic pericardial biopsy was repeated , and malignant mesothelioma was diagnosed ( fig . 5 ) . pericardiectomy was initially considered , but operative findings during the pericardial biopsy suggested myocardial invasion . most common symptoms of acute pericarditis are pleuritic chest pain and fever , but symptoms may vary according to underlying disease . friction rub may have a diagnostic value , while electrocardiography and echocardiography also useful for the diagnosis . if etiology is identified , treatments according to the underlying disease are applied , although etiology of acute pericarditis can not be identified in most of cases . in case of idiopathic acute pericarditis , non - steroidal anti - inflammatory drugs including high - dose aspirin or ibuprofen and colchicines are the mainstay of treatment . despite of treatment , corticosteroids are treatment of option in this case.3 ) malignant mesothelioma has various symptoms but dyspnea is most common symptom.1 ) because there is no pathognomonic symptom or sign in this disease , diagnosis is hard to obtain and diagnostic consideration of other disease such as idiopathic acute pericarditis or acute myocardial infarction is common . but , the possibility of this disorder may be considered in pericardial effusion and pericarditis , especially in recurrent cases . thomason et al.2 ) described 28 cases of primary pericardial mesothelioma from 1972 to 1992 , and there are only 1 case of mediastinal mass on chest x - ray among 24 patients whose chest x - ray results were available . pericardial mass on echocardiography or ct also revealed low sensitivity , which were 12% and 44% . echocardiography has limited value when the tumor is diffusely infiltrating , rather than mass forming . gssinger et al.4 ) suggested possible role of cardiac mri on diagnosis of mediastinal mesothelioma . malignant mesothelioma shows high signal intensity on t2 weighted image and expresses higher signal after gadolinium enhancement on cardial mri , and it appears to be helpful in establishing the diagnosis.5 ) there are some features suggesting malignancy , which are infiltration of deep tissues , severely atypical cytoplasm and necrosis . immunohistochemistry also provide a diagnostic clue.6 ) prognosis is very poor , with little effects of chemo- or radiotherapy .	we report on a 21-year - old man with fever , dyspnea , and pleuritic chest pain . an electrocardiography showed st elevation in multiple lead and thoracic echocardiography revealed moderate pericardial effusion . he was initially diagnosed with acute pericarditis , and treated with nonsteroidal anti - inflammatory drugs and colchicines with clinical and laboratory improvement . after 1 month of medication , his symptoms recurred . an echocardiography showed constrictive physiology and the patient was treated with steroid on the top of current medication . the patient had been well for 7 months until dyspnea and edema developed , when an echocardiography showed marked increased pericardial thickness and constriction . pericardial biopsy was performed and primary malignant pericardial mesothelioma was diagnosed . malignancy should be considered in the differential diagnosis of recurrent pericarditis .
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Proceed to summarize the following text: there have been many unusual types of pituitary tumors that might neglect by radiologists and clinicians . a 62-year - old male has complained from sudden frontal headache , nausea , vomiting , decreased vision , blurred vision and double vision . in skull radiographic , there was an expansion of sellaturcica and a lesion with clear border on t1 ; heterogeneous ; iso - to hypotense ; size about 3/5 2/5 cm with no surrounding edema that it has homogeneously attracted contrast lesion , that shown a signal hypointensity on t2 with heterogeneous enhancement . in the coronal sections , the tumor expanded to the suprasellar region and optic chiasmatic that has compressed especially on the left side . there have been many unusual types of pituitary tumors that might neglect by radiologists and clinicians . pituitary tumors have mainly originated from the anterior part of the pituitary , and pituitary adenomas have been the most common types of these tumors ( 1 ) . posterior pituitary cells have contained specialized glial cells which called pituicytes ( 2 ) . most of the posterior pituitary astrocytoma has reported as low grade tumors with a slow progression ( 2 ) . in addition to these tumors , several tumors which have located in the sellar and parasellar region , might originate the different sources . some examples of these tumors include : craniopharyngioma , meningioma , choristomas , dermoid or epidermoid cysts , hamartoma , chordoma , chondrosarcoma , metastasis and germinoma ( 3 , 4 ) . besides , the location or perspective of the tumors might be erroneously made with adenoma or sellar and parasellar tumors . for example , optic nerve glioma could mimic the view of supra sellar cystic lesions or craniopharyngioma ( 3 , 4 ) . diagnosis of sellar and supra sellar masses : it might seem an easy work to diagnose preoperatively these tumors , although the majority of clinical aspects of some lesions wouldbe similar to the pituitary macroadenomas , but some of them , such as glioblastoma have shown more aggressive behavior than adenomas . approximately , half of these tumors have caused mri contrast uptake but sometimes a number of them have shown rim enhancement ( 5 ) . it might be a focal hemorrhage in imaging findings of pituitary macroadenoma , then although it can help for diagnosis but there is bleeding in many high - grade gliomas . other imaging techniques such as scientgraphy of somatostatin receptors might be suitable to differentiate adenomas from other pathologies in the sellar and parasellar regions ( 6 ) . a 62-year - old male without any history of previous disease , has complained from sudden frontal headache , nausea , vomiting , decreased vision in the left eye , blurred vision and double vision . we have observed in the cranial nerves examination paralysis of the left abducens nerve ( impaired movement in his left eye to the outside ) , visual impairment in the right eye , and a severe defect in the nasal and temporal field in the left eye . the vision activity of the left eye was 7/10 , and then , 10/10 for the right eye . in fundoscopy , the retina has normal but partial atrophy that has observed in optic nerves . in skull radiographic , there was an expansion of sellaturcica and a lesion with clear border on t1 ; heterogeneous ; iso - to hypotense ; size about 3/5 2/5 cm with no surrounding edema that it has homogeneously attracted contrast lesion , that shown a signal hypointensity on t2 with heterogeneous enhancement . in the coronal sections , the tumor expanded to the suprasellar region and optic chiasmatic that has compressed especially on the left side . also , it has found an enhancement of the carotid artery without obstructive symptoms and brief extension into the left cavernous sinus ( figures 1 and 2 ) . the serum levels of gh = 0.1 ; igf = 300 ; prolactin = 7.8 ; tsh = 0.5 were normal . serum levels for lh and fsh were in normal range , 1.3 and 10 miu / ml , respectively . we have faced to a creamy soft tissue tumor in sella turcica that was relatively hypervascular with tendency to bleeding . we have found a soft creamy soft tissue tumor in sella turcica without any evidence of bone invasion . partial resection has done and no complications have found after surgery . the pathologist has diagnosed a glioblastoma multiform due to neoplastic tissue with severe atypia proliferation and extensive necrosis in the microscopic slides . microscopic examination has revealed proliferation of mitotically active highly atypical cells in a fibrillary background with tumoral coagulative necrosis and capillary proliferation , some with glomeruloid feature ( figures 3 - 6 ) . regarding localization of the tumor , immunohistochemical staining has performed to establish the true nature of the neoplastic cells . these cells have shown strong positive reactivity with gfap ( glial fibrillary acidophilic protein , figure 4 ) and were negative for epithelial markers of cytokeratin ( ck ) and epithelial membrane antigen ( ema ) . pituicytes and modified glial cells have located in the pituitary stalk and neurohypophysis and originated from empendymal cells . there would be five types of glial cells : major cells , dark cells , oncocyte cells , ependymal cells and granular cells ( 7 , 8) . a review of the literature has revealed only a few case astrocytoma of hypophysis , and there has been scanty discussion of the imaging findings of pituitary astrocytomas . we have found only a handful number of suprasellar malignant gliomas have been published and only one case report of sellar and supracellar gb masquerading as a pituitary macroadenoma ( 9 ) . although these tumors were indistinguishable from other pituitary tumors , but pituitary adenomas would be distinctive due to the spindled view of the cells . on the other hand , immunohistochemistry studies have shown presence of gfap immunoreactivity , neuroendocrine markers and pituitary hormones in these tumors ( 10 ) . regarding the pituitary adenomas were the most common types of pituitary tumors , and mri images for non - adenoma tumors were similar to adenoma tumors ; it should consider for a correct diagnosis . in the other words , posterior pituitary tumors should consider by radiologist and clinician before treatment ( 11 , 12 ) . because of their scarcity , it was hard to ascertain the prevalence of adenoma tumors . reports have shown that it has happened in the young and middle - aged subjects with signs of headaches , an intracranial focal lesion with a reduction in hypophysis hormones . visual symptoms have not expected in these tumors , however , in our patient there was a visual symptom because of a compression of the optic chiasm . weather the behaviors of hypophysis gb would be different from other part of cns , that mimicked a slow growing features was a question to be proved . these tumors have shown a signal hypointensity on t1 ; low to moderate on t2 . in our patients , the tumor was hyperintense in t2-weighted images but unlike glioblastoma that has characterized with central necrosis , this tumor has absorbed dye in the central part , and caused to happen an error diagnosis by radiologist . although it has not been possible to differentiate the anterior or posterior origin of the tumor based on imaging findings , authors recommend a specific attention to the posterior pituitary tumors for diagnosis . information about this tumor should consider by the clinicians , radiologists and pathologists in the diagnosis of sellaturcica region for a correct diagnosis and treatment and less likely to recognize the error of tumors of the sellaturcica region .	introduction : pituitary adenomas have been the most common sellaturcica tumors . there have been many unusual types of pituitary tumors that might neglect by radiologists and clinicians . one of these tumors would be the malignant glioma.case presentation : a 62-year - old male has complained from sudden frontal headache , nausea , vomiting , decreased vision , blurred vision and double vision . in skull radiographic , there was an expansion of sellaturcica and a lesion with clear border on t1 ; heterogeneous ; iso - to hypotense ; size about 3/5 2/5 cm with no surrounding edema that it has homogeneously attracted contrast lesion , that shown a signal hypointensity on t2 with heterogeneous enhancement . in the coronal sections , the tumor expanded to the suprasellar region and optic chiasmatic that has compressed especially on the left side . partial tumor resection has conducted . the surgery has done by a transsphenoidal approach . the pathologist has diagnosed a glioblastoma . this diagnosis has confirmed using immunohistochemistry technique.conclusions:there have been many unusual types of pituitary tumors that might neglect by radiologists and clinicians . one of these tumors would be the malignant glioma .
You are an expert at summarizing long articles. Proceed to summarize the following text: web- and mobile - phone - based applications are now commonly used to improve coverage and quality of health services . in india , the mother and child tracking system ( mcts ) is one such web - based applications that is used nation - wide to improve the coverage of maternal , newborn , and child health ( mnch ) services . similarly , the use of mobile - phone application such as mobile health ( mhealth ) for improving health services is becoming popular in india . the first step of utilizing any e - health initiative is registration of eligible beneficiaries ; however incomplete registration is a major concern . only 35% of pregnant women were found to be registered through the mcts in india in 2011 . the purpose of the current study was to assess an innovative mhealth intervention to ensure completeness of pregnancy , delivery , and death registration by the accredited social health activists ( ashas ) in the tribal areas of gujarat . this cross sectional study was undertaken by a local voluntary organization , society for education welfare and action rural ( sewa rural ) among randomly selected five predominant tribal villages of two primary health centers ( phcs ) of bharuch district for a period of 2 months ( april - may 2015 ) . sewa rural and the department of health and family welfare , government of gujarat , india implemented an innovative mhealth application called the innovative mobile - phone technology for community health operations ( imtecho ) since 2013 . the purpose of the imtecho is to improve the coverage and quality of the mnch services through improving support and supervision of ashas and phc staff . some of the strategies used in the imtecho to ensure completeness of registration were as follows : point of care registration using the imtecho mobile - phone application , linkage of ashas performance - based incentives to electronic record in the imtecho , regular feedback to ashas regarding completeness of registration , and demand generation among the community by the use of mobile - based videos . to find the actual number of events ( reference ) in the sampled villages , trained and independent data collectors conducted house - to - house survey to enumerate every household . using a pretested questionnaire , the members of each household were asked to identify current events after obtaining written informed consent from them . a detailed account of each event was collected including last menstrual period ( lmp ) , date of delivery , native village , etc . information was collected from a key informant as well such as asha and anganwadi workers . after completing information collection in every village , asha 's record was checked in her mobile to identify the women who were not registered through the imtecho application ; subsequently , asha was interviewed to identify reasons behind the nonregistration of the event through the imtecho application . every event found during household survey was compared with the imtecho system to identify those events which were missed by asha using the imtecho application . to detect 10% difference in sensitivity between registration of deliveries in the imtecho and reference , the required sample size calculation was 98 deliveries in both groups considering 90% power and 5% two - sided alpha error . those villages were included where single ashas had been using the imtecho application since january 1st 2014 . native currently pregnant women having lmp more than 3 months from the day of survey were included in the study . the data of native women who delivered between january 2014 and april 2015 and all infant who died between january 2014 and april 2015 were studied . the villages were selected through stratified random sampling so that equal numbers of villages are chosen from two study phcs . the study was approved by the scientific committee of sewa rural and written informed consent was taken from participants after explaining the whole procedure of our study . primary outcome of interest for the study was the proportion of pregnancies , deliveries , and infant deaths registered through the imtecho application compared to actual number of events . there were 844 household enumerations , 39 pregnancies , 102 deliveries and 15 infant deaths that were recorded . the median age of the study participants was 23 years ( range : 18 to 36 years ) . table 1 tabulates data on pregnancies ( n = 39 ) , deliveries ( n = 102 ) and infant deaths ( n = 5 ) found during the household survey , 38 ( 97.43% ) , 101 ( 99.01% ) , and 5 ( 100% ) were already registered by the ashas through the imtecho application . the asha missed one case of pregnancy and delivery because she was under the impression that the women did not belong to the study village as they spent majority of time in another village . for the pregnancies , deliveries , and infant deaths found in household survey , the sensitivity of the imtecho application with the household survey was 97% ( 95% ci 0.85 - 1.00 ) , 99% ( 95% ci 0.93 - 0.99 ) , and 100% ( 95% ci 0.46 - 1.00 ) , respectively . number of pregnancies , deliveries , and infants deaths found during the household survey and in the imtecho among sampled villages ( n = 5 villages ) * 95% cis for sensitivity was calculated using the wilson method on vassarstats online statistics software . the study found that the proportion of pregnant women registered through imtecho was similar to the enumeration . a survey done in india in 2011 - 2012 showed 95% of districts reporting data on pregnant women and 93% on children enrolled under the mcts , while in gujarat , india only 51% of pregnant women among estimated gujarat had 7.69% of data on pregnant women and 10.27% of data on children that were not reported appropriately by sub - centres under the mcts . a similar study carried out in rural ahmedabad shows only 79% of the women have a health card with 82.5% women having documentation of treatment and advice for the antenatal details and only 3.9% of women had been given advice regarding postnatal details . a possible explanation for the difference in findings is the registration being made into this innovative mhealth application . near - complete registration is ensured via numerous safeguards incorporated into the program that includes linking performance - based incentives with digital record , demand generation , regular feedback , and point of care registration . as documented in another publication , the ashas found the use of mobile application quite acceptable , feasible , and useful ; therefore , they were more inclined to timely register pregnant women and deliveries . one of the limitations of the study is its small sample size : using a larger population sample would be helpful , as it would demonstrate the effects of the imtecho application in a more appropriate manner . also , sewa rural has been active in the study area for more than a decade , which might have positive influence on the results . however , similar findings were noticed in another new project area that is far from the reach of sewa rural . to conclude , the strategies used to ensure complete registration in the imtecho application could be applied in other e - health initiatives . similar mhealth initiatives might be considered for scaling up in other tribal areas to improve the coverage and quality of the mnch services .	background : the innovative mobile - phone technology for community health operation ( imtecho ) is a mobile - phone application that helps accredited social health activists ( ashas ) in complete registration through the strategies employed during implementation that is linking ashas incentives to digital records , regular feedback , onsite data entry , and demand generation among beneficiaries.objective:to determine the proportion of pregnancies , deliveries , and infant deaths ( events ) being registered through the imtecho application against actual number of events in a random sample of villages.materials and methods : five representative villages were randomly selected from the imtecho project area in the tribal areas of gujarat , india to obtain the required sample of 98 recently delivered women . a household survey was done in the entire villages to enumerate each family and create a line - listing of events since january 2014 ; the line - listing was compared with list of women registered through the imtecho application . the proportion of events being registered through the imtecho application was compared against the actual number of events to find sensitivity of the imtecho application.result:a total of 844 families were found during household enumeration . out of actual line - listing of pregnancies ( n = 39 ) , deliveries ( n = 102 ) , and infant deaths ( n = 5 ) found during household enumeration , 38 ( 97.43% ) , 101 ( 99.01% ) , and 5 ( 100% ) were registered by ashas through the imtecho application.conclusion:the use of mobile - phone technology and strategies applied during the imtecho implementation should be upscaled to supplement efforts to improve the completeness of registration .
You are an expert at summarizing long articles. Proceed to summarize the following text: the therapeutic drug monitoring ( tdm ) of immunosuppressive drugs in blood of organ - transplanted patients is of utmost importance to prevent intoxication or rejection due to incorrect dosage . commonly used immunosuppressive agents are calcineurin inhibitors such as cyclosporin a and tacrolimus or the mtor inhibitors sirolimus and everolimus . recently a new therapeutic strategy has been to combine immunosuppressive drugs with different mechanisms of action in order to reduce undesirable side effects . analytical problems occur in the detection of clinically relevant low levels of immunosuppressive agents , which in turn requires a more sensitive analytical measurement procedure at low analyte concentrations for all the drugs used . several lc - ms / ms methods for routine measurement of immunosuppressive drugs in whole blood have been described , , . these routine methods have been designed for high throughput combined with a short turnaround time . methods without chromatographic separation of the analytes require cycles of only a few minutes per sample . unfortunately , co - elution of the compounds usually leads to a bias in the analytical results , due to a lack of specificity of the mass transition in the mrm mode of the lc - ms / ms measurements , . avoidance of this phenomenon requires separation and detection of the parent substances from the different metabolites with biological activity . lc - ms / ms procedures for determination of serum levels of digoxin and digitoxin have been recently published from this laboratory , using cs adduct formation , which allows potential improvement of both analytical sensitivity and specificity . the aim of the present study was to develop a method for determination of immunosuppressive agents , by utilising the principle of measurement of cs adducts , thus forming the basis of a reliable method for defining target values for immunosuppressive drugs in external quality assessment schemes ( eqas ) . this article describes a lc - ms / ms method with complete chromatographic separation of the immunosuppressive drugs cyclosporin a ( cyclosporin a , sandimmune ) , tacrolimus ( fk-506 , prograf ) , sirolimus ( rapamycin , rapamune ) and everolimus ( certican ) , with cyclosporin d , ascomycin and 32-desmethoxy - rapamycin as internal standards . rapamycin ( > 99% ) , tacrolimus ( fk-506 , > 99% ) and cyclosporin a ( > 99% ) were obtained from lc laboratories , woburn , ma , usa . davids , pa , usa . ascomycin , formic acid ( 98% , puriss . p.a . ) and cesium hydroxide ( purity 99.97% ) , were purchased from sigma - aldrich , taufkirchen , germany . water was prepared using the purification system direct - q 5 ( millipore gmbh , eschborn , germany ) . the hplc system used was from shimadzu ( duisburg , germany ) consisting of a scl-10a system controller , three lc-10advp pumps ( a , b , c ) , a dgu-14a degasser ; a sil-10ad autoinjector ; a ctu-10as column oven ; and a fcu-12a flow switch ( rotary valve ) . the mass spectrometer was a 4000 qtrap equipped with a turbov esi source with turboion spray probe from applied biosystems ( mds - sciex , darmstadt , germany ) . the chromatographic separation was performed on a phenyl - hexyl - rp column ( luna , 2 x 150 mm , 5 m , phenomenex , aschaffenburg , germany ) . a ternary gradient was used for elution consisting of eluent a ( pump a ) : 0.1% v / v hcooh in water + 0.1 mmol / l csooch ; eluent b ( pump b ) : 0.1% v / v hcooh in methanol + 0.1 mmol / l csooch and eluent c ( pump c ) : 0.1% v / v hcooh in ch3cn + 0.1 mmol / l csooch . the column temperature was set at 50c . in order to avoid contamination of the esi source a switching valve system ( rotary valve a ) was introduced . at position 0 of rotary valve the ms detection was performed in the positive ion mode . the mass transitions ( m / z ) for ms detection are given in table 2 ( tab . ( the abbreviations for settings are specific to the 4000 q trap software used to control the system . ) rapamycin ( > 99% ) , tacrolimus ( fk-506 , > 99% ) and cyclosporin a ( > 99% ) were obtained from lc laboratories , woburn , ma , usa . davids , pa , usa . ascomycin , formic acid ( 98% , puriss . p.a . ) and cesium hydroxide ( purity 99.97% ) , were purchased from sigma - aldrich , taufkirchen , germany . water was prepared using the purification system direct - q 5 ( millipore gmbh , eschborn , germany ) . the hplc system used was from shimadzu ( duisburg , germany ) consisting of a scl-10a system controller , three lc-10advp pumps ( a , b , c ) , a dgu-14a degasser ; a sil-10ad autoinjector ; a ctu-10as column oven ; and a fcu-12a flow switch ( rotary valve ) . the mass spectrometer was a 4000 qtrap equipped with a turbov esi source with turboion spray probe from applied biosystems ( mds - sciex , darmstadt , germany ) . the chromatographic separation was performed on a phenyl - hexyl - rp column ( luna , 2 x 150 mm , 5 m , phenomenex , aschaffenburg , germany ) . a ternary gradient was used for elution consisting of eluent a ( pump a ) : 0.1% v / v hcooh in water + 0.1 mmol / l csooch ; eluent b ( pump b ) : 0.1% v / v hcooh in methanol + 0.1 mmol / l csooch and eluent c ( pump c ) : 0.1% v / v hcooh in ch3cn + 0.1 mmol / l csooch . the column temperature was set at 50c . in order to avoid contamination of the esi source a switching valve system ( rotary valve a ) was introduced . at position 0 of rotary valve the ms detection was performed in the positive ion mode . the mass transitions ( m / z ) for ms detection are given in table 2 ( tab . ( the abbreviations for settings are specific to the 4000 q trap software used to control the system . ) as shown in previous publications , the generation of cs ion adducts combined with lc - ms / ms offered great advantages in analysis of digoxin and digitoxin , both in sim and in mrm mode . it was observed , that in the mrm mode , the cs adduct as parent ion decomposes in a way that the cs ion becomes detectable as product ion . this principle of measurement was investigated for the immunosuppressive agents tacrolimus , sirolimus , everolimus , cyclosporin a together with the analogues ascomycin , 32-desmethoxy - rapamycin and cyclosporin d used as internal standards . the generation of cs adducts is examplified for cyclosporin a in figure 2 ( fig . the q1 scan was performed by using a standard solution , dissolved in eluent b , which contained 0.1 mm cs . under these conditions the singly charged cs adduct generates the major signal ( 1335 amu ) . only small amounds of the protonated form ( 1203 amu ) , the na adduct ( 1225 amu ) and the k adduct ( 1241 amu ) are visible . na and k probably originate from the manufacturing procedure and can be separated by hplc . fragmentation of the cyclosporin a cs adduct was studied using an 4000 q trap in the enhanced product ion scan mode ( figure 3 ( fig . the ion trap option offers a high mass resolution . in the product ion scan of cs adduct of cyclosporin a with a parent mass of 1335 amu , the mass of 132.9 amu is the main signal . the isotope pattern of this signal and the defined mass indicates that this signal originates solely from cs . during the mass transition the mass of 1202 amu thus , it can be considered , that cyclosporin a is split off in form of a neutral molecule in the fragmentation process . this scan demonstrates , that the only source of the neutral loss of the unprotonated cyclosporin a molecule is the cs adduct of cyclosporin a with the mass of 1235 amu . the adduct formation with cs and the subsequent collision induced dissociation with cs as the product ion was observed for all seven analytes . this principle of detection was used for the development of the chromatographic separation of all immunosuppressive drugs . a baseline separation was realized by using a ternary gradient ( figure 5 ( fig . 9 ) ) and the analogues cyclosporin d ( figure 10 ( fig . 10 ) ) , ascomycin ( figure 11 ( fig . 11 ) ) and 32-desmethoxy - rapamycin ( figure 12 ( fig . 12 ) ) are shown in separate runs . in addition to the declared components , these standard solutions contain additional substances ( figures 6 - 10 ( fig . 9 ) , ( fig . 10 ) ) , which are separated during the chromatographic procedure . tacrolimus , sirolimus , everolimus , ascomycin and 32-desmethoxy - rapamycin contain isobaric components , which probably represent isomers , as has been described elsewhere . for the cyclosporins a and d cross contamination is observed ( figures 11 - 12 ( fig . cyclosporin a contains about 5% cyclosporin d and cyclosporin d contains about 1% cyclosporin a. this is of importance , as cyclosporin d is used as internal standard for quantification of cyclosporin a. for quantification of tacrolimus , ascomycin was used as internal standard , for sirolimus and everolimus the internal standard used was 32-desmethoxy - rapamycin . the validity of this method was tested during the course of this study , in which the inter - run precision was measured using standard solutions . the precision measured expressed as the coefficient of variation ( cv ) - under these conditions was 2.57% for sirolimus , 2.11% for everolimus , 2.31% for tacrolimus and 2.11% for cyclosporin a ( table 4 ( tab . recent lc - ms / ms measurements of immunosuppressive drugs have often used nh4 adducts , which give rise to several different product ions . addition of cs ions reduced the number of product ions to one single product ion , namely the cs ion , which is measurable with high specificity and high signal intensity . it is of utmost importance to have a highly specific analytical method with high accuracy and precision for use as an intended reference measurement procedure . the specificity of current lc - ms / ms methods should be improved by chromatographic separation . in the method presented here , different internal standards listed in the text have been used for the quantification of the four immunosuppressive drugs . the best results were obtained using the corresponding analogues , namely ascomycin for tacrolimus , 32-desmethoxy - rapamycin for sirolimus and everolimus and cyclosporin d for cyclosporin a. this has been discussed elsewhere by vogeser et al . a baseline separation of cyclosporin d from cyclosporin a has been realized , as well as the separation of sirolimus from 32-desmethoxy - rapamycin . if needed , separation of everolimus from the internal standard 32-desmethoxy - rapamycin or tacrolimus from the internal standard ascomycin can be made by slight modifications of the ternary gradient profile . until now analytes used have been extracted from biological sources and contain sodium and potassium ions ( see figure 2 ( fig . 2 ) ) which can be removed by chromatographic separation so that the presence of na and k adducts was less than 2% . the high throughput routine methods which have been published for determination of immunosuppressive drugs do not separate the different immunosuppressive agents chromatographically . as a result , the routine methods currently in use are not suitable for setting target values in eqas . . in patient samples problems may occur during quantification , due to the presence of metabolites . the metabolites are usually more hydrophilic than the parent substance , due to hydroxylation , and can be separated by modifying the ternary gradient system used here . for optimal quantification when stable - labelled compounds become available the method presented here can be developed into a reference measurement procedure for immunosuppressive drugs .	in this study a new principle of measurement in lc - ms / ms ( liquid chromatography mass spectrometry ) for determination of the immunosuppressive drugs sirolimus , everolimus , tacrolimus , and cyclosporin a has been introduced by using the cs+ ion as the product ion in the multiple reaction monitoring mode ( mrm).separation of the immunosuppressive agents was achieved using a phenyl - hexyl - rp column together with a ternary gradient elution profile , consisting of water , methanol and acetonitrile combined with 0.1% v / v formic acid and 0.1 mmol / l cs+ . quantification was performed using cyclosporin d , ascomycin and 32-desmethoxy - rapamycin as internal standards . the inter - run precision of this new method , expressed as the coefficient of variation , was 2.57% for sirolimus , 2.11% for everolimus , 2.31% for tacrolimus and 2.11% for cyclosporin a.
You are an expert at summarizing long articles. Proceed to summarize the following text: natural menopause is the permanent cessation of menstruation which is determined 12 months after the last menstrual period.1 the number of postmenopausal women has been increasing in recent years due to the increase of life expectancy.23 nowadays , most women spend more than one - third of their life beyond their menopause.4 the etiology of menopause is not well - known . however , it seems that it is related to ovarian dysfunction and hormonal changes.5 nearly , 50% to 80% of women complain of menopausal symptoms such as hot flashing , sweating , sleep disturbance , tiredness , and depression.6 menopause can have psychological , physical , and vasomotor symptoms along with sexual dysfunction.578 these symptoms can affect the quality of life ( qol ) . according to the world health organization ( who ) , qol is defined as " an individual 's perception of their position in the context of the culture and value systems in which they live and in relation to their goals".910 a review study in 2015 reported that different sociodemographic factors such as body mass index ( bmi ) , marital dimension , economic dimension , educational level , working status , duration of menopause , parity , and race affected the qol in menopausal women.11 iranian culture give special value and importance on the elderly and older women have a significant place in the culture because of their extensive experience as mothers and wives . in addition , some of the values women hold in iran might be at risk due to mood fluctuations and other challenges might occur related to qol.12 only a few studies showed the association between socio - demographic factors and qol among postmenopausal women.13 therefore , the purpose of this cross - sectional research was to determinate the effective factors on qol among postmenopausal iranian women . we enrolled eligible postmenopausal women in hamadan city located in the west of iran from june 2015 to october 2015 . this research was approved in the ethics committee of hamadan university of medical sciences . according to the results of a cross - sectional study conducted by makvandi et al.14 in iran , the mean ( standard deviation [ sd ] ) qol in postmenopausal women was 9.72 ( 3.27 ) . based on these results , we arrived at a total sample size of 280 at 95% significance level . anticipating an 8% rejection due to failure in completing the questionnaire , we increased the sample size to a maximum of 300 women . for this purpose , we prepared a list of all the 20 health centers in hamadan city and categorized them into four different areas based on socio - demographic status . eligible women were selected randomly based on quotas set for each health center and their names and numbers were listed . if a woman was absent for the interview , she was given a second chance . in case . inclusion criteria consisted of women ( a ) from whose last menstrual period at least 1 year had passed ( b ) had intact uterus and ovaries ( c ) had no history of physical and mental disorders ( d ) and had not used hormone replacement therapy . we used the menopause - specific qol ( menqol ) questionnaire for measuring the qol in postmenopausal women . this questionnaire consists of 29 items in vasomotor ( 3 items ) , psychosocial ( 7 items ) , physical ( 16 items ) and sexual ( 3 items ) domains.15 the validity and reliability tests for this questionnaire were conducted in iran.16 in this study , test - retest ( intraclass correlation coefficients ) was used for the menqol questionnaire reliability . the scores of menqol questionnaire were 0.93 , 0.88 , 0.87 , and 0.92 for the physical , psychological , sexual and vasomotor domains , respectively . a " zero " is equivalent to a woman responding " no " , showing she has not experienced this symptom in the past month . score " one " shows that the woman experienced the symptom , but it was not bothersome at all . scores " two " through " seven " show increasing levels of bother experienced from the symptom and correspond to " 1 " through " 6 " check boxes on the menqol . moreover , some socio - demographic variables were asked from these women including age , menopause duration , marital status , education , employment status , number of children , income level , weight , and height . bmi is divided into underweight ( < 18.5 kg / m ) , normal weight ( 18.5 - 24.9 kg / m ) , overweight ( 25 - 29.9 k / m ) and obese ( 30 kg / m).17 the postmenopausal women were interviewed by female interviewer and on average , each interview lasted 19 minutes . spss version 16 ( spss inc . , chicago , il , usa ) was used for data analysis . the relation of demographic variables with qol was investigated using analysis of variance ( anova ) , t - test and logistic regression analysis . overall , 11 women refused to participate in this study and were replaced by other eligible women . the age range of women was 45 to 65 years and the mean age was 55.46 ( sd = 5.49 ) . the mean ( median ) scores of qol for vasomotor , psychosocial , physical and sexual domains were 11.65 5.93 ( median : 12 ) , 19.36 1.20 ( median : 18 ) , 39.12 1.95 ( median : 39 ) and 11.02 5.66 ( median : 12 ) , respectively . according to the menqol questionnaire most mean scores in vasomotor , psychosocial , physical and sexual domains belong to the items of " night sweats " ( 4.17 2.08 ) , " feeling anxious or nervous " ( 3.34 2.14 ) , " ache in muscles and joints " ( 3.41 2.04 ) and " change in your sexual desire " ( 3.77 2.11 ) , respectively . the mean score of qol in four domains have been presented in table 2 . sixty to 65 years old women had significantly lower qol scores ( better qol ) in psychosocial , physical and sexual domains compared with the other age group and 45 to 49 years old women had significantly higher scores in all the domains of qol . women with university degrees had the lowest scores in the physical domain and the highest scores of sexual , physical and vasomotor domains belonged to women with guidance school degrees . women with fewer than four live children had lower qol scores in psychosocial domain ( p = 0.017 ) . compared to married women , divorced / financial status was associated with physical domain and women with higher incomes had lower scores in qol compared with other groups . menopausal duration was not associated with all the domains of the qol . in vasomotor domain employed women had lower scores than housewives ( p = 0.012 ) . according to the results of the logistic regression analysis after adjusted variables employment and incoming decreased the risk of vasomotor and physical scores above the median , respectively ( table 3 ) . in our study , 45 to 49 years old women had significantly higher scores in all the qol domains . aging in women decreased the scores of qol except for 60 to 65 years old women whose scores increased again in psychosocial , physical and sexual domains . aging in vasomotor domain decreased qol scores . fallahzadeh 's study16 shows the lowest scores for 60 to 65 years old women in the vasomotor domain which is in agreement with our study , while blumel et al . some studies have shown that age is a predictor of reduction in qol.1920 in our study , women with university degrees had lower scores in all domains but this association was significant in vasomotor , psychological and physical domains . similar results have been reported in different studies.212223 in contrast , fallahzadeh 's study16 showed that women with university degrees had the highest mean scores in vasomotor domain . these studies show that knowledge and education are the reason for having fewer problems and a better qol . having an education makes a positive contribution to women 's lives in that they reach a higher level of income . in this study , this is similar to the study of blumel et al.18 another study13 reported that working women had lower scores of qol compared to housewives which was not significant . women with more than three children obtained higher scores of qol compared to those who had fewer than four children , this difference was significant in psychosocial domain . this is similar to other studies.13 fallahzadeh16 in iran reported that the number of children had a significant impact on physical domain scores . a study defined that higher number children could increased parental stress and responsibility as well as financial problems.24 women with lower incomes had significantly higher mean scores in physical and psychosocial domains . this is similar to fallahzadeh 's study.16 daley et al.25 reported that obese women had significantly higher vasomotor and somatic symptoms than women of normal weight . a review study reported that obesity was associated with a poor health - related qol in postmenopausal women.26 in our study , a high bmi predictor higher scores in vasomotor , psychosocial and physical domains of qol . obesity is a health problem that can decrease qol in postmenopausal women.22 based on the present study , duration of menopause did not have any impact on qol , and this finding has been confirmed by another study.13 in contrast , caylan et al.27 reported that duration of menopause was associated with higher scores in psychosocial , physical and sexual domains in qol . it seems that low qol more than four years after menopausal duration may be due to various factors such as race , age and culture.28 this study assessed qol and related factors in postmenopausal women in iran . our study had a few limitations : ( a ) the women self - reported their weight and height . the literature shows that overweight and obese women are more likely to under - report their weight while those who are underweight are more likely to over - report their weight.2930 ( b ) our design was a cross - sectional study . we had no information about the women 's qol in their premenopausal period which could affect their postmenopausal qol . therefore , more evidence based on large prospective cohort studies is required for a more precise investigation of effective factors on postmenopausal women 's qol . menopause causes a decrease in qol , which is dependent to age , work , bmi , financial status and number of children variables . therefore , it is necessary to develop effective intervention programs to improve qol in postmenopausal women .	objectivesmenopause can have psychological , physical , and vasomotor symptoms along with sexual dysfunction and these symptoms can affect the quality of life ( qol ) . the purpose of this research was to determine and association the effective factors on qol among postmenopausal iranian women.methodsthis cross - sectional study was conducted in 2015 on 300 postmenopausal women in hamadan , iran . we used the menopause - specific qol questionnaire ( menqol ) for measuring qol in postmenopausal women . spss version 16 was used for data analysis.resultsthe mean scores of qol for vasomotor , psychosocial , physical , and sexual domains were 11.65 5.93 , 19.36 1.20 , 39.12 1.95 and 11.02 5.66 , respectively . higher scores had worse qol . using menqol scores , our study showed significant differences in qol based on age , education level , financial status , number of children , employment , and body mass index ( bmi).conclusionmenopause causes a decrease in qol , which is dependent to age , work , bmi , financial status and number of children variables . therefore , it is necessary to develop effective intervention programs to improve qol in postmenopausal women .
You are an expert at summarizing long articles. Proceed to summarize the following text: thyroid eye disease ( ted ) is an autoimmune process which involves abnormal proliferation and activation of orbital fibroblasts , increased glycosaminoglycans , and proinflammatory cytokine production . cytokines can modulate the immune reaction in ted by increasing major histocompatibility complex class ii , adhesion molecules , prostaglandin , and heat shock protein expression in the orbit [ 1 , 2 ] . glucocorticoid therapy is effective in ted as it has anti - inflammatory and immunosuppressive actions , inhibits release of immune mediators such as cytokines , interferes with function of t and b lymphocytes , and decreases glycosaminoglycan production by orbital fibroblast [ 3 , 4 ] . pulsed intravenous methylprednisolone ( ivmp ) therapy is an established treatment of active moderate to severe ted and sight - threatening dysthyroid optic neuropathy . ivmp therapy is also more efficacious and associated with fewer side effects than oral steroids [ 6 , 7 ] . the favourable response rates of pulsed ivmp are about 80% , as compared with about 60% for oral steroids . however , there are concerns regarding the safety profile of pulsed ivmp , especially on the cardiovascular system . minor effects include flushing , mild hypertension , gastritis , weight gain , depression , hyperglycemia , insomnia , palpitations , sinusitis , and urinary tract infections [ 7 , 912 ] . major adverse events reported include cardiovascular events , acute liver damage , and even death [ 1318 ] . there is no established consensus on the monitoring of patients during and after ivmp administration . previous studies had reported ivmp resulting in hypertension in ted patients [ 9 , 19 ] or bradycardia [ 20 , 21 ] , but none looked at the extent and pattern of blood pressure and heart rate fluctuations at various time points after the steroid infusions . in addition , literature detailing the risk factors for such fluctuations is scarce [ 9 , 21 ] . the purpose of this study is to analyze the changes in vital signs ( including heart rate ( hr ) , systolic ( sbp ) , and diastolic blood pressure ( dbp ) ) during and after ivmp infusion ( 0 min , 30 min , 60 min , and 90 min ) , as well as changes in electrocardiogram ( ecg ) , potassium levels , glucose levels , and any other adverse effects . we performed a retrospective review of the charts of ted patients who underwent pulsed methylprednisolone therapy from 2004 to 2010 in singapore national eye centre . numerous different parenteral regimes for ivmp have been reported , with no clear consensus on the optimum dosage , dosing intervals , and duration of treatment [ 8 , 22 ] . the dosing regimen used in our institution during the study period consisted of 1 g hourly per day over 3 days given in the outpatient setting [ 11 , 23 , 24 ] . the study was carried out in accordance with the tenets of world medical association 's declaration of helsinki . all subjects were examined for best correctable visual acuity ( bcva ) , colour vision , visa scoring , and slit - lamp examination prior to pulsed methylprednisolone therapy . baseline blood investigations including full blood count , liver function test , renal panel , glucose , hepatitis b and hepatitis c screening , and ecg were performed . patients who had any symptoms or signs of infection , severe gastric disease , unstable cardiac disease , active liver disease , and poorly controlled diabetes mellitus or hypertension were excluded from ivmp treatment . all subjects had their vital signs ( hr , sbp , and dbp ) charted before infusion ( 0 min ) , at 30 min ( during infusion ) and 60 min ( end of infusion ) , and after infusion ( 90 min ) . all subjects had to complete a questionnaire at the end of infusion which documented any subjective complications experienced . subjects were reviewed after each cycle of 3 doses of ivmp for bcva , colour vision , visa score , and full slit - lamp examination . subjects were reviewed at interval of 1 to 3 months after treatment depending on their condition and physician 's discretion . subjects may undergo repeated cycles of ivmp if the ted was considered active or patients developed signs of compressive optic neuropathy . statistical analysis was performed with spss software ( ibm corp . , released 2010 , ibm spss statistics for windows , version 19.0 , armonk , ny ) . a linear mixed model was utilized to analyze and account for the cumulative effect of the multiple doses of each cycle of methylprednisolone . three candidate covariance structures were checked for minimizing the mean squared error of predictions , first - order autoregressive [ ar(1 ) ] , compound symmetry ( cs ) , and unstructured ( un ) , to estimate the fixed effects and we have selected the structure with the smallest akaike 's information criterion ( aic ) and bayesian 's information criterion ( bic ) . the study included 38 subjects who underwent a total of 242 infusions of ivmp ( table 1 ) . the mean age ( standard deviation , sd ) was 48.4 8.8 years and 65.8% were male . the subjects underwent a mean ( sd ) of 2.3 1.5 cycles of ivmp ( range : 1 to 7 cycles ) . twenty - six subjects required adjuvant therapy of methotrexate , and 10 subjects required radiotherapy . more than half ( 57.9% ) of the subjects showed hyperthyroidism ( defined as raised free thyroxine with a suppressed thyroid stimulating hormone ) on thyroid function test at baseline . there was a small but statistically significant percentage drop in mean ( sd ) sbp at 30 min and 60 min compared to baseline sbp ( 3.31 9.91% , p < 0.001 , and 2.25 12.18% , p = 0.03 , resp . ) but no significant difference after stopping infusion at 90 min ( 0.67 11% , p = 0.126 ) . there was also small but statistically significant drop in mean percentage change dbp at 30 min , 60 min , and 90 min compared to baseline dbp ( 2.92 10.6% , p < 0.001 , 2.1 11.82% , p = 0.001 , and 1.34 12.63% , p = 0.02 , resp . ) ( figure 1 ) . the ivmp resulted in a significant decrease in mean percentage change hr ( p < 0.001 ) from 0 min to 30 min ( 5.73 10.14% ) , 60 min ( 5.58 11.36% ) , and 90 min ( 5.92 12.12% ) ( figure 2 ) . after comparing the sbp , dbp , and hr between the 3 doses of ivmp , there were no statistically significant cumulative effects of methylprednisolone on percentage change of sbp , dbp , and hr between doses 1 , 2 , and 3 within each ivmp cycle . there was a significant decrease ( p = 0.045 ) in mean ( sd ) potassium levels from 4.14 0.36 mmol / l to 4.08 0.37 there was no significant cumulative effect on potassium levels between each dose of the cycle ( p = 0.837 ) . the mean ( sd ) random capillary glucose level was significantly increased from 7.76 3.28 mmol / g to 11.87 5.87 mmol / g after infusion ( p < 0.001 ) . there was also no significant cumulative effect on glucose levels between each dose ( p = 0.563 ) . there were 4 patients ( 10.5% ) who required subcutaneous insulin therapy to correct the hyperglycemia , and all these subjects had preexisting type 2 diabetes mellitus . overall , there was improvement in both bcva and colour vision after treatment after each cycle of 3 doses of 1 g ivmp , although it was not statistically significant ( p = 0.348 and p = 0.685 for bcva right and left eye and p = 0.459 and p = 0.641 for colour vision right and left eye , resp . ) . however , if only the subjects with compressive optic neuropathy were analyzed , there was a significant improvement in both visual acuity and colour vision ( table 2 ) . there was also significant improvement ( p < 0.001 ) of visa inflammatory score from mean score ( sd ) of 4.29 1.14 before treatment to 1.92 1.12 after treatment . examining the overall adverse effects of ivmp , there were 6 ( 2.5% ) episodes of bradycardia ( hr less than 50 bpm ) , 8 ( 3.3% ) episodes of tachycardia ( hr more than 100 bpm ) , and 12 ( 5% ) episodes of moderate to severe hypertension or stage 2 hypertension ( sbp > 160 mmhg or dbp > 100 mmhg ) . these 12 episodes occurred in 4 subjects ; one of them was hyperthyroid with no underlying hypertension and two were hypothyroid and hypertensive while one was euthyroid with underlying hypertension . three subjects ( 1.2% ) had severe hypertension ( as defined by sbp 180 mmhg or dbp 110 mmhg ) ( table 3 ) . one patient developed urticarial rash and the pulsed methylprednisolone was stopped immediately and hence did not complete the cycle of 3 doses of therapy . two patients ( 5.3% ) had mildly deranged liver enzymes , which all reverted to normal by 1 year after the administration of ivmp . two subjects complained of breathlessness and 1 had chest tightness , with no accompanying vital sign or ecg changes . these symptoms did not recur during the subsequent doses of ivmp in the two subjects . pulsed methylprednisolone infusion for treatment of active ted has been shown to be relatively safe and effective . ivmp therapy is established to be effective in improving inflammation in active ted , more efficacious than oral steroids with less adverse events [ 8 , 27 ] . consistent with the current literature , our study showed that these patients had improved inflammatory score and those with compressive optic neuropathy had improvement in visual acuity . steroids result in sodium retention and volume expansion and act on nitric oxide synthase pathway . steroids may also mediate vascular vasoconstrictor sensitivity to catecholamines and other vasoconstrictor hormones systems [ 30 , 31 ] . our results showed that ivmp had a general trend in lowering the sbp and dbp ; however the mean change was small and may not be clinically significant . although the effects of ivmp on bp were not cumulative in our study , we found 12 ( 5% ) episodes of moderate to severe hypertension ( sbp 160 mmhg or dbp 100 mmhg ) , and three subjects had sbp 180 mmhg or more at the end of infusion . all these patients ' blood pressure returned to normal at 90 minutes and remained asymptomatic . nonetheless , ivmp should be used with caution in those with poorly controlled hypertension since sudden fluctuation in blood pressure may still exert stress on the cardiac system and result in pulmonary oedema and acute heart failure . there were three patients with thyroid dysfunction at baseline who developed moderate to severe hypertension during ivmp infusion . both hyperthyroidism and hypothyroidism are associated with increased blood pressure ; hypothyroidism is a recognised cause of secondary hypertension , and hyperthyroidism results in higher risk of elevated sbp , widened pulse pressure , sinus tachycardia , and atrial fibrillation and may even lead to heart failure [ 3335 ] . ivmp administration has been associated with cardiac arrhythmias in some studies [ 20 , 36 ] . although ivmp resulted in decrease in hr that remained low at 90 min after ivmp infusion in our study all the subjects who developed bradycardia in our study were on beta - blockers , while the subjects who developed tachycardia were hyperthyroid and were already tachycardic at baseline . our study suggests that patients on beta - blockers were more susceptible to developing bradycardia . bradycardia after steroid administration had been reported , but the mechanisms were not well studied [ 20 , 21 ] . one explanation for the occurrence of reversible bradycardia is by the pharmacokinetics of steroid operating through the hypothalamic - pituitary axis suppression . ivmp may also cause hyperglycemia , especially in diabetic patients and transient deranged liver enzymes ; thus careful screening of patient 's glucose levels and liver function should be performed prior to ivmp administration [ 10 , 38 ] . in our study , the four subjects who required insulin therapy for correction of hyperglycemia were all diabetics . similarly in a large study by feldman - billard et al . , the authors found that methylprednisolone therapy resulted in raised glucose levels , but the effects were different in nondiabetics and diabetics . in nondiabetics , the hyperglycemia was maximum after first pulse and returned to baseline with subsequent pulses , but diabetic patients had cumulative effects of the hyperglycemia with repeated pulses , and a quarter of the diabetic patients required insulin . our study showed that four out of seven diabetic patients required insulin at some point during their cycles but did not show any overall cumulative effects of glucose with repeated pulses . however , our study did not analyze the cumulative effects on diabetics alone due to the small numbers . although the hyperglycemic effects may be transient and correctable , acute hyperglycemia is an independent risk factor of cardiovascular events and should be prevented [ 39 , 40 ] . hypertension and hyperglycemia may also result in pathophysiological changes in the retina , such as arteriolar narrowing , retinal haemorrhages , microaneurysms , exudates , and optic disc swelling [ 41 , 42 ] . although the hypertensive effects of methylprednisolone may be transient , the acute increase of blood pressure may lead to vascular necrosis , resulting in necrosis of smooth muscle and endothelial cells . acute retinopathy changes associated with hypertension may be generally reversible , but potential worsening of diabetes control induced by repeated pulsed high dose methylprednisolone therapy may exert irreversible damage to end target organs through the metabolic memory . they postulated that ivmp caused liver damage by either direct damage on liver cells , precipitation of virus - induced hepatitis , or reactivation of immune system after ivmp - induced immune suppression resulting in autoimmune hepatitis our subjects underwent a large range of ivmp courses ( one to seven cycles ) , and seven patients had cumulative doses of more than 9 g. of note , six of these subjects were treated between 2004 and 2007 , which were before the recommendation that ivmp should not exceed cumulative dose of 8 g . none of our patients developed liver failure , and the two subjects who had mild increase in liver enzymes ( less than three times of the upper reference limit ) had less than 8 g administered . their liver function tests returned to normal within 1 year after the ivmp pulses were stopped . in our study , one subject developed urticarial rash near the end of the 60-minute infusion . anaphylactic shock had been reported in ivmp administration [ 45 , 46 ] ; hence it is recommended that the drug be infused slowly over 60 min . despite demonstrating a decrease in potassium levels after ivmp infusion , this may suggest that in relatively well patients with normal potassium at baseline , there is no need to recheck their potassium levels after ivmp administration . the caveat is that these patients should not have any underlying medical conditions that may result in them having electrolyte abnormalities , such as renal failure or use of medications such as diuretics . the main strengths of our study were the large number of 242 infusions analyzed and recorded patients who had multiple infusions administered with the long follow - up duration . the main limitations were the small number of study subjects and the large variance of dosage administered ( cumulative dose : 1 g to 21 g ) instead of a fixed cumulative dosage administered . however , such variation is expected in the management of patients with active ted where response to ivmp may be variable and other forms of second - line treatment may be required at different time points during the course of treatment . ivmp is a relatively safe and efficacious therapy for active moderate to severe ted and optic neuropathy . overall , ivmp administration resulted in mild and noncumulative effects on heart rate and blood pressure . however , patients with poorly controlled hypertension , diabetes , or active liver disease should have their underlying conditions optimised before undergoing pulsed methylprednisolone therapy . severe hypertension may occur during ivmp treatment in susceptible individuals such as those with underlying hypertension and uncontrolled thyroid dysfunction , whereas bradycardia may be more likely in those on beta - blockers . patients whose potassium levels are normal at baseline and are not predisposed to hypokalemia from drugs or medical conditions may not require close monitoring of their potassium levels after every dose .	objective . to analyze changes in vital signs ( heart rate ( hr ) , systolic ( sbp ) , and diastolic blood pressure ( dbp ) ) during and after intravenous methylprednisolone ( ivmp ) and any other adverse effects . methods . retrospective review of charts of patients who received ivmp as treatment regime for thyroid eye disease . all subjects had vital signs charted during and after infusions . results . this study included 38 subjects and a total of 242 infusions administered . ivmp resulted in a small but significant percentage drop in mean sbp at 30 min ( p < 0.001 ) and 60 min ( p = 0.03 ) but no difference at 90 min . there was also small but significant percentage drop in mean dbp and hr ( dbp : p < 0.001 for 30 min , p = 0.001 for 60 min , and p = 0.02 for 90 min and hr : p < 0.001 for 30 min , 60 min , and 90 min ) . there were no cumulative effects on change of blood pressure or hr . there were 6 episodes of bradycardia ( 2.5% ) and 12 episodes of moderate to severe hypertension ( 5% ) . no significant cardiovascular or hepatic toxicity was found . conclusion . ivmp is relatively safe and efficacious . ivmp demonstrated mild and noncumulative effects on vital signs . severe hypertension may occur in susceptible individuals such as those with underlying hypertension and uncontrolled thyroid dysfunction , whereas bradycardia may be more likely in those on beta - blockers .
You are an expert at summarizing long articles. Proceed to summarize the following text: a balance between energy intake and energy expenditure is responsible for body weight maintenance ; disturbances in this balance may cause individuals to be underweight , anorexic , overweight , or obese [ 1 , 2 ] . signals secreted by peripheral tissues , such as fat or the digestive tract , are active participants in body weight regulation by acting at central level . skeletal muscle has recently been shown to function as a peripheral endocrine organ by releasing peptide signal molecules known as myokines , which are implicated in the regulation of several physiological and metabolic pathways [ 4 , 5 ] . myokines secreted during muscle contraction may exert a protective role against illnesses associated with sedentary lifestyles . the muscle - secreted peptide irisin has been recently described as a cleavage product of the type i membrane protein fibronectin type iii domain containing 5 ( fndc5 ) . in muscle , fndc5 is induced by the peroxisome proliferator - activated receptor ( ppar ) transcriptional coactivator pgc1- , which is described as an essential mediator in the exercise - induced energy expenditure cascade and stimulates several beneficial effects of exercise in muscle tissue [ 7 , 8 ] . after physical activity , fndc5 has been reported to promote white adipose tissue browning by increasing thermogenesis and energy expenditure . thus , augmentation of circulating irisin levels by forced liver expression of fndc5 using adenoviral vectors improves glucose tolerance and reduces obesity of mice fed on a high - fat diet . under this context , irisin was rapidly postulated to be beneficial in the treatment of obesity , diabetes , and a wide range of pathological conditions characterized by a variable imbalance of energy demand and expenditure [ 4 , 913 ] . unexpectedly , some studies have indicated that the circulating irisin levels in humans correlate positively with parameters of adiposity such as bmi and are the highest in obese individuals [ 1417 ] . on the other hand , a recent paper reports that irisin correlates negatively with bmi , waist - hip ratio , and fat mass in men and that circulating irisin is lower in nondiabetic overweight and obese men [ 18 , 19 ] . our group has established a new perspective for fndc5 by describing that fndc5/irisin is also an adipokine expressed and secreted by adipocytes from white adipose tissue ( wat ) in rats and humans . we have observed that short - term endurance exercise increases fndc5/irisin secretion by adipose tissue , while food restriction decreased secretion . accordingly , anorectic animals showed discrete secretion of fndc5/irisin , while fat from obese animals oversecretes fndc5/irisin . thus , we postulated that adipose tissue may contribute to circulating fndc5/irisin and that the contribution may vary depending on the physiological state or the pathological situation . the objective of this study was to evaluate the circulating levels of irisin in conditions of extreme bmi , such as anorexia and obesity , and the correlations of irisin with basal metabolism and daily activity . a total of 145 females participated in this study after being informed about the research and signing informed consent . thirty patients were anorexic ( an ; bmi 17.3 1.8 kg / m ; 28.1 9.4 years ) , 66 were obese ( ob ; bmi 42.8 6.7 kg / m ; 29.0 6.2 years ; 21.2% with type 2 diabetes mellitus ) , and 49 were healthy , normal - weight controls ( nw ; bmi 21.7 1.6 kg / m ; 44.5 11.4 years ) . all consecutive patients were referred to seven centers from six spanish sites ( all involved in the ciberobn spanish research network ) : the eating disorders unit ( department of psychiatry , university hospital of bellvitge - idibell , barcelona ) , the department of endocrinology at the university hospital of santiago ( santiago de compostela ) , the department of diabetes , endocrinology and nutrition ( clinic university hospital virgen de victoria , mlaga ) , the department of endocrinology ( university of navarra , pamplona ) , the diabetes , endocrinology and nutrition department , biomedical research institute of girona ( idibgi - doctor josep trueta hospital , girona ) , the hospital del mar research institute ( imim - hospital del mar , barcelona ) , and the department of basic psychology , clinic and psychobiology ( university jaume i , castelln ) . the an patients were diagnosed by experienced clinicians ( according to dsm - iv - tr diagnostic criteria ) using the structured clinical interview for dsm iv axis i disorders ( scid - i ) . these an patients at extreme bmi were hospitalized , hence showing weaken health and very limited physical activity . the following exclusion criteria were applied to the clinical cases groups ( an and obese patients ) : ( 1 ) male ; ( 2 ) age under 18 or over 60 years , and ( 3 ) a comorbid binge eating disorder in the obese patients ( according to dsm - iv - tr criteria , using scid - i ) . healthy controls were recruited by several methods including word - of - mouth recruitment and advertisements in the local university . prior to assessment , the healthy controls were asked about lifetime or current presence of an endocrine disease or obesity . the lifetime history of health or mental illnesses profile was based on the general health questionnaire ( ghq)-28 . the following exclusion criteria were applied to the healthy control groups ( nw ) : ( 1 ) individuals who have suffered a lifetime eating disorder ( assessed using scid - i ) , ( 2 ) age under 18 and over 60 years , ( 3 ) lifetime obesity ( bmi > 30 ) , and ( 4 ) being male . anthropometric , body composition , and resting energy expenditure ( ree ) measurements were performed by bioelectrical impedance ( tanita mc-180 multifrequency ; tanita corporation of america , inc , arlington heights , il , usa ) , equipped with 8 tactile electrodes with a platform with 2 electrodes for each foot and 2 handgrips with 2 electrodes each . the bioelectrical impedance analysis was chosen since it is able to predict the ree without bias with respect to more costly equipment and invasive procedures needed to directly measure ree . , cambridge neurotechnology , cambridge , uk ) , which is a small ( 39 32 9 ) and light - weight ( 10.5 g ) accelerometer that measures sleep - wake physical activity . the actiwatch is worn on the nondominant wrist for 6 days ( 4 week days and 1 weekend ) , from 00:00 hr on day 1 to 00:00 hr on day 7 . the data were collected over a 1-minute epoch length , and a 6-day average was calculated . the analysis was performed using the actiwatch 7 software ( camntech ltd . and cambridge neurotechnology ltd . , cambridge , uk ) . the actiwatch aw7 originates from the actiwatch aw4 , which has similar reliability to other accelerometers and is an adequate instrument for assessing daily physical activity . the quantitative measurement of irisin in human plasma samples was performed using a commercial enzyme - linked immunosorbent assay ( elisa ) kit directed against amino acids 31143 of the fndc5 protein ( irisin elisa kit ek-067 - 52 ; phoenix pharmaceuticals inc . the absorbance from each sample was measured in duplicate using a spectrophotometric microplate reader at wavelength of 450 nm ( versamax microplate reader ; associates of cape cod incorporated , east falmouth , ma ) . insulin was analysed by an immunoradiometric assay ( biosource international , camarillo , ca , usa ) in a beckman coulter ( fullerton , ca , usa ) , showing 0.3% cross - reaction with proinsulin . glucose was measured using a dimension autoanalyzer ( dade behrng , deerfield , il , usa ) . the homeostatic model assessment index ( homa - ir ) was calculated following the formula ( fasting plasma glucose ( mg / ml ) fasting plasma insulin ( mu / l)/405 ) , as described elsewhere . the human adipose tissue specimen was obtained with written informed consent approved for this particular study by the comit tico de investigacin clnica de galicia ceic de galicia ( spain ) according to the declaration of helsinki . adipose tissue was obtained from nw ( body mass index < 35 ) who underwent cholecystectomy surgery and ob patients ( body mass index > 35 ) who underwent laparoscopic gastrectomy surgery . the visceral fat was located in the hypogastric region around the internal organs , and the subcutaneous fat was located in the mesogastric region . the tissues were transported from the operating room to the laboratory in sterile krh buffer with penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) . secretome and tissues were collected and processed for immunodetection as previously described [ 5 , 16 ] . the sample size of the current trial was estimated by calculating the differences in irisin between the ob and nw patients according to the equation reported by mera et al . . the sample size was established at a minimum of 26 volunteers per group to detect differences according to the adiposity levels . the normal distribution of the variables was tested using the kolmogorov - smirnov and the shapiro - wilk tests . however , based on the sample size of > 60 subjects included in the current study , a one - way anova was used to study the differences between an , nw , and ob subjects adjusted by age . the potential association between anthropometric and biochemical parameters with and irisin levels was evaluated using the pearson coefficient test . multivariate linear regression models were fitted to explain the variations in the irisin plasma levels . data are reported as the mean se , and confidence intervals ( 95% ci ) are used to describe the linear coefficient ( b ) values . as expected , the ob subjects exhibited higher body weight with higher fat mass and fat - free mass compared with the an and nw subjects , with the differences being statistically significant after adjusting by age ( figure 1(a ) ) . the nw subjects exhibited statistically higher body weight compared with the an subjects accompanied by higher fat mass and fat - free mass . considering the body composition as a percentage of the total body weight , the an subjects exhibited higher fat - free mass than the nw and ob subjects ( figure 1(a ) ) . the differences in the anthropometrical parameters were consistent with the significantly increased plasma irisin concentrations in the ob subjects compared with the an and nw groups ( figure 1(b ) ) . the nw subjects exhibited a slight but not statistically significant increase compared with the an subjects ( p > 0.05 ) . further analysis showed that the irisin levels were positively correlated with two parameters of adiposity ( i.e. , body weight ( r = 0.52 ; p < 0.001 ) and bmi ( r = 0.52 ; p < 0.001 ) ) . the irisin plasma levels were not correlated with height ( r = 0.05 ; p > 0.05 ) . the correlations indicated that irisin strongly reflects the body fat mass component , which is further supported by the bioelectrical impedance analysis of body composition in which the irisin plasma levels correlated with the fat mass measured in kg ( r = 0.52 ; p = 0.001 ; figure 2 ) and the percentage of fat mass ( r = 0.48 ; p < 0.001 ) . however , the muscle component may also contribute to irisin circulating levels as shown by the positive correlation between irisin and fat - free mass ( r = 0.43 ; p = 0.001 ; figure 2 ) . an inverse correlation was observed between the irisin levels and the percentage of fat - free mass ( r = 0.53 ; p < 0.001 ) . the associations between irisin and fat mass and fat - free mass were maintained after adjusting for the studied groups ( data not shown ) . in addition , a direct association was observed between plasmatic levels of irisin and glucose ( r = 0.22 ; p = 0.0026 ) , insulin ( r = 0.34 : p < 0.001 ) , and homa - ir ( r = 0.33 ; p = 0.001 ) . this positive association was maintained especially for insulin and homa - ir after adjusting for the studied groups . because irisin secretion was shown to be induced by exercise and energy expenditure , the daily physical activity and resting energy expenditure ( ree ) were evaluated in the three bmi groups . the actiwatch analysis in the ob subjects exhibited statistically lower daily physical activity with higher resting energy expenditure ( ree ) compared with the nw subjects adjusted by age ( figures 3(a ) and 3(b ) , resp . ) . therefore , the ob subjects exhibited higher daily physical activity compared with the extreme an hospitalized subjects displaying very limited activity but lower activity compared with the nw subjects ( p < 0.05 ) . obesity was accompanied by increased ree compared with the an and nw subjects ( p < 0.05 ) . the plasma irisin levels were inversely correlated with daily physical activity ( r = 0.22 ; p = 0.001 ) and directly correlated with ree ( figures 3(c ) and 3(d ) ) . after adjusting by the bmi group , only the association between irisin and ree was maintained ( r = 0.34 ; p = 0.001 ) , and the daily physical activity was not statistically significant ( r = 0.06 ; p = 0.432 ) . to further investigate the potential factors involved in the changes in the irisin plasma levels , a multiple regression model was performed including ree and physical activity , adjusting for age , and bmi group ( table 1 ) . < 0.001 ) of the irisin levels variability , mainly depending on ree , whereas physical activity was not statistically associated . when fat free mass was included in the regression model , the association between ree and irisin plasma levels persisted . however , when the same analysis was performed including fat mass in the multivariate model , ree becomes significantly unassociated with irisin circulating levels , whereas that fat mass was the main factor and explained approximately 30% ( p < 0.05 ) of the irisin concentration variability independently of age , bmi group , fat - free mass , daily physical activity , and ree ( table 1 ) . an increase in 1 kg of fat mass was associated with a 2-fold increase in the irisin plasma levels ( p = 0.003 ) . these results indicate that ree influence the irisin circulating levels but in a lesser extent than fat mass . in order to assay the participation of adipose tissue on irisin circulating levels , the immunodetection of this protein was tested on adipose tissue biopsies from healthy and obese individuals . as shown in figure 4 , fndc5/irisin was detected on visceral and subcutaneous adipose tissue secretion from nw and ob individuals ( figure 4(a ) ) and in ob at both intracellular and secretion level ( figure 4(b ) ) . previous studies are rather controversial , and there is no general agreement about circulating irisin levels and their correlation with bmi [ 14 , 15 , 17 , 18 ] . the current study performed with patients in conditions of extreme bmi confirms the positive correlation of circulating irisin and different parameters of adiposity including body weight , bmi , and fat mass . this work contributes to novel valuable information by showing that circulating irisin levels correlate positively with resting energy expenditure and negatively with daily physical activity . therefore , the inverse correlation of irisin with daily physical activity and the direct relationship between irisin and adiposity indicate that adipose tissue may be a primary inducer of irisin secretion , especially in obesity . the recent discovery of the pgc1--dependent myokine fndc5/irisin , which mediates brown - fat development and thermogenesis in white fat , has opened a new field of research [ 4 , 7 , 913 ] . the soluble form of fndc5 binds to unidentified receptors on the surface of wat to induce the expression of ucp1 and other bat - associated genes partly via increased ppar- expression . as a result , irisin may act as a muscle - derived energy - expenditure signal that directly communicates with adipose tissue and induces browning . this effect may improve the wat metabolic profile and enhance whole - body energy expenditure , making irisin a potential new target for the treatment of metabolic diseases . the possible beneficial effect of irisin on the treatment of obesity has been challenged by recent reports showing unexpected high levels of irisin in obese animals and humans [ 1417 ] as well as its association with the insulin resistance onset in association with weight regain . moreover , it was observed that in obese individuals , body weight and bmi reduction by bariatric surgery or by nutritional intervention significantly reduced irisin circulating levels [ 15 , 17 ] and its depletion after an energy restricted program is associated with the reduction of important lipid and carbohydrate metabolism biomarkers in patients with metabolic syndrome . other reports have described conflicting data indicating a lack of correlation between the circulating irisin concentration and the adiposity parameters . this discrepancy could be due to methodological differences because there is no consensus concerning the identity of the soluble portion of the fndc5 membrane protein , the mechanism of secretion , or the presence of variations on target epitopes among manufacturers . however , most published reports indicate correlations between irisin and adiposity levels . the present work further corroborates these findings ; irisin plasma levels were elevated in obesity , and a strong relationship was found between irisin and several adiposity parameters . in the current work , the obese subjects exhibited higher ree compared with the normal weight and anorexia nervosa subjects and it was directly related to irisin levels , independently of daily physical activity . this association between irisin and energy expenditure was previously reported in a cohort of postmenopausal women and it was proposed that this association could be explained by the irisin action on the brown adipose tissue thermogenesis . however , the association between irisin and ree appears to be masked by the strong association between fat mass and irisin plasma levels . fat mass was revealed as the main factor explaining approximately 30% of the variability in the irisin plasma levels independently of age , fat free mass , daily physical activity , ree , and bmi group . the fact that daily physical activity was found to be independent of plasma irisin levels in our work may seem contrary to what was expected . bostrom 's results have shown the expression and secretion of irisin after endurance exercise , which can not be compared to normal daily activity assayed in our paper . moreover , there are studies that , in contrast to the initial report by bostrom , have shown no variations of fndc5 mrna expression in muscle or at circulating level after aerobic or strength exercise training [ 15 , 33 , 34 ] . on the other hand , an increase of irisin circulating levels was observed after short - term exercise ( sprints ) which suggest that irisin secretion is dependent on acute or chronic sorts of exercise with a possible counter - regulation / adaptation over time . therefore , our results in relation to daily activity are coherent with previous evidences in the bibliography . our group has previously described that fndc5/irisin is a myokine and an adipokine expressed and secreted by wat . interestingly , we observed that explants of adipose tissue from obese animals or humans oversecreted irisin compared with the control subjects . therefore , we suggest that the muscle / adipose secretion ratio may vary and may be affected by the physiological situation ; during exercise , muscle tissue strongly participates in the fndc5 circulating levels , while in atypical bmi cases , such as obesity , the adipose tissue would actively increase the circulating fndc5/irisin . the current work further reinforces this hypothesis , because the main difference between the obesity and normal weight and anorexia nervosa subjects is the higher fat mass ; therefore , adipose tissue could be involved in the circulating irisin concentration differences observed depending on the bmi as reinforced in preliminary experiments assaying fndc5/irisin immunodetection in adipose tissue from nw and ob individuals in figure 4 . the results in the present work with previous reports [ 1417 , 36 , 37 ] might suggest a possible mechanism of resistance to irisin in situations of elevated bmi as previously observed in other adipokines ( e.g. , leptin ) . the elevated concentration of circulating irisin in overweight and obese subjects suggests that a great amount of bat would be expected in these patients . although it is now clear that bat is present and functional in adult humans , significant low amounts of bat have been described in overweight and obesity , which supports the irisin - resistance hypothesis [ 39 , 40 ] . however , the current work demonstrates that irisin circulating levels is associated with energy expenditure independently of daily physical activity but in a lesser degree than fat mass , which was revealed as the main factor influencing the circulating irisin concentration in obese women with extreme bmi from anorexia nervosa to obesity . therefore , these findings reinforce the positive relationship between circulating irisin and adiposity and the relevance of wat - secreted irisin in situations of elevated bmi , such as obesity . further studies are needed to elucidate whether irisin is involved in obesity development or irisin resistance that exist in an obesity state to counteract the metabolic disturbances related to excess adiposity .	fndc5/irisin has been recently postulated as beneficial in the treatment of obesity and diabetes because it is induced in muscle by exercise , increasing energy expenditure . however , recent reports have shown that wat also secretes irisin and that circulating irisin is elevated in obese subjects . the aim of this study was to evaluate irisin levels in conditions of extreme bmi and its correlation with basal metabolism and daily activity . the study involved 145 female patients , including 96 with extreme bmis ( 30 anorexic ( an ) and 66 obese ( ob ) ) and 49 healthy normal weight ( nw ) . the plasma irisin levels were significantly elevated in the ob patients compared with the an and nw patients . irisin also correlated positively with body weight , bmi , and fat mass . the ob patients exhibited the highest ree and higher daily physical activity compared with the an patients but lower activity compared with the nw patients . the irisin levels were inversely correlated with daily physical activity and directly correlated with ree . fat mass contributed to most of the variability of the irisin plasma levels independently of the other studied parameters . conclusion . irisin levels are influenced by energy expenditure independently of daily physical activity but fat mass is the main contributing factor .
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Proceed to summarize the following text: renal cell carcinoma ( rcc ) accounts for about 2% of all cancer cases worldwide and represents the sixth leading cause of all cancer deaths [ 1 , 2 ] . one - third of patients present at advanced stages of disease , and up to 40% of patients who underwent local surgical resection will have disease recurrence [ 3 , 4 ] . despite its relatively low incidence , rcc presents itself as one of the most common sources of brain metastases along with lung and breast cancer , melanoma , and colorectal carcinoma . approximately 1,200 to 1,500 cases of brain metastases from rcc are diagnosed annually , and 4% to 17% of all patients with rcc will develop brain metastases during their clinical course of disease . the median survival of patients with untreated brain metastases from primary rcc is reported to be approximately 1 to 2 months , whereas the median survival time after radiotherapy and corticosteroid treatment for patients with this type of malignancy was reported to be 2 to 8 months . since surgical resection is not always possible , wbrt has played an important role in the treatment of patients with rcc brain metastasis but has yielded unsatisfactory results in terms of overall survival and local tumor control in these patients due to the relative radioresistant nature of rcc to conventional radiation therapy . due to the potential neurotoxic effects of wbrt as well as the radioresistant features of this primary , wbrt may not be the treatment of choice in these patients , particularly with oligometastatic disease . stereotactic radiosurgery ( srs ) is a minimally invasive radiation technique that delivers a highly conformal , high dose of radiation to a prescribed target volume [ 11 , 12 ] . this procedure can be completed in one up to five treatment sessions and offers the possibility to treat multiple tumor sites during one treatment session . stereotactic radiosurgery is increasingly used for the treatment of brain metastases with or without prior microsurgical resection [ 13 , 14 ] , as tumors traditionally considered to be radioresistant such as renal cell carcinoma have shown favorable response rates in various studies [ 7 , 1520 ] . however , the optimal treatment of these patients still remains controversial . in this study , our aim was to analyze the outcomes after srs for the treatment of brain metastases from rcc . furthermore , we examined potential prognostic factors that correlate with improved survival and local tumor control in these patients . this is a retrospective evaluation of all patients treated with srs for brain metastases from primary rcc at our institution . patients ' medical records were reviewed to obtain patient , tumor , and treatment characteristics and follow - up data . neuroimaging studies for each individually treated lesion were reviewed prior to radiosurgical treatment and at regular intervals ( 1 , 3 , 6 , 9 , 12 , and 24 months ) after completion of srs . data were collected by personnel not directly involved in either direct patient care or any related treatment decision - making process . the design and analysis of this study were approved by the institutional review board ( irb ) of dana farber / harvard cancer center ( df / hcc ) ( irb#09 - 451 ) . the study cohort consists of 76 patients with rcc brain metastases treated with srs at bidmc between august 2005 and december 2013 . for 10 ( 13.2% ) patients , evaluation of overall survival , local and distant brain tumor control were performed for the remaining 66 patients with a total of 207 lesions , for which all follow - up data sets were available and analysis was completed . in 65/66 patients ( 98.5% ) , brain metastases from rcc were diagnosed by magnetic resonance imaging ( mri ) . in one patient ( 1.5% ) , the diagnosis was based on computed tomography ( ct ) alone , since the patient harbored a contraindication to undergo mri scanning . each patient 's performance status was assessed at each visit using the karnofsky performance status ( kps ) and was further classified by prognosticators for assessment of their outcomes . all patients were treated in the cyberknife ( accuray inc . , sunnyvale , california ) robotic frameless stereotactic radiosurgery system . diagnostic thin slice ( 1 mm ) gadolinium enhanced axial mri images were fused with ct scan obtained in an immobilization mask at planning . patients were followed up from the time of srs with clinical examination and neuroimaging with contrast enhanced mri 1 month after treatment and every 2 - 3 months thereafter until the last follow - up appointment or until the date of death . overall survival , local control , distant brain control , local progression - free survival , and distant brain progression - free survival were assessed after srs . overall survival was calculated as the time in months from srs until the date of death . in case of censored data , the patients ' last date of clinical follow - up a determination of the cause of death was attempted for all patients who died during the observation period . patients were considered to have died due to neurologic causes if they had either absent or stable systemic disease and progressive neurologic dysfunction . if patients had developed fatal organ failure , infection , or hemorrhage , in the setting of a stable neurological examination at the last clinic visit , they were considered to have died from progression of systemic disease or intercurrent disease ( e.g. , pulmonary embolus ) and not due to neurological causes . treatment response was evaluated according to the updated response evaluation criteria in solid tumors ( recist ) . local control ( lc ) was defined as no further tumor growth after treatment , subdivided into complete response ( cr ) , partial response ( pr ) , or stable disease ( sd ) on follow - up ct and/or mri scans . in lesions which underwent resection prior to srs , lc was defined as the absence of new nodular contrast enhancement adjacent to the resection cavity on mri . local failure ( lf ) was defined as tumor recurrence at the site of the targeted lesion and was further classified as progressive disease ( pd ) . distant brain control ( dc ) was defined as the absence of new intracranial lesions after treatment , whereas distant brain failure ( df ) was defined by the appearance of new brain metastases or leptomeningeal disease outside the lesions previously treated with srs . actuarial local progression - free survival ( lpfs ) and distant brain progression - free survival ( dpfs ) were calculated in months from the date of srs to the date of ct / mr - imaging showing local or distant brain failure . otherwise , patients were censored at the time of their last mri scan . for patients receiving wbrt for salvage , descriptive statistics were obtained for a variety of patient and treatment characteristics in this study . actuarial os , lc , and df rates were calculated using the kaplan - meier method . multivariate analysis was performed using cox proportional hazards regression for continuous variables and in order to identify prognostic factors for os and lpfs . for both univariate and multivariate analyses , kaplan - meier curves for os , lpfs , and dpfs and univariate analysis were conducted using graph pad prism version 6.00 software for mac ( graph pad software , san diego , ca ; windows ; microsoft , seattle , wa ) . descriptive statistics and multivariate analyses were performed using the stata 13 software package ( stata corp . in a total of 66 patients with 207 brain metastases , the median follow - up after srs was 10 months ( mean , 15.8 months ; range , 684 months ) . of the analyzed 66 patients , 51 were male ( 77.3% ) and 15 were female ( 22.7% ) . the patients ranged in age from 31 to 85 years ( mean age of 58.9 years ) at the time of their initial brain metastasis diagnosis . the median karnofsky performance status ( kps ) was 90 ( range of 60100 ) . thirty - nine patients ( 59.1% ) presented with a single brain metastasis ; 27 patients ( 40.9% ) had two or more brain metastases at time of diagnosis . at the time of srs treatments , 56 patients ( 84.8% ) were found to have uncontrolled systemic disease and 10 patients ( 15.2% ) were found to have controlled systemic disease . according to the recursive partitioning analysis ( rpa ) by the radiation therapy oncology group ( rtog ) , 3 patients ( 4.5% ) were classified as rpa class i , 59 patients ( 89.4% ) as rpa class ii , and 4 patients ( 6.1% ) as rpa class iii . patients were also classified into subgroups according to the score index for radiosurgery ( sir ) and the basic score for brain metastases ( bsbm ) to allow a prognostic determination of patients with brain metastasis who underwent srs and to make this data set comparable to other available literature . according to the sir , 35 patients ( 53% ) were found to have a score less than 6 , and 31 patients ( 47% ) were found to have a score equal to and more than 6 . according to the bsbm , 12 patients ( 18.2% ) had a score of 0 , whereas 44 patients ( 66.7% ) had a score of 1 and 8 patients ( 12.1% ) were found to have a score of 2 . two patients ( 3% ) had a score of 3 . according to the disease - specific graded prognostic assessment ( ds - gpa ) , 27 patients ( 40.9% ) were classified as ds - gpa 4 , 18 ( 27.3% ) as ds - gpa 3 , 14 ( 21.2% ) as ds - gpa 2 , 6 ( 9.1% ) as ds - gpa 1 , and 1 ( 1.5% ) as ds - gpa 0 . in 51 patients ( 77.3% ) , the histologic subtype was defined as clear cell carcinoma , but also two cases of papillary rcc ( 4.5% ) and one case of chromophobe rcc ( 1.5% ) were observed . in 12 patients ( 18.2% ) with brain metastases from rcc , the histologic subtype remained unclassified . at the time of diagnosis of the first brain metastasis , 63 patients ( 95.5% ) also had extra cranial metastases . stereotactic radiosurgery with the cyberknife ( accuray , sunnyvale , ca ) technique was used to treat all patients in this cohort with brain metastases from rcc . a total of 207 lesions were treated in 179 separate sessions via a total of 132 treatment plans . an average of 1.2 lesions was irradiated in each treatment session and an average of 1.6 lesions was irradiated in each treatment plan ( range , 16 ) . the median prescription dose was 22 gy , the median conformality index was 1.3 ( range of 1.036.96 ) , and the median homogeneity index was 1.32 ( range , 1.121.72 ) . the median prescribed isodose line was 76% ( range of 5889% ) and the median coverage of each individual lesion was 96.39% ( range , 84.2%100% ) . all patients received prophylactic corticosteroids ( dexamethasone ) and anticonvulsants ( levetiracetam ) during and after the srs treatment . of those , 25 patients ( 44.6% ) received standard systemic therapy ( e.g. , high dose il2 ; sunitinib and pazopanib ) , 5 patients ( 9% ) were treated with irb - approved experimental study therapy regimens ( e.g. bevacizumab + interferon ; bevacizumab versus erlotinib ; and pazopanib versus sunitinib , or tivozanib ) , and 26 patients ( 46.4% ) were treated with a combination of both . 10 patients ( 15.2% ) had received no systemic therapy at all at the time of srs treatment . as an initial treatment , 24 patients ( 36.4% ) underwent surgical resection before srs , 36 patients ( 54.5% ) were treated with srs only , and 6 patients ( 9.1% ) had received prior wbrt ( median dose , 30 gy ; range , 2030 gy ) . six patients ( 9.1% ) were treated with wbrt for salvage after srs and 2 patients ( 3% ) had resection due to progression after treatment with srs . in patients initially treated with surgical resection prior to srs , gross total resection could be achieved in 22 patients ( 91.7% ) . ( 6 months after the last srs treatment ) , 48 patients were dead ( 72.7% ) and 18 were alive ( 27.3% ) . most of the deceased patients ( 24 ; 50% ) died from documented progression of systemic disease ( nonneurological death ) , whereas in 21 patients ( 43.75% ) , the specific cause of death was unknown in the setting of a stable neurological examination at last visit and 3 patients ( 6.25% ) died from progression of intracranial disease ( neurological death ) . the median overall survival was 72.2 months ( 95% ci 45.295.5 months ) from the diagnosis of the primary tumor , 17.5 months ( 95% ci 11.522.5 months ) from the diagnosis of the first brain metastasis , and 13.9 months ( 95% ci 9.721.3 months ) from the time of srs for the analyzed study population . actuarial survival rates for the analyzed patient cohort calculated from the time of srs were 98.5% ( n = 65 ) at 1 month , 87.4% ( n = 55 ) at 3 months , 77.8% ( n = 49 ) at 6 months , 68% ( n = 41 ) at 9 months , 54.8% ( n = 33 ) at 12 months , and 34.1% ( n = 18 ) at 24 months ( figure 1 ) . the median overall survival from the time of srs for the 39 patients with a single brain metastasis was 20.3 months ( 95% ci 13.629.2 months ) compared to 5.4 months ( 95% ci 1.310.2 months ) in 8 patients with multiple ( > 3 ) brain metastases ( p = 0.0022 ) ( figure 2 ) . no statistically significant difference in median overall survival was found when comparing patients with a single brain metastasis to patients with two ( 11.2 months ) or three brain metastases ( 9.7 months ) at initial presentation ( p = 0.1853 ) . the median os was significantly different for the three different rpa classes ( p = 0.0001 ) . in patients stratified into rpa class i , median os was not reached because all 3 patients were still alive at the time of analysis . patients in rpa class ii and iii only had a median survival of 14.1 months and 4.1 months respectively ( figure 3 ) . stratifying the patients cohort by their initial treatment modality ( surgery prior to srs , srs , and wbrt prior to srs ) resulted in a median survival for patients initially treated with srs only ( n = 36 ) of 13.6 months ( 95% ci 6.923.5 months ) and a median survival of 21.9 months ( 95% ci 10.570.4 months ) for patients who underwent surgical resection ( n = 24 ) as an initial treatment . patients who underwent wbrt ( n = 6 ) before treatment with srs had a median survival of 5.9 months after srs ( figure 1 ) . the actuarial one - year overall survival rates for patients treated with those different approaches were 55.9% for patients who underwent srs as a sole treatment , 67.8% for patients who underwent surgical resection prior to srs , and 16.7% for patients treated with wbrt prior to srs ( no significant difference in overall survival was detected between patients treated with srs only and patients treated with surgery + srs ( p = 0.1141 ) . in univariate analysis of the entire cohort age ( p = 0.0000 ) , prior surgery ( p = 0.0486 ) , rpa class ( p = 0.0000 ) , kps ( 70 versus < 70 , p = 0.0000 ) , sir ( 6 versus < 6 , p = 0.0093 ) , bsbm ( p = 0.0027 ) , number of brain metastases ( > 3 versus 3 , p = 0.0009 ) , initial tumor volume ( p = 0.0000 ) , and ds - gpa ( p = 0.0002 ) were associated with significantly better overall survival . prior wbrt ( p = 0.0097 ) was found to be significantly associated with poor overall survival . sex , systemic and intracranial disease status at the time of srs , and whether the patients had received systemic treatment during their course of systemic disease were not found to be significantly associated with a difference in overall survival . in multivariate cox analysis , factors associated with a significantly better overall survival were age ( p = 0.038 ) , rpa class ( p = 0.000 ) , kps ( 70 versus < 70 , p = 0.000 ) , and the initial number of brain metastases ( > 3 versus 3 , p = 0.002 ) . again , prior wbrt was significantly associated with poorer overall survival ( p = 0.014 ) . prior surgery ( p = 0.053 ) was only found to be borderline significant in multivariate cox regression . factors not found to be significantly associated with better overall survival were sex , systemic and intracranial disease status at the time of srs , whether the patients had received systemic treatment during their course of systemic disease , and initial tumor volume ( table 3 ) . in univariate analysis of the two subgroups initially treated with srs and surgical resection followed by srs , age ( p = 0.0000 ) , rpa class ( p = 0.0001 ) , kps ( 70 versus < 70 , p = 0.0001 ) , sir ( 6 versus < 6 , p = 0.0385 ) , bsbm ( p = 0.0147 ) , ds - gpa ( p = 0.0001 ) , initial tumor volume ( p = 0.0000 ) , and the initial number of brain metastases ( > 3 versus 3 , p = 0.0002 ) were found to have a significant impact on overall survival . in multivariate cox analysis of these two subgroups , again rpa class ( p = 0.000 ) , kps ( 70 versus < 70 , p = 0.001 ) , sir ( 6 versus < 6 , p = 0.043 ) , ds - gpa ( p = 0.003 ) , bsbm ( p = 0.008 ) , and the initial number of brain metastases ( > 3 versus 3 , p = 0.001 ) were found to be prognostic for better overall survival . in multivariate analysis , the initial tumor volume was not found to be a prognostic factor for overall survival . over the course of the entire follow - up period , of the 138 lesions treated with srs only in a total of 51 patients , local failure was noted in 10 lesions ( 7.2% ) of 8 patients ( 15.7% ) during the entire follow - up period . in a total of 25 lesions treated with surgery and srs as an adjunct in 24 patients , local failure of the 44 lesions treated with wbrt prior to srs in a total of 6 patients , local failure was observed in 3 lesions ( 6.8% ) in 1 patient ( 16.7% ) . actuarial 1-year local control rates for lesions treated with srs as a sole treatment , surgical resection + srs , and wbrt + srs were 84% , 94% , and 88% , respectively ( figure 4 ) . in univariate analysis , no significant difference in local control was found between lesions treated with the three different approaches ( p = 0.445 ) . furthermore , no statistically significant difference in local control could be detected comparing srs with surgical resection + srs ( p = 0.3422 ) , srs with wbrt + srs ( p = 0.445 ) , and wbrt+ srs with surgical resection + srs ( p = 0.333 ) . tumor volume was found to be the only significant variable in univariate log - rank analysis ( p = 0.0000 ) ; however , in multivariate cox analysis , neither tumor volume nor surgical resection or the number of brain metastases was found to be prognostic for local progression - free survival . the median time until distant brain failure was 7 months after srs ( 95% ci 615 months ) . actuarial freedom from distant brain failure was 90.9% at 1 month , 76.3% at 3 months , 48.9% at 6 months , 41.7% at 9 months , 35.2% at 12 months , and 23.8% at 24 months after srs . the distant brain progression - free survival for all patients is shown in figure 5 . median distant brain progression - free survival for patients who initially received srs alone , surgery + srs , and wbrt + srs was 19 , 7 , and 3.5 months , respectively . in univariate as well as in multivariate analysis , prior wbrt was significantly associated with better distant tumor control ( p = 0.007 in univariate analysis and p = 0.014 in multivariate analysis ) . of the 24 patients initially treated with surgical resection , 7 had side effects : among this group , 6 patients developed fatigue , one patient additionally experienced worsening of his left - sided weakness after srs ( 1/24 acute grade 3 toxicity ) . among 36 patients who received srs only as an initial treatment , 7 patients had side effects . 5 patients developed fatigue , and among those , one patient had seizures due to expanding vasogenic edema after treatment . of the remaining two patients , one patient experienced worsening edema causing mass effect and midline shift ( 2/24 acute grade 3 toxicity ) ; the other presented with symptomatic radiation necrosis causing left hemiplegia . in the group of patients who received upfront wbrt , one patient developed nausea after treatment with wbrt , before undergoing srs . overall , there were 4/66 ( 4.5% ) acute grade 3 toxicity and 1/66 ( 1.5% ) grade 3 long term toxicity . the mean volume in patients experiencing toxicity was 12.6 and the median dose was 20 gy . brain metastases from rcc are reported with a frequency of approximately 4 to 17% of patients during their course of disease . as newer therapies for the management of rcc emerge and standard treatments are further refined , these patients will live longer and , as a consequence , are more likely to develop brain metastases during their course of disease . in addition , brain metastases from rcc are known for their high propensity of intratumoral hemorrhage and their extensive surrounding edema which is profound when compared to other metastatic brain lesions from other primaries . surgery may often not be feasible due to location of the lesion and studies on wbrt as a sole treatment in the treatment process of these patients have shown disappointing results regarding overall survival [ 2227 ] . srs is proving to be a useful modality in the treatment of brain metastasis from rcc . in this retrospective cohort analysis , we evaluated the effectiveness , safety , and potential prognostic factors of srs for the treatment of brain metastases from rcc on survival and local and distant tumor control at our institution . stereotactic radiosurgery for brain metastases from rcc has been suggested to prolong overall survival when compared to patients treated with modalities such as wbrt only [ 15 , 20 , 28 ] with median survival rates of 5.1 to 17.8 months . in our study , the median survival of the entire cohort was 13.9 months . the groups treated with srs only , surgical resection plus subsequent srs , and wbrt plus srs achieved a median overall survival of 13.6 , 21.9 , and 5.9 months , respectively . the results for survival among the three groups were found to be statistically significant . a significant difference in median overall survival this result confirms findings from previously published series evaluating the role of srs for patients with brain metastases from this primary [ 21 , 29 , 30 ] . the majority of patients in our series were graded into rpa class ii due to the presence of active extracranial disease or advanced age . multiple studies have reported the effectiveness of srs for brain metastases from rcc have reported local control rates ranging from 60.9 to 100% ( table 4 ) [ 7 , 8 , 1416 , 1821 , 24 , 2842 ] . the majority of patients in these studies were treated with linac - based or gamma knife - based devices , but comparable data on treatment outcomes for these patients undergoing cyberknife srs is lacking . to date , our retrospective study is the largest study conducted so far to evaluate the outcome of patients and potential prognostic factors in the treatment of cyberknife radiosurgery for brain metastases from rcc . in our study , actuarial 1-year local control rates for lesions treated with the three different initial treatment approaches were 84% for patients treated with srs as a sole treatment , 94% for patients who underwent surgical resection plus subsequent srs , and 88% for patients initially treated with wbrt followed by srs which is comparable to that in literature . except for a single report , srs as a sole treatment or in combination with wbrt has shown favorable results comparable to those of surgery plus subsequent wbrt in patients with a single brain metastasis in the current literature [ 4951 ] . several important factors such as tumor size , location , number of brain metastases , presence of symptomatic peritumoral edema , and mass effect have to be taken into consideration when it comes to the decision whether a patient should undergo surgical resection or srs . in regard to the tumor size , the rtog protocol 90 - 05 established srs dose - volume prescription criteria , recommending a maximum dose of 24 , 18 , and 15 gy in a single fraction for tumors ranging up to 2 cm , between 2 and 3 cm , and greater than 3 cm in diameter , respectively [ 6 , 10 ] . other reported optimal doses range from 15 up to 22 gy , with a median dose of 20 gy . the treatment of patients with brain metastases from rcc has several advantages : stereotactic radiosurgery is a minimally invasive procedure , usually performed in the outpatient setting , with the potential to treat multiple lesions during one treatment session , and can be performed repeatedly in case of local or distant brain tumor recurrence [ 12 , 52 ] . however , despite the noted improvements in local tumor control , some questions still remain to be investigated in the field of this treatment modality , such as an appropriate selection of patients for srs versus surgery , the development of validated prognostic factors after treatment with srs , and the role of adjuvant wbrt . the role of adjuvant treatment options such as wbrt and targeted systemic treatments especially needs to be further investigated in brain metastases from rcc . during the course of treatment , a total of 4 ( 6% ) patients of the analyzed cohort presented with grade 3 side effects related to srs . especially among the patients with severe complications such as symptomatic radiation necrosis , worsening symptoms , and seizures related to the treated lesion , there was trend towards larger initial tumor volume . the more severe side effects were more common among the patients with larger lesions treated with srs only than in the surgical group . in patients with a single brain metastasis , with good performance status , and limited to controlled systemic disease , surgical resection of brain metastasis has shown survival benefit in randomized data , but , however , there is currently no class i evidence available for the optimal surgical treatment of patients with 2 or more brain metastases . since local failure rates as high as 60% after surgical resection have been reported , adjuvant srs or wbrt is recommended [ 54 , 55 ] . two randomized trials [ 53 , 56 ] have furthermore shown that the addition of wbrt to either surgery or srs results in significantly improved local and distant brain tumor control , although improved overall survival has only been reported for surgery plus subsequent wbrt . in addition , bindal and colleagues reported equivalent survival time of patients with up to three brain metastases and good performance status who had all lesions removed to that of similar patients undergoing surgery for a single brain lesion . to date , randomized trials reporting significantly improved survival , local and distant brain control after treatment with surgery , and/or srs have only been including patients with brain metastases from different primary tumors , but no such trials have been conducted on the surgical treatment for patients with brain metastases from renal cell carcinoma . in our study , the group of patients initially treated with surgical resection followed by srs had a median survival of 21.9 months and the highest 1-year local tumor control rate ( 94% ) , although these results failed to show statistically significant difference compared to the outcome of patients initially treated with srs only , which may be due to the relatively low number of patients in our cohort . this observation should hence be reexamined in a larger sample which can be achieved by pooling data or when patients are accrued in a multicenter trial . based on the available data in the current literature , the first - line treatment for accessible brain metastases from rcc has been surgical resection followed by wbrt . to date , six studies have been conducted to evaluate the outcome and prognostic factors of patients who underwent surgical resection for brain metastases from rcc [ 5863 ] . in aggregate , the results of these investigations including our findings support the role of surgery for brain metastases from rcc in selected patients with good prognostic factors , limited or controlled systemic disease , and a single brain metastasis in a surgically accessible location , as surgery usually results in immediate relief of symptoms and can contribute to achieving excellent local tumor control . however , in terms of quality of life , even patients with poor prognostic factors may also benefit from surgical intervention if a lesion causing significant mass effect can be removed . the question whether surgical resection only or a combined approach is more favorable still remains unclear as prospective randomized trials for patients with brain metastases from rcc are lacking . historically , whole - brain radiation therapy ( wbrt ) has been the mainstay of treatment in the management of patients with brain metastases , although this treatment modality is potentially associated with neurocognitive dysfunction and with suboptimal control rates , especially for larger tumors [ 10 , 52 ] . outcomes after wbrt appear especially poor for patients with metastatic rcc , as this tumor has traditionally been considered to be relatively radioresistant compared to brain metastases from other primaries , such as lung or breast [ 9 , 10 ] . in our study , patients treated with wbrt prior to srs had the second highest local control rate . furthermore , upfront wbrt was significantly associated with improved distant brain tumor control in univariate and multivariate analysis , although this treatment combination revealed a median survival of 5.9 months in these patients , a significantly worse result compared to the overall survival of patients initially treated with srs only and patients treated with surgery and srs as an adjunct . the outcomes of patients with brain metastasis from rcc treated with wbrt were evaluated : the first study conducted by wroski et al . revealed a median survival of 3.3 months calculated from the last day of wbrt , with death from neurologic causes in 76% of patients . one year later , another study to further investigate the question whether wbrt is a suitable treatment for patients with metastatic brain lesions from rcc was published by culine et al . . the median survival of patients who received radiotherapy alone was 7 months , compared to a median survival of 1 month of patients who did not undergo any specific treatment and 10 months of patients who underwent surgery . in 2004 , cannady and colleagues published another study with comparable survival rates to those of wroski et al . , with a median survival after wbrt of 3.3 months in a total of 46 patients who received wbrt as their initial treatment for brain metastasis . furthermore , the median survival rates for the different rpa classes were evaluated : the median survival for rpa classes i , ii , and iii was 8.5 , 3 , and 0.6 months , respectively , but no statistically significant difference was observed among the three classes . in addition , some studies also revealed the potential benefit of dose escalation [ 22 , 6567 ] . , in which higher doses of radiation ( 40 gy in 20 fractions or 45 gy in 15 fractions ) compared to standard treatment regimens resulted in improved local control and overall survival rates . patients who were treated with higher doses had a median overall survival of 12 months and 6 months ' local control rates of 57% , compared to patients treated with lower doses , who had a median overall survival of 4 months and 6 months ' local control rates of 21% . these rather unsatisfactory results of treatment or rcc metastasis with wbrt only led to the implementation of more aggressive treatment approaches for brain metastasis from rcc , such as surgical resection and srs . in 1987 , gay et al . analyzed the median survival rates of 25 patients who received radiation therapy only ( 13 weeks ) and 7 patients who underwent surgical resection and postoperative radiation ( 66 weeks ) . however , interpreting the results of this study , it has to be kept in mind that the patients who underwent surgery were preselected because of stable systemic disease , an accessible single metastatic lesion , and the belief that the tumor burden could be completely resected . ikushima et al . extended the available data with their retrospective analysis of the effect of adjuvant fractionated stereotactic radiotherapy ( fsrt ) after surgery compared to surgical resection with adjuvant wbrt and wbrt alone . the different treatment groups achieved median survival times of 25.6 , 18.7 , and 4 months , respectively . the results in this study , however , are confounded by the fact that the patients included had a relatively good performance status compared to the patients of other studies conducted on this topic before and frst was only indicated in patients with a good performance status and a tumor diameter of 3 cm and if patients presented with less or equal to 3 lesions . a recent study published by fokas and colleagues evaluated the role of the treatment with srs and wbrt in brain metastasis from rcc in a total of 88 patients . fifty - one patients were treated with srs , and 17 were treated with srs plus adjuvant wbrt , whereas the remaining 20 patients were treated with wbrt only . the median overall survival for these different treatment groups was 12 , 16 , and 2 months , respectively . statistically significant difference was found in overall survival rates of patients treated with srs only as well as patients treated with a combination of srs and wbrt compared to patients treated with wbrt only . taking everything into account , the results of these studies , including our retrospective analysis , suggest improved local and distant brain tumor control for brain metastases from rcc when wbrt is administered . although rcc is considered to be a radioresistant tumor , these results suggest that there might be an effect of wbrt on microscopic metastases from rcc within the brain or a potential delay in the appearance of new brain metastases . this result might be partially explained by selection bias , because wbrt was more commonly used in patients with a larger number of brain metastases . our results suggest that more aggressive treatment options like surgical resection and srs , possibly in combination with wbrt , might be beneficial for patients with favorable performance status and a limited number of brain lesions . however , wbrt and supportive care continue to be the treatment of choice in patients with multiple brain metastases , poor performance status , uncontrolled systemic disease , and a short life expectancy [ 52 , 68 ] . as more aggressive treatment options may be associated with an increased risk in these patients , it is important to take into account the prognosis of each patient in order to individualize the treatment approach and to offer the best possible treatment modality for an improved outcome of these patients . the present study has inherent limitations based on its retrospective nature , and the obtained results may be somewhat influenced by clinical selection bias . in light of varying treatment regimen during the course of disease of the analyzed patients , reliable prognostic factors remain difficult to assess . furthermore , complete follow - up was only available for 86.8% of all patients . the other patients were transferred to other facilities for further follow - up and could not be analyzed in this study . therefore , despite the fact that this cohort is the largest reported series to date , the analyzed cohort is a rather heterogeneous group of patients with a variety of different systemic treatment regimens , prior wbrt , and prior surgery , or patients treated with srs only . due to this fact , it is difficult to analyze the exact impact of the different treatment options as well as potential prognostic factors on the outcome of this patient cohort . randomized controlled trials are needed to further evaluate the impact of srs and possible combination approaches with surgery or wbrt as well as reliable prognostic factors on survival and tumor control in the future . stereotactic radiosurgery is a safe and effective treatment option in patients with brain metastases from rcc and results in excellent local control rates . in case of a limited number of brain metastases , surgery or srs might be appropriate , depending on the individual characteristics of the patients and the number , size , and location of brain metastases . further investigations such as randomized controlled trials are necessary for a reliable evaluation of prognostic factors and for a comparison of the outcome of patients treated with srs alone versus combined treatment approaches .	background . renal cell carcinoma is a frequent source of brain metastasis . we present our consecutive series of patients treated with stereotactic radiosurgery ( srs ) and analyse prognostic factors and the interplay of wbrt and surgical resection . methods . this is a retrospective study of 66 patients with 207 lesions treated with the cyberknife radiosurgery system in our institution . the patients were followed up with imaging and clinical examination 1 month and 2 - 3 months thereafter for the brain metastasis . patient , treatment , and outcomes characteristics were analysed . results . 51 male ( 77.3% ) and 15 female ( 22.7% ) patients , with a mean age of 58.9 years ( range of 3185 years ) and a median karnofsky performance status ( kps ) of 90 ( range of 60100 ) , were included in the study . the overall survival was 13.9 months , 21.9 months , and 5.9 months for the patients treated with srs only , additional surgery , and wbrt , respectively . the actuarial 1-year local control rates were 84% , 94% , and 88% for srs only , for surgery and srs , and for wbrt and additional srs , respectively . conclusions . stereotactic radiosurgery is a safe and effective treatment option in patients with brain metastases from rcc . in case of a limited number of brain metastases , surgery and srs might be appropriate .
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Proceed to summarize the following text: mood disorders such as depression and anxiety are among one of the most common complications during pregnancy and the postpartum period . researchers emphasized that the prevalence of ppd varies in different part of the world , while usa women fell at the midpoint , the lowest levels of ppd were reported for european and australian women , on the contrary , the highest depressive symptom scores were seen for women from asia as well as south america . research finding also shows the prevalence of ppd is nearly twice in developing countries in comparison to developed ones . researchers reviewed 59 studies from north america , europe , australia , and japan and estimated the prevalence of ppd to be 13% . for instance , the prevalence of 37% in bahrain , 21% in lebanon , and 36% in pakistan was reported . in iran , the reported rates of ppd ranged in different cities of the country , 34.8% in ilam , 20.3% in shiraz , 22% in sari , 34% in tabriz , 23% in tehran , and 32% in hamadan . a recent meta - analysis of 41 studies indicated that ppd prevalence in iran was 25.3% with the highest prevalence among a woman with a history of depression 45.2% and unwanted delivery 43.4% . a literature review on ppd indicated that cultural forces and socioeconomic elements play a pivotal role in the manifestation of ppd symptoms . researchers also had expressed that although ppd is a phenomenon that affects women from a wide range of society and cultures , the manifestation of symptoms could be different . recently , a growing number of researches have suggested some factors such as previous history of depression , anxiety or stress , poor social support , social relationships , low self - esteem , limited community resources , marital conflict , physical abuse , and postpartum physical complications are related with ppd and could modify depressive symptoms . in addition , authors highlighted the role of cultural - based factors , including negative belief , poor quality of life , lack of family support , and mutual understanding among family members in the occurrence of ppd . in spite of the importance of cultural factors of ppd , there is a little study considering differences in the cultural context of western and non - western societies . cross - cultural comparisons of the depressive symptoms among people of swiss , iran , canada , and japan indicated that symptom of depression experienced differently . for example , guilt feelings were present in 68% of the swiss sample and 32% of the iranian sample ; suicidal ideas were present in 70% of the canadian subjects and 40% of the japanese subjects and somatization was present in 57% of the iranian cohort and 27% of the canadian cohort . in addition to the diversity of the western and non - western country in terms of prevalence and symptomatic variation of ppd , some studies were found different risk factors of ppd in the non - western country . for example , noted that low level of education and undesired gender of the child to be risk factors of ppd in iranian sample . satisfaction of living with spouse family and contentment of husband 's family about the gender of the child showed significant correlation with ppd in iranian 's mothers . speaking of the potential interaction of different risk factors and ppd , some studies suggest that emergency cesarean sections were associated with a higher risk for ppd . while , only in the recent years , a significant increase has been reported in the rate of cesarean section in iran , which became six - fold over the past three decades . finally , in the cultural context of ppd , some researchers noted the role of religion in ppd symptoms and highlighted the role of religious and cultural beliefs as a cause of depression . for example , in some muslim 's society depression reported to be caused by lack of faith or failure to pray regularly or as a result of moral transgression . a number of studies have found that the religiosity and spirituality have an inverse association with depressive symptoms . especially , mann et al . , reported the protective effects of antenatal religiosity / spirituality in ppd . iran is an islamic country and majority of its social and institutional structures are formally organized in religious structure , and according to the iranian religious beliefs , giving birth to a child clears mother 's sin and it is like praying for god 's . these cultural and religious expectations may prevent help - seeking behavior and hinder diagnosis of ppd . recently , several studies on ppd have been conducted in iran , the majority of them were not experimental studies , and examine only the relationship between maternal fatigue and ppd , effects of ppd on life quality , or studying risk factors and prevalence of ppd . few experimental studies addressed psychosocial treatments aimed at improving psychological health of iranian woman with ppd . one experimental study only examines the effect of interpersonal psychotherapy on marriage and pdd . to our best knowledge , no previous study to date investigated the effects of an antenatal psychological intervention to prevent ppd in iranian samples . therefore , in the present study , we designed a psycho - educational program based on cognitive behavioral treatment . this quasi - experimental study was conducted in 2013 . the design of the study was pre- and post - test with control group and random assignment . this design is one of the vigorous designs that promote external validity while enhancing internal validity . since individuals were randomly divided into two groups and randomly assigned into two groups , all extraneous variable including maturation , history , and selection have been controlled . five hundred and thirty - four women referred to two obstetric clinics in tehran , iran , between august 2012 and march 2013 were screened for the study . the women were enrolled in the study if they had scored 13 or higher on the beck depression inventory ( bdi ) . women were eligible for the trial if they were aged 1832 years ; had a single birth ; an uncomplicated pregnancy , ability to read and write . exclusion criteria included using antidepressant / anti - anxiety medications and personal history of psychiatric disorder . according to the sample size formula , [ ( z + z)]/(d ) , z1 = 1.96 , confidence interval = 95% , z1 = 0.85 , d = 0.20 , 196 participants were selected for both study group . to determine sample size , 196 participants took part in the study , but 61 ( 34 in the intervention group and 27 in the control group ) were excluded due to failure in cooperation and not reliable responses to the questionnaires . they were placed randomly into the intervention group ( n = 64 ) and control group ( n = 71 ) based on their numbers . this proposal was approved by the research council of baghiatallah university of medical sciences , tehran , iran . in terms of ethical points and the intervention program as well as the questionnaires was given to the research council in order to assure confidentiality . all participants affirmed their consent to participate in the study before collection of the data . these will be measured as follows : as the primary outcome assessed postnatal distress , it consists of 10 questions scored on a 4-point likert scale to determine the presence and severity of the ppd symptoms . it has been translated and validated in iran and has shown desirable validity for identifying ppd with satisfactory sensitivity ( 95% ) and specificity ( 88% ) . it has 21 items and widely used for measuring the severity of depression or to screen depressive symptoms . bdi has a sufficient reliability for many population and its common psychometric properties reported to be acceptable . the iranian version of bdi considered having high reliability and sound instrument to measure depression in iranian population . beck anxiety inventory ( bai ) is a good tool to use as a severity indicator . the research concluded that the iranian version of bai with good internal consistency ( 0.92 ) , validity ( 0.72 ) , and reliability of ( 0.83 ) , is within the acceptable range . religious attitude scale questionnaire of khodayarifard et al . , was utilized to measure religious attitude . the scoring for the religious attitude scale questionnaire is computed as following ; 484 is a mild level of religious attitude ; 85165 moderate level of religiousness ; 166200 high level of religious attitude . the content of questionnaire categories was ethics , values , the effect of religion on personal and social lifestyle and behavior , ideology , and religious beliefs . stated the questionnaire had a high reliability of cronbach 's alpha coefficient ( = 0.89 ) . the test - retest stability correlations for all subscale ranged from 0.91 to 0.97 . in this study , outcomes were measured at two points : in pretest before and in posttest after the intervention which was 2 weeks after delivery . the intervention package , enhancing cognitive behavioral skills program ( ecbsp ) , was designed and implemented for participants in the intervention group while the control group received routine antenatal care by obstetrics nurses . a multidisciplinary team of therapists with a variety of professional backgrounds including midwives , health psychology , psychotics , and licensed clinical psychologist with a concentration of cognitive - behavioral therapy designed the ecbsp . the intervention package included 8 , 4060 min sessions of cognitive behavioral therapy ( cbt ) intervention delivered individually to participants by recorded film and interactive workbook . the essence of the ecbsp is to identify and test automatic negative thoughts in subjects . the rationale provided was that when negative thoughts were triggered , the person would expect negative and aversive event about him or herself , the world , and the future . the treatment consisted of training in self - monitoring , self - focused attention , relaxation , understanding of the problem , and setting up an alternative view of the problem , revising automatic thoughts , behavioral approach tasks , and exposure to worry cues . other themes of the ecbsp included education on depressive and anxiety symptomatology , positive communication , realistic expectation about pregnancy , delivery , and parenting , recording their mood , pleasurable activities , and relaxation exercise . the patients received the homework to ensure transfer of skills from therapeutic contexts to daily life . the characteristics of the participants on the baseline variables were evaluated using chi - square analysis and independent t - tests to detect any significant differences between the two groups before introducing the ecbsp . multivariate analysis of covariance ( mancova ) was performed to analyze possible differences between the experimental and the control group at posttest . this quasi - experimental study was conducted in 2013 . the design of the study was pre- and post - test with control group and random assignment . this design is one of the vigorous designs that promote external validity while enhancing internal validity . since individuals were randomly divided into two groups and randomly assigned into two groups , all extraneous variable including maturation , history , and selection have been controlled . five hundred and thirty - four women referred to two obstetric clinics in tehran , iran , between august 2012 and march 2013 were screened for the study . the women were enrolled in the study if they had scored 13 or higher on the beck depression inventory ( bdi ) . women were eligible for the trial if they were aged 1832 years ; had a single birth ; an uncomplicated pregnancy , ability to read and write . exclusion criteria included using antidepressant / anti - anxiety medications and personal history of psychiatric disorder . according to the sample size formula , [ ( z + z)]/(d ) , z1 = 1.96 , confidence interval = 95% , z1 = 0.85 , d = 0.20 , 196 participants were selected for both study group . to determine sample size , 196 participants took part in the study , but 61 ( 34 in the intervention group and 27 in the control group ) were excluded due to failure in cooperation and not reliable responses to the questionnaires . they were placed randomly into the intervention group ( n = 64 ) and control group ( n = 71 ) based on their numbers . this proposal was approved by the research council of baghiatallah university of medical sciences , tehran , iran . in terms of ethical points and the intervention program as well as the questionnaires was given to the research council in order to assure confidentiality . all participants affirmed their consent to participate in the study before collection of the data . these will be measured as follows : as the primary outcome assessed postnatal distress , it consists of 10 questions scored on a 4-point likert scale to determine the presence and severity of the ppd symptoms . it has been translated and validated in iran and has shown desirable validity for identifying ppd with satisfactory sensitivity ( 95% ) and specificity ( 88% ) . it has 21 items and widely used for measuring the severity of depression or to screen depressive symptoms . bdi has a sufficient reliability for many population and its common psychometric properties reported to be acceptable . the iranian version of bdi considered having high reliability and sound instrument to measure depression in iranian population . beck anxiety inventory ( bai ) is a good tool to use as a severity indicator . the research concluded that the iranian version of bai with good internal consistency ( 0.92 ) , validity ( 0.72 ) , and reliability of ( 0.83 ) , is within the acceptable range . religious attitude scale questionnaire of khodayarifard et al . , was utilized to measure religious attitude . the scoring for the religious attitude scale questionnaire is computed as following ; 484 is a mild level of religious attitude ; 85165 moderate level of religiousness ; 166200 high level of religious attitude . the content of questionnaire categories was ethics , values , the effect of religion on personal and social lifestyle and behavior , ideology , and religious beliefs . stated the questionnaire had a high reliability of cronbach 's alpha coefficient ( = 0.89 ) . the test - retest stability correlations for all subscale ranged from 0.91 to 0.97 . in this study , outcomes were measured at two points : in pretest before and in posttest after the intervention which was 2 weeks after delivery . the intervention package , enhancing cognitive behavioral skills program ( ecbsp ) , was designed and implemented for participants in the intervention group while the control group received routine antenatal care by obstetrics nurses . a multidisciplinary team of therapists with a variety of professional backgrounds including midwives , health psychology , psychotics , and licensed clinical psychologist with a concentration of cognitive - behavioral therapy designed the ecbsp . the intervention package included 8 , 4060 min sessions of cognitive behavioral therapy ( cbt ) intervention delivered individually to participants by recorded film and interactive workbook . the essence of the ecbsp is to identify and test automatic negative thoughts in subjects . the rationale provided was that when negative thoughts were triggered , the person would expect negative and aversive event about him or herself , the world , and the future . the treatment consisted of training in self - monitoring , self - focused attention , relaxation , understanding of the problem , and setting up an alternative view of the problem , revising automatic thoughts , behavioral approach tasks , and exposure to worry cues . other themes of the ecbsp included education on depressive and anxiety symptomatology , positive communication , realistic expectation about pregnancy , delivery , and parenting , recording their mood , pleasurable activities , and relaxation exercise . the patients received the homework to ensure transfer of skills from therapeutic contexts to daily life . as the primary outcome assessed postnatal distress , it consists of 10 questions scored on a 4-point likert scale to determine the presence and severity of the ppd symptoms . it has been translated and validated in iran and has shown desirable validity for identifying ppd with satisfactory sensitivity ( 95% ) and specificity ( 88% ) . it has 21 items and widely used for measuring the severity of depression or to screen depressive symptoms . bdi has a sufficient reliability for many population and its common psychometric properties reported to be acceptable . the iranian version of bdi considered having high reliability and sound instrument to measure depression in iranian population . beck anxiety inventory ( bai ) is a good tool to use as a severity indicator . the research concluded that the iranian version of bai with good internal consistency ( 0.92 ) , validity ( 0.72 ) , and reliability of ( 0.83 ) , is within the acceptable range . religious attitude scale questionnaire of khodayarifard et al . , was utilized to measure religious attitude . the scoring for the religious attitude scale questionnaire is computed as following ; 484 is a mild level of religious attitude ; 85165 moderate level of religiousness ; 166200 high level of religious attitude . the content of questionnaire categories was ethics , values , the effect of religion on personal and social lifestyle and behavior , ideology , and religious beliefs . stated the questionnaire had a high reliability of cronbach 's alpha coefficient ( = 0.89 ) . the test - retest stability correlations for all subscale ranged from 0.91 to 0.97 . in this study , outcomes were measured at two points : in pretest before and in posttest after the intervention which was 2 weeks after delivery . the intervention package , enhancing cognitive behavioral skills program ( ecbsp ) , was designed and implemented for participants in the intervention group while the control group received routine antenatal care by obstetrics nurses . a multidisciplinary team of therapists with a variety of professional backgrounds including midwives , health psychology , psychotics , and licensed clinical psychologist with a concentration of cognitive - behavioral therapy designed the ecbsp . the intervention package included 8 , 4060 min sessions of cognitive behavioral therapy ( cbt ) intervention delivered individually to participants by recorded film and interactive workbook . the essence of the ecbsp is to identify and test automatic negative thoughts in subjects . the rationale provided was that when negative thoughts were triggered , the person would expect negative and aversive event about him or herself , the world , and the future . the treatment consisted of training in self - monitoring , self - focused attention , relaxation , understanding of the problem , and setting up an alternative view of the problem , revising automatic thoughts , behavioral approach tasks , and exposure to worry cues . other themes of the ecbsp included education on depressive and anxiety symptomatology , positive communication , realistic expectation about pregnancy , delivery , and parenting , recording their mood , pleasurable activities , and relaxation exercise . the patients received the homework to ensure transfer of skills from therapeutic contexts to daily life . the characteristics of the participants on the baseline variables were evaluated using chi - square analysis and independent t - tests to detect any significant differences between the two groups before introducing the ecbsp . multivariate analysis of covariance ( mancova ) was performed to analyze possible differences between the experimental and the control group at posttest . the mean age of sample was 25.8 years ( standard deviation [ sd ] = 3.7 ) ; 33.8% hold bachelor 's or higher degrees , 81% of women were unemployed , and over 74% had a cesarean delivery . no statistically significant differences were identified between participants in the intervention and control group in terms of demographic characteristics [ table 1 ] . participant demographic characteristics at baseline table 2 presents the means and sds on the dependent variables . descriptive results indicated that the average of depression and anxiety scores changed in posttest in comparison with those in pretest for the intervention group ; however , in the control group , there were changes only in the average of self - esteem and religious attitude . in order to study the significance of differences between two groups , mancova was used . the result of the mancova reported that two groups had a significant difference in at least one of the dependent variables ( p < 0.01 ) . box 's test of equality of covariance matrices showed that the correlation between the dependent variables does not differ significantly between the groups ( box 's m = 19.73 , f = 1.2626 , p > 0.217 ) . furthermore , the results of mancova after controlling of pretest scores showed that there were significant differences between intervention and control groups in scores of bdi and epds ( p < 0.05 ) . we did not find any statistically significant differences in mean anxiety symptom , self - esteem , and religious attitude score when comparing participants of the intervention group with the control group ( p > 0.05 ) [ table 2 ] . comparison of the two groups means and sd before and after intervention by t - test and between groups by mancova analysis each year , about 25% of iranian women are diagnosed with ppd . other mood disorders such as postpartum stress and anxiety increase each year but the little attention the problem has received . therefore , the need to early identification and intervention to prevent postpartum disorder has been growing . in this study , researchers tried to investigate whether a randomized controlled trial of ecbsp program based on cbt considering the religious and cultural context of iran , would be effective in preventing postpartum mood disorders and improve self - esteem of at - risk pregnant mothers for pdd . to our best knowledge , this is the first trial of cbt aiming to reduce depression and anxiety by the end of pregnancy as well as increasing the mother 's self - esteem . the findings from this study suggest that ecbsp can partly prevent the worsening of mood disorders . this finding was consistent with previous studies that reported a positive effect of antenatal cbt intervention on reduction and prevention of ppd . one characteristic that motioned study had in common and could explain the reduction in the depression scores is early identification of at - risk mother for pdd and introducing cbt intervention in earlier phase of the disorder that could lead to more improvement of psychological health . other researchers had consistently revealed that by teaching participants to identify and challenge negative thought , the individual will be able to positively shift their perceptions about themselves and the world around them . although some of the studies reported that antenatal intervention had not effected on the reduction of pdd . improvements in depression were not accompanied by changes in anxiety , self - esteem , and the religious attitude . these latter findings confirm those of reynolds and coats ( 1986 ) and misri et al . reynolds and coats , studied the effectiveness of cbt on the reduction of depression symptoms and found no significant change in self - esteem although there was a positive significant change in depression . one possibility of not changing the level of self - esteem was that the intervention period may be too short to affect the self - esteem . although the current study did find improvements in ppd symptoms following the treatment , it failed to show any benefits in terms of anxiety . previous research has highlighted the importance of involving women 's partners in the treatment of postpartum mood disorders . one explanation for our failure to observe a change in self - esteem may be due to the fact that self - esteem is formed through a complex interaction of the factors , and it is a complex concept to operationalize and evaluate . the participants of the present research reported the high score on religious attitude scale and considered to be highly religious people . the results of the study did not show any significant differences between two groups and changes as a result of intervention on participant 's religious attitudes . this finding is not consistent with that from moradi et al . about positive effects of religious on depression . many other researchers reported the positive effect and relation between religious attitude and mental health , self - esteem , depression , suicide , and anxiety in persian sample . it is important to note that our intervention was designed to address negative though and lack of the skills underlings the postpartum mood disorder not changing in the religious attitude of participants . another possible explanation related to the failure of indicating the role of religious attitude in ppd may be results of making no discrimination between intrinsic and extrinsic religious orientation . for instance , bazmi and allahvirdiyani reported that while there is a negative significant relationship between inward religious orientation with depression , anxiety , and stress in iranian population , outward religious orientation indicated positive association with these disorders . hence , the findings about the religious attitude should be considered with a substantial cautious as dein et al . reported that a great controversy has surrounded the relationship between religious involvement and mental health . the refusal to participate rate was calculated 31% , which is lower than similar research with nearly 50% drop out . one of the main limitations of the study was the absence of follow - up stage . therefore , replication study to verify the effectiveness of the cbt on ppd in long - term is crucial . risk factors of ppd may be different among iranian women . cultural context and religious attitude early identification of women at - risk for ppd may aid in the prevention and alleviation of depressive symptoms and comorbidities . finally , the findings have demonstrated the feasibility of early identification of at - risk mothers by antenatal screening and the application of cbt intervention . in conclusion , this study once again clears the evidence that preventive cbt intervention can reduce the severity of ppd .	background : the current study was conducted to examine the effect of cognitive behavior therapy on the reduction postpartum mood disorder and increasing the self - esteem of at - risk iranian mothers.methods:in this quasi - experimental study , 135 at - risk mothers were selected from the population by means of cluster sampling and randomly assigned into one of two groups : intervention ( n = 64 ) , or control ( n = 71 ) . the control group received usual medical care , and the intervention group received an eight sessions cognitive behavior program during pregnancy . assessments were administered at two time points ( pretest at the beginning of the third trimester and posttest at 2 weeks postpartum ) . beck anxiety , beck depression , edinburgh postpartum depression , ( ppd ) coopersmith self - esteem , and religious attitude questionnaire were used to collect data.results:the mean age of participants was 25.8 3.7 years . one - third of them had either bachelor or higher degrees in education ( 33% ) . about two - third of participants were unemployment with similar distribution in both the groups ( intervention = 80% , control = 83% ) . the majority ( 70% ) of the participants had cesarean section deliveries . there were no statistically significant differences respects to sociodemographic characteristics between the control and intervention groups ( p > 0.05 ) . the multivariate analysis of covariance results showed that the average scores of ppd were reduced significantly in the intervention group ( p < 0.001 ) . also while the mean score of anxiety in the intervention group decreased from 23.31 ( standard error [ se ] = 12.11 ) to 16.64 ( se = 8.33 ) and self - esteem increased from 29.09 ( se = 3.51 ) to 31.81 ( se = 2.76 ) , no change was statistically significant in comparison to the control group.conclusions:according to the findings of the present study , cognitive behavior intervention is effective in reducing ppd in at - risk mothers .
You are an expert at summarizing long articles. Proceed to summarize the following text: cardiovascular ( cv ) disease is the main cause of morbidity and mortality in patients with end - stage renal disease ( esrd ) . the increased risk is partly due to a higher prevalence of traditional cv risk factors . nevertheless , these patients also present other nontraditional cv risk factors related with the setting of uremic background that result in functional and structural alterations of the arterial wall , leading to an increase in arterial stiffness . as in the general population , arterial stiffening has been described as an independent predictor of both cv and overall mortality in haemodialysis ( hd ) patients [ 28 ] . another component that has been proposed to play a role in cv risk is the hd session per se . the dialysis procedure may induce acute functional alterations of the arterial wall through several mechanisms , the most remarkable being the intermittent immunoactivation state induced by dialysis [ 911 ] , and the acute intravascular volume drop produced during the hd session [ 1217 ] . it has been proposed that these acute functional alterations could be detected by noninvasive pulse wave analysis ( pwa ) . as pwa measurements are being increasingly introduced in the clinical setting , the main objective of our study was to separately analyze the acute effects on pwa of bioincompatibility and ultrafiltration ( uf ) during the dialysis session . we separately analyzed the acute effect on pwa of membrane bioincompatibility ( study i , n = 11 ) and ultrafiltration ( study ii , n = 19 ) during the hd session . patients were eligible for entry into the study when ( 1 ) they had been on hd for at least 3 months , ( 2 ) they were in a stable clinical situation , and ( 3 ) they did not suffer cardiovascular instability on dialysis . all selected patients agreed to participate in the study , which was approved by the hospital ethics committee . radial arterial waveform in the nonfistula arm was measured by applanation tonometry with the sphygmocor device , using the pwa software package . studied variables included augmentation index corrected by heart rate ( ai@75 ) , subendocardial viability ratio ( sevr ) , ejection duration ( ed ) , and aortic systolic and diastolic blood pressure . measurements were taken by a single observer in triplicate and averaged . as a biological demonstration of membrane bioincompatibility , successive white blood cell counts in the advia autoanalyzer and c3a generation ( enzyme immunoassay quidel ) were obtained . the analysis was performed by leukocyte count and c3a levels monitored before , at 15 , 30 , 60 minutes , and at the end of the hd session . pwa was performed by sphygmocor system before , at 15 and 30 minutes of starting hd session . all patients were dialyzed with the same cellulose diacetate membrane ( baxter ) with surfaces of 170 or 210 m and dialysate calcium concentrations of 1.5 mm . in order to avoid the effects of intravascular volume changes on pwa , fluid removal was completely avoided ( ultrafiltration = 0 ) during the 30 minutes of pwa monitoring . pwa was performed before and after a conventional hd session with the same cellulose diacetate membrane ( baxter ) and dialysate calcium concentrations than in study i. the procedure was carried out in normal conditions , with programmed uf as clinically needed based on their dry weight . a total of 19 patients were studied , and the mean ultrafiltration was 2.43 1.12 kg . data are expressed as mean sd . the effect of hd session on the measured variables was tested by means of the paired t - test and repeated measures anova . eleven patients were included in study i , with a mean age of 76.9 10.5 years , 54.6% men , with a mean time on dialysis of 33.9 37.9 months . mean predialysis peripheral blood pressure measured in triplicate and averaged was 135.1 18.1/61.7 7.5 mmhg . membrane bioincompatibility results related with leukocyte count , c3a , peripheral and central blood pressure and stiffness parameters are displayed in table 1 . a significant decrease in leukocyte count occurred at the beginning of dialysis : 6801 1186 versus 4412 1333 at 15 minutes ( p < 0.001 ) , while c3a levels sharply increased from 427 269 to 3501 1638 ng / ml at 15 minutes ( p < 0.000 ) , both parameters being inversely correlated ( r = 0.74 , p = 0.01 ) . no changes were demonstrated in any of the variables analysed in pwa : ai@75 ( 26.1 11.1 versus 26.6 12.4 ) , ed ( 37.1 5.9 versus 35.8 5.1 ) , and sevr ( 129.1 30.3 versus 141.1 29.5 ) . only aortic systolic bp was lower at 15 minutes : 120.1 17.7 versus 110.4 25.8 mmhg ( p = 0.009 ) , in correlation with a reduction of brachial systolic bp : 135.1 18.1 versus 122.7 27.4 mmhg ( p = 0.01 ) , without changes in aortic or brachial diastolic bp . nineteen patients were included in study ii , with a mean age of 71.4 12.2 years , 63.2% were men , and the mean time on dialysis was 36.2 47.4 months . important changes in all the pwa parameters evaluated were observed at the end of the haemodialysis session : ai 29.9 10.1 versus 18.6 15.0 ( figure 1 ) , ed 37.6 3.6 versus 32.8 4.6 , sevr 124.6 19.9 versus 171.7 37.1 , central aortic systolic bp 139.8 25.5 versus 119.4 28.5 mmhg ( all p < 0.00 ) , without changes in aortic diastolic bp 75.8 13.9 versus 75.1 17.9 mmhg ( p : ns ) . however , there was no good correlation between the amount ultrafiltered and the changes in pwa parameters ( r = 0.65 ) , suggesting that arterial stiffness improvement was not caused by fluid removal alone . arterial stiffness measurement in esrd patients has gained importance in the last years , especially for its relationship with increased left ventricular hypertrophy and decreased coronary perfusion during diastole . nevertheless , the acute effects of the hd session on arterial stiffness have been sparsely studied . the results of previous publications are controversial , and it is not clear whether hd per se can induce acute functional changes in the arterial wall [ 18 , 19 ] . this study was designed to separately evaluate the two seemingly and opposite main determinants of arterial stiffness changes during the hd session . in study i , we demonstrate the existence of immunoactivation assessed by considerable changes in leukocyte count and c3 levels . however , such bioincompatibility was not detectable by pwa , since we did not observe any changes in any arterial stiffness parameters . previous studies have shown changes in vascular function using other functional studies as pulse wave velocity ( pwv ) , flow - mediated dilation , or endothelium - independent vasodilation [ 9 , 10 ] . to our knowledge , no previous studies have analyzed the effect of the membrane bioincompatibility on ai@75 . when the dialysis procedure was performed with the normal pattern of uf , and pwa was measured at the end of the dialysis session ( study ii ) , a significant effect on arterial stiffness parameters was shown . we observed a decrease in both aortic systolic bp and ai@75 at the end of the hd session . contrary to expected , we found no correlation between changes in arterial stiffness parameters and the magnitude of fluid removal . previous publications have analysed the effect of the hd session on arterial stiffness yielding different results . some of them defend a lack of improvement of arterial stiffness arguing that vascular changes in esrd are structural rather than functional , that oxidative stress counteracts the effect of uf , or that the beneficial effect of acute volume reduction may be obscured by the activation of the rennin - angiotensin system [ 21 , 22 ] . other studies agreed that pwv remains unchanged as a more structural parameter [ 13 , 14 ] , while ai@75 decreases with the hd session ( as a more functional one ) . this improvement has mainly been attributed to volume correction [ 1215 ] , although some authors have already questioned this association [ 1618 ] . given the lack of association with the uf , we analysed possible relationships with pre - hd brachial or aortic bp , pre - hd ai@75 , bp decrease during hd or dialysis vintage , but we did not find correlation with any of these variables . these discrepancies in the literature can be justified by two main reasons . on the one hand , the large number of variables that can influence during a hd session , not only immunoactivation and intravascular volume drop , but also bp changes , oxidative stress , activation of the rennin - angiotensin system , changes in calcium or magnesium levels during the session , or the differences between patients in intravascular refilling due to individual nutritional status , among others [ 2126 ] . it is , therefore , very difficult to analyze each of them separately . on the other hand , there is a lack of uniformity in the way arterial stiffness is measured in the different studies . we can conclude that hd procedure with the diacetate cellulose membrane induces a clear immunoactivation effect easily demonstrable by leukocyte cell count and c3a generation that is not detectable by pwa . the improvement of arterial stiffness observed after the hd session assessed by pwa was not related with ultrafiltration nor with changes in bp . further studies with control of the different variables should be carried out in order to determine how each of these factors affects arterial stiffness during the hemodialysis session .	membrane bioincompatibility was demonstrated by successive white blood cell counts and c3a generation . pulse wave analysis was obtained by applanation tonometry ( sphygmocor ) in a sequential way : basal , after 30 minutes with nul ultrafiltration , and after a complete dialysis with ultrafiltration . at 15 minutes of haemodialysis , significant decrease in leukocyte count occurred : 6801 1186 versus 4412 1333 ( p < 0.001 ) , while c3a levels sharply increased from 427 269 to 3501 1638 ng / ml ( p < 0.000 ) . no changes were observed in augmentation index without ultrafiltration : 26.1 11.1 versus 26.6 12.4 . only aortic systolic blood pressure was lower at 15 minutes : 120.1 17.7 versus 110.4 25.8 mmhg ( p = 0.009 ) , in agreement with a reduction in brachial systolic blood pressure : 135.1 18.1 versus 122.7 27.4 mmhg ( p = 0.01 ) , without changes in aortic or brachial diastolic blood pressure . important changes in pulse wave analysis were observed after a complete haemodialysis session : augmentation index 29.9 10.1 versus 18.6 15.0 , aortic systolic blood pressure 139.8 25.5 versus 119.4 28.5 mmhg ( p < 0.00 ) , without changes in aortic diastolic blood pressure . in summary , haemodialysis with cellulose diacetate acutely induced a transient state of immunoactivation due to bioincompatibility , this phenomenon was nondetectable by pulse wave analysis . complete haemodialysis session led to important changes in pulse wave analysis .
You are an expert at summarizing long articles. Proceed to summarize the following text: we report a case of 77 year old woman who was found to have a bronchial occlusion caused by a thoracic aortic aneurysm and the relief of this obstruction by the implantation of expandable metallic stents is described . clinical examination was unremarkable apart from a low grade temperature of 37.8 oc and breath sounds were equal bilaterally . after admission the patient 's condition deteriorated with increasing dyspnoea and a worsening pao2 / fio2 ratio . her chest radiograph showed a complete white - out of her left lung field ( fig 1 ) . chest radiograph on admission to intensive care unit ct scanning revealed a large descending thoracic aortic aneurysm measuring 8 cm in maximum diameter ( fig 2 ) . the left main bronchus was compressed between the aneurysm and the aortic trunk ( fig 3 ) . the neck of the aneurysm was markedly conical immediately distal to the left subclavian artery , so proximal stent graft placement would have necessitated left common carotid artery and left subclavian artery bypass grafting to provide a parallel proximal landing zone . the distal neck was also markedly conical with a diameter of 42 mm at the coeliac axis , with extensive mural thrombus . these features rendered a successful endovascular repair unlikely , and therefore endovascular interventional was discounted . sagittal ct section showing large thoracic aneurysm ct chest showing complete compression of left main bronchus by large aortic aneurysm flexible bronchoscopy with fluoroscopic guidance under general anaethesia was used to deploy two uncovered expandable metallic stents ( ultraflex ; boston scientific , ballybrit business park , galway , ireland ) . one stent was placed into her left main bronchus ( proximal release , length , 4 cm ; diameter , 10 mm ) and the second into her right main bronchus ( proximal release , length , 4 cm ; diameter , 12 mm ) . prior computed tomography in this case proved useful in order to provide detailed anatomical information of the occluded airway and thus allowing an effective , uncomplicated , stent insertion . a chest drain was inserted after the procedure for a right sided pneumothorax which had developed while on mechanical ventilation . arterial oxygenation improved quickly afterwards , allowing the inspired fraction of oxygen to be reduced to 0.3 , and there were no complications from the procedure . a subsequent radiograph confirmed re - inflation of the left lung field ( fig 4 ) . chest radiograph post stenting to left main bronchus a multidisciplinary team meeting including vascular surgeons , radiologists and intensivists to discuss this complex case proved pivotal in guiding further treatment options . definitive treatment of the aneurysm was unanimously deemed to be extremely difficult and so after discussion with the patient 's family it was agreed that the risk of the procedure and the burden of care required afterwards would not be in this patient 's best interests . despite what initially appeared to be a successful stent insertion , the patient 's condition deteriorated , and she died 23 days later from a ventilator - associated pneumonia . the use of endobronchial stenting as a means to relieve extrinsic bronchial compression caused by a thoracic aortic aneurysm is rare , with only seven cases reported in the medical literature . six of these patients , akin to our patient , had an expandable metallic stent deployed in either the left main bronchus , the left lower lobe bronchus or bilateral endobronchial stent insertions when surgical treatment was not feasible . ( 1 - 6 ) the remaining patient , after an initially uneventful tracheal and left lower lobe bronchus stent insertion , required a repeat procedure after 2 days due to significant extrinsic compression which had deformed the original stainless - steel balloon - expandable palmaz stent ( johnston & johnston interventional systems , warren , nj ) . a metallic wallstent ( schneider , minneapolis , mn ) was used at this second intervention in the same sites , with additional stenting in the right main stem bronchus , bronchus intermedius and right lower lobe bronchus.(6 ) the palmaz stent , easily compressed by an external force , has since become unpopular.(7 ) in this series of cases , two patients had a fatal massive haemoptysis , caused by erosion of the stent transbroncially resulting in an aorto - bronchial fistula . these complications occurred 18 months after the insertion of a gianturco - z stent ( cook europe , bjaerverskov , denmark ) and 20 days from an ultraflex stent respectively.(1,2 ) the remaining patients had no complications related to the stent itself . of these , one died of bronchopneumonia 8 days after stent insertion , another died after one year from a myocardial infarction and another after 14 days from presumed rupture of the aneurysm.(3,4,6 ) a further patient was discharged after 9 days with no complications reported , and the remaining patient was alive after 23.5 months.(5,6 ) expandable metallic stents placement offers both a rapid and effective approach to restoring airway patency , improving ventilatory function and serving to address symptoms . despite the recognised complications such as airway inflammation , stent migration , airway erosion and perforation , stent fracture and collapse , our patient had no feasible alternative intervention to address her respiratory compromise.(8 ) in view of the fact that vascular stenting was contraindicated , bronchial stenting was in this case a palliative procedure in order to facilitate weaning from mechanical ventilation . metallic stents can be categorized into two types : balloon expandable and self - expanding stents . balloon - expandable stents depend on balloon - mediated deployment in order to expand the stent adequately and do not exert continuous radial pressure after placement . on the other hand , ultraflex stents used in this case are either bare or covered , with either proximal or distal release systems . these are popular because of ease of insertion , their flexibility , and dynamic expansiveness . ( 9 ) tracheobronchial stenting is an established and safe method for the palliative treatment of mechanical central airways obstruction . it provides immediate symptomatic relief in patients with a contra - indication to open surgery . ( 7 ) although permanent application of a self - expanding stent to relieve extrinsic compression of a left main bronchus by an aortic aneurysm in this context is a high risk procedure , it can have a role in certain patients . the long term outcome is not known for the ultraflex stents when used in this context . in addition to highlighting the uncertainties of ultraflex stenting in certain non - malignant conditions , this case illustrates the benefits of a multidisciplinary approach to complex surgical cases . the diverse experience of the multidisciplinary team meeting facilitated a considered and balanced approach to ongoing treatment . while initial treatment was instituted in a particularly effective manner , a limit was agreed upon using the recommendations of the multidisciplinary team . most importantly , family members were involved and were comforted in the knowledge that treatment was guided by experienced surgeons , anaesthetists and radiologists .	a case of bronchial occlusion caused by a thoracic aortic aneurysm and the relief of this obstruction by the implantation of expandable metallic stents is described . stent deployment provided an immediate improvement in lung ventilation and chest radiograph appearances . stent insertion was uncomplicated , but weaning from mechanical ventilation was unsuccessful and the patient died from a ventilator - associated pneumonia , unrelated to the procedure . endobronchial stenting should be considered as a non - invasive therapy for the treatment of bronchial obstruction , with respiratory compromise , caused by a thoracic aortic aneurysm when vascular surgery is not an option . the medium to long term survival of this patient group is poor . this can be attributed to complications related to the stent and also to the poor performance status of these patients .
You are an expert at summarizing long articles. Proceed to summarize the following text: tuberculosis ( tb ) is the second leading cause of death from an infectious disease worldwide . susceptibility to tb can be increased by several comorbidities , one of which is type 2 diabetes mellitus ( dm ) . dm patients present with an overall threefold increased risk of developing active tb . globally , 15% of tb cases are estimated to be attributable to dm and thus with a predicted increase of dm by 155% over the next 20 years , dm will become an increasingly important factor challenging tb control [ 57 ] . dm patients exhibit alterations in the immune response against mycobacterium tuberculosis ( mtb ) , making them more susceptible to infection or progression towards active tb disease and less responsive to treatment [ 811 ] . whilst proinflammatory cytokines are necessary for protection against mtb , anti - inflammatory cytokines may counteract these effects . possible factors that may impact the host response in patients with dm are short - chain fatty acids ( scfas ) , the main metabolic products of fermentation of nondigestible dietary fibres by the gut microbiota . numerous reports have demonstrated that dm patients present with an altered composition of their gut microbiota , which subsequently alters their scfa levels [ 1824 ] . scfas strongly modulate immune and inflammatory responses [ 22 , 2531 ] , thereby influencing the host response to mtb . scfas , of which butyrate ( c4 ) is the most thoroughly studied , act on immune and endothelial cells via at least two mechanisms : activation of g - protein coupled receptors ( gpcrs ) and inhibition of histone deacetylase ( hdac ) . they affect the function of various cell types such as lymphocytes [ 33 , 34 ] , neutrophils [ 25 , 31 , 35 ] , and macrophages [ 28 , 3638 ] . in light of the emerging role of the microbiota in inflammation and immunity , we hypothesized that scfas , and in particular butyrate , may affect the immune response and susceptibility to mtb in type 2 dm patients . in this study we investigated the role of physiological concentrations of scfas on the cytokine response against mtb in human peripheral blood mononuclear cells ( pbmcs ) . we subsequently examined a number of possible mechanisms via which altered concentrations of one particular scfa , c4 , might affect the host immune response to mtb in dm patients . to this purpose , we studied the influence of physiological concentrations of c4 on hdac activity , immune signalling pathways , the eicosanoid pathway , and cellular metabolism . to our knowledge , this is the first study reporting on the effects of physiological plasma concentrations of c4 on mtb - induced cellular responses . physiological plasma concentrations of c4 are in the micromolar range , whilst in previous studies c4 has been used in the millimolar range . thus , this study substantially adds to our knowledge of scfas as possible mediators of altered immune responses to mtb in dm patients . pbmcs were isolated from buffy coats donated after written informed consent by healthy volunteers to the sanquin blood bank ( http://www.sanquin.nl/en/ ) in nijmegen . experiments were conducted according to the principles expressed in the declaration of helsinki . since blood donations were anonymous no tuberculosis skin test or ifn- release assay was performed . however , the incidence of tb in the dutch population is extremely low ( 4/100,000 ) , and bacillus calmette - gurin ( bcg ) vaccination is not part of the routine vaccination program . blood donors were not screened for dm as prevalence of dm among people under 45 years of age ( median age of blood donors ) is about 1.5% and therefore dm is unlikely to be a confounding factor . h37rv mtb was grown to mid - log phase in middlebrook 7h9 liquid medium ( difco , becton dickinson ) supplemented with oleic acid / albumin / dextrose / catalase ( oadc ) ( bbl , becton dickinson ) , washed three times in sterile saline , heat killed , and then disrupted using a bead beater , after which the concentration was measured using a bicinchoninic acid ( bca ) assay ( pierce , thermo scientific ) . isolation of pbmcs was performed by differential centrifugation over ficoll - paque ( ge healthcare ) . cells were adjusted to 5 10 cells / ml ( beckman coulter ) and suspended in rpmi 1640 ( gibco ) supplemented with 10 g / ml gentamicin ( lonza ) , 10 mm l - glutamine ( life technologies ) , and 10 mm pyruvate ( life technologies ) . 100 l of pbmcs was incubated in round - bottom 96-well plates ( greiner ) , pretreated with scfas for 1 h , and stimulated with 1 g / ml of h37rv lysate or 10 ng / ml lps ( sigma - aldrich , e. coli serotype 055:b5 ) . cells were incubated for 24 h or 7 days at 37c in a 5% co2 environment ( n = 6 to 11 ) . alternatively , pbmcs were pretreated for 1 hour ( 37c , 5% co2 ) with ranolazine ( itk diagnostics ) , trimetazidine ( sigma ) , pertussis toxin ( enzo life sciences ) , etomoxir ( sigma ) ( inhibitors of -oxidation , n = 3 ) , aspirin ( aspgic injection powder , n = 3 ) , cycloheximide ( sigma , n = 6 to 7 ) , anti - il-10 antibody igg2a ( biolegend , n = 10 to 12 ) , or igg2a isotype control ( biolegend , n = 10 to 12 ) prior to stimulation . cell culture supernatants were collected and stored at 20c for cytokine measurements , performed by elisa : tnf- , il-1 , il-17a , il-22 , and il-1ra ( r&d systems ) and il-6 , ifn- , and il-10 ( sanquin ) . for quantitative real - time pcr ( qpcr ) analysis rna was isolated from pbmcs using trizol reagent ( invitrogen life technologies ) according to the manufacturer 's protocol . rna was transcribed into complementary dna ( cdna ) by reverse transcription using iscript cdna synthesis kit ( bio - rad , hercules , ca ) . power sybr green pcr master mix ( applied biosystems ) was used for qpcr on an ab steponeplus real - time pcr system ( applied biosystems ) . qpcr data were normalized to the housekeeping gene human 2 m ( n = 3 to 10 ) . western blotting was carried out using a trans - blot turbo system ( bio - rad ) according to manufacturer 's instructions . equal amounts of protein were separated by sds - page on 415% polyacrylamide gels ( bio - rad ) and transferred to pvdf ( bio - rad ) membranes . membranes were blocked for 1 h and then incubated overnight with primary antibody ( dilution 1 : 1000 ) in 5% ( w / v ) bsa or milk in tbs - tween buffer ( tbs - t ) . blots were washed in tbs - t 3 times and incubated with hrp - conjugated anti - rabbit antibody ( 1 : 5000 ; sigma ) in 5% ( w / v ) milk in tbs - t for 1 h at room temperature ( rt ) . after washing , blots were developed with ecl ( bio - rad ) following manufacturer 's instructions . primary antibodies used were rabbit anti - p38 mapk , rabbit anti - phospho - p38 mapk , rabbit anti - erk1/2 ( p44/p42 mapk ) , rabbit anti - phospho - erk1/2 ( p44/42 mapk , t202/y204 ) , and rabbit anti - phospho - jnk ( t183/y185 ) ( all cell signalling ) ( n = 2 ) . lactate was measured from cell culture supernatants using a coupled enzymatic assay in which lactate was oxidised and the resulting h2o2 was coupled to the conversion of amplex red reagent to fluorescent resorufin by hrp ( horseradish peroxidase ) . 30 l of lactate standard or 200x diluted sample was added to 30 l of reaction mix . the 30 l of reaction mix consisted of 0.6 l of 10 u / ml hrp ( sigma ) , 0.6 l of 100 u / ml lactate oxidase ( sigma ) , 0.3 l of 10 mm amplex red reagent ( life technologies ) , and 28.5 l pbs . samples were incubated for 20 min at rt and fluorescence ( excitation / emission maxima = 570/585 nm ) was measured on an elisa reader ( biotek ) ( n = 3 to 5 ) . briefly , 1.5 million stimulated pbmcs were lysed in 75 l of homogenization buffer ( 10 mm nicotinamide ( sigma ) , 10 mm tris - cl ( sigma ) , and 0.05% ( w / v ) resulting supernatants two 18 l aliquots were removed and either 2 l of 0.2 m hcl or 0.2 m naoh was added to each aliquot . the samples were heated for 30 min at 65c and after incubation 2 l of opposite reagent ( naoh or hcl ) was added to each aliquot . 5 l of sample or nad ( -nicotinamide adenine dinucleotide hydrate ; sigma ) standard was then mixed with 85 l of reaction mix and 60 l of fluorescence mix in a black 96-well plate . n - bis(2-hydroxyethyl)glycine ; sigma ) , 0.6 mm ethanol ( sigma ) , and 5 mm edta ( life technologies ) . the fluorescence mix consisted of 0.5 mm pms ( phenazine methosulfate ; sigma ) , 0.05 mm resazurin ( sigma ) , and 0.2 mg of adh ( alcohol dehydrogenase ; sigma ) . the reaction was incubated for 15 min at rt and fluorescence ( excitation / emission maxima = 540/586 nm ) was measured on an elisa reader ( biotek ) ( n = 3 to 5 ) . hdac fluorometric cellular activity assay bml - ak503 ( fluor de lys , enzo life sciences , inc . , farmingdale , ny ) was used to determine hdac activity in pbmcs pretreated with c4 ( 30 min ) and then stimulated with h37rv ( 30 min ) . subsequently pbmcs were incubated with acetylated substrate for 2 hours , after which a developer was added to generate a fluorescent signal from the deacetylated substrate . trichostatin a ( tsa ) was used as a positive control for hdac inhibition ( n = 5 to 6 ) . pbmcs were treated with 50 mol c4 for 1 h and stimulated with 1 g / ml h37rv or 10 ng / ml lps for 7 days . subsequently cells were restimulated with 200 l rpmi supplemented with 10% serum , golgi - plug inhibitor ( gpi brefeldin a ; 1 g / ml , bd pharmingen ) , pma ( phorbol 12-myristate 13-acetate ; 50 g / ml , sigma - aldrich ) , and ionomycin ( 1 g / ml , sigma - aldrich ) for 46 h at 37c and 5% co2 . cells were then washed with pba ( pbs 1% bsa ( albumin from bovine serum ) ) and stained extracellularly for 30 min with cd4-pecys7 ( itk ) for t - helper 17 ( th17 ) cells at 4c . next , cells were washed and permeabilized by fix and perm buffer ( ebioscience ) according to the manufacturer 's protocol for 4560 min at 4c . finally cells were washed and resuspended in 300 l pba to be measured using the cytomics fc500 ( beckman coulter ) cell death was measured by staining pbmcs with annexin v - fitc ( biovision ) and propidium iodide ( pi ) ( invitrogen molecular probes ) . cells were incubated in the dark on ice with annexin - v staining solution ( rpmi supplemented with 5 mm cacl2 and 0.1 l / ml annexin - v ) for 15 minutes . cells were measured with the cytomics fc500 ( beckman coulter , woerden , netherlands ) , and data were analysed using cxp analysis software v2.2 ( beckman coulter ) ( n = 3 to 5 ) . all data were analysed using a paired nonparametric wilcoxon signed - rank test , as the data were not normally distributed . data are shown as cumulative results of levels obtained in all volunteers ( means sem ) . dm is associated with altered gut microbiota and consequently altered scfa levels [ 1822 ] . in line with current literature [ 22 , 2531 ] , we hypothesized that scfas have the potential to influence the host inflammatory response against mtb . in particular we investigated the effects of varying doses of acetate ( c2 ) , propionate ( c3 ) , and butyrate ( c4 ) on h37rv - induced cytokine responses , with rpmi as negative control and lps as positive control ( figure 1 ) . scfas themselves did not induce cytokine production ( results not shown ) but significantly affected h37rv - induced cytokine release . c2 , c3 , and c4 significantly , dose - dependently decreased h37rv - induced production of proinflammatory cytokines tnf- , il-1 , and il-17 , while nonsignificant effects were found for il-6 , ifn- , and il-22 production . in contrast , c3 and c4 induced a significant increase in h37rv - induced production of the anti - inflammatory cytokine il-10 . similarly , c3 and c4 but not c2 decreased lps - induced production of tnf- and il-6 , while the release of il-1 was significantly decreased in response to all three scfas ( results not shown ) . moreover , all three scfas incurred a dose - dependent , nonsignificant decrease in lps - induced il-10 production ( results not shown ) . overall , c4 resulted in some of the most significant changes in cytokine responses ( figure 1(b ) ) . moreover , the potency of butyrate in reducing cytokine responses to h37rv and lps was greater than that for the other scfas . importantly , changes in cytokine levels could not be explained by altered ph levels or cell death ( supplementary figure 1 a and b in supplementary material available online at http://dx.doi.org/10.1155/2016/6014631 ) . therefore , following this screen , we continued our study with c4 at a concentration of 50 m , which is physiologically relevant because it is comparable to human plasma concentrations . since this might account for its anti - inflammatory effects [ 4144 ] , we examined the effect of c4 on hdac expression and activity . c4 significantly decreased hdac8 but not hdac1 gene expression upon h37rv stimulation of pbmcs ( figure 2(a ) ) . consistent with previous reports [ 36 , 4244 ] , c4 at a high dose of 1 mm decreased hdac activity upon both rpmi and h37rv stimulation . however , different from its effect on gene expression , c4 at a physiological dose of 50 m had no effect on actual hdac activity ( figure 2(b ) ) , while trichostatin a ( tsa , positive control ) strongly decreased hdac activity . these data suggest that butyrate 's inhibition of hdac activity is unlikely to play a role in the effects of low doses of c4 on mtb - induced inflammatory responses and stresses the importance of studying the effects of butyrate at physiologically relevant concentrations . signalling of toll - like receptors ( tlrs ) , important receptors for mtb recognition [ 4547 ] , is controlled by feedback mechanisms regulated by several intracellular kinases [ 48 , 49 ] . because impaired mtb recognition and insufficient tlr signalling may account for the anti - inflammatory effects of c4 , we examined whether c4 affected these feedback loops . however , c4 had no effect on phosphorylation of the map kinases p38 , erk ( figure 3(a ) ) , or jnk ( supplementary figure 2 ) . c4 has also been reported to induce expression of inhibitors of tlr signalling pathways , but we found that c4 significantly decreased mrna expression of tlr signalling inhibitors socs1 and tollip and did not affect expression of socs3 or st2 ( figure 3(b ) ) . of note , these results were not explained by cell death ( supplementary figure 1 b ) . aside from tlr signalling , c4 possibly exerts its anti - inflammatory effects through modulation of the eicosanoid pathway . eicosanoids , oxygenated metabolites of arachidonic acid , modulate the host immune response to mtb [ 5155 ] . c4 has been reported to upregulate key enzymes of the eicosanoid pathway upon lps stimulation , but a reverse effect has also been described . we did not observe a significant impact of c4 on transcript levels of cyclooxygenase 2 ( cox-2 ) , one of the main eicosanoid enzymes , upon h37rv or lps stimulation ( supplementary figure 3 a ) . alternatively , c4 has been described to induce release of the anti - inflammatory prostaglandin pge2 [ 26 , 30 , 57 ] . inhibition of pge2 with aspirin could not counteract the inhibitory effects of c4 on tnf- and il-1 cytokine responses upon either h37rv or lps stimulation ( supplementary figure 3 b ) . the eicosanoid pathway is therefore unlikely to be the mediator pathway through which c4 exerts its anti - inflammatory effects . another possible explanation for butyrate 's anti - inflammatory effects is its influence on cellular metabolism . a recent paper described that microbiota have a strong effect on energy homeostasis in the mammalian colon and showed that c4 regulates different aspects of energy metabolism acting as an important energy source for colonocytes . contrary to this previous study , we observed no effects of c4 on cellular lactate production , the nad / nadh redox ratio , tca cycle gene expression ( figure 4 ) , or -oxidation ( supplementary figure 4 ) . these data strongly suggest that c4 modulates the immune response to mtb independently of cellular metabolism . we next examined whether the inhibitory effect of c4 on mtb - induced proinflammatory cytokine responses , with a concomitant increase in anti - inflammatory il-10 production ( figure 1 ) and decrease in th17 proliferation ( supplementary figure 5 a ) , was also present at the level of gene transcription . c4 led to a decrease in tnf- , il-12 , and il-23 mrna levels upon h37rv stimulation and a parallel increase in il-10 mrna ( figure 5(a ) ) , while no effect on production of the anti - inflammatory cytokine il-1ra was observed ( supplementary figure 5 b ) . these data point to il-10 as a possible intermediary mediator of the anti - inflammatory effects of c4 . we therefore assessed whether removing il-10 protein from the cellular environment could counteract the inhibitory effects of c4 . to this end , we pretreated pbmcs with cycloheximide ( chx ) , an inhibitor of translation . stimulation of pbmcs with h37rv in the presence of c4 in combination with chx resulted in higher tnf- responses , as compared to incubation with h37rv and c4 alone . upon lps stimulation , this effect was not present ( figure 5(b ) ) . we subsequently examined whether blocking il-10 specifically using an anti - il-10 antibody could counteract the inhibitory effects of c4 on proinflammatory cytokine response . blocking il-10 completely restored il-6 cytokine responses in response to h37rv and c4 , while tnf- and il-1 production was partly restored ( figure 5(c ) ) . this suggests an important role for intermediary protein synthesis , specifically il-10 , in mediating the anti - inflammatory effects of c4 . dm is associated with a threefold increased risk of active tb , but the underlying immunological mechanisms remain largely unknown [ 3 , 12 , 13 ] . alterations in the gut microbiota of dm patients are associated with changes in plasma scfa concentrations . multiple papers have reported a decrease in c4-producing bacteria in type 2 dm patients [ 18 , 19 , 21 , 23 , 24 ] . we here show that scfas , especially c4 , exhibit anti - inflammatory properties ; low doses of c4 decreased mtb - induced proinflammatory cytokine responses on both the transcriptional level and the translational level , while production of il-10 was increased . this anti - inflammatory effect was independent of hdac activity , toll - like receptor signalling , the eicosanoid pathway , or cellular metabolism . we observed a general anti - inflammatory effect of c2 , c3 , and c4 on mtb - induced cytokine production . c4 induced some of the most significant and most potent changes in cytokine responses , which is in line with published results , although our study is the first to examine the effects of physiological concentrations of scfas on mtb - induced cytokine responses in vitro . firstly , the inhibitory effect of all three scfas on production of tnf- and il-1 was comparable for mtb and lps stimulation . however , while c3 and c4 had a clear effect on lps - induced il-6 release , this was not found for mtb . this suggests that scfas do not affect mtb - induced il-6 , although il-6 has been assigned an important role in mtb host responses [ 5962 ] . secondly , c2 , c3 , and c4 had a much stronger inhibitory effect on t - cell derived cytokine il-17 than on t - cell derived cytokines ifn- and il-22 . because c4 also strongly decreased th17 proliferation ( supplementary figure 5 a ) , scfas may affect th17 subsets more than other t - cell subsets . this may be of great relevance since th17 cells , and il-17 in particular , have been reported to be essential in protective immunity against mtb [ 63 , 64 ] but inversely associated with dm complications [ 6567 ] . lastly , the stimulatory effect of c3 and c4 on anti - inflammatory il-10 release was mtb - specific and was not seen with lps stimulation . il-10 has been delineated as an important mediator in mtb infection : it has been reported to block bacterial killing in mtb - infected macrophages , suppress multinucleated giant cell formation and cytokine production , and inhibit the development of protective immunity [ 6874 ] . in contrast to tb , il-10 may have a protective role in type 2 dm by reducing insulin resistance and obesity [ 7577 ] . therefore , the increase in il-10 production we see as induced by c4 is very relevant for the course of both dm and tb disease . we examined several possible mechanisms underlying the effect of c4 on cytokine production , starting with hdac activity , which is known to be inhibited by scfas . c4 at a physiological low dose of 50 m had little effect , while millimolar concentrations of c4 ( as used in other studies [ 36 , 4144 ] ) decreased hdac activity upon h37rv stimulation . this is expected as ic50 values of hdac inhibition by c4 are > 100 m , depending on the class of hdac . the strongest effect was noted for hdac8 , which is reported to be most sensitive to c4 . this argues that physiological c4 concentrations in human plasma do not exert hdac inhibition and underlines the importance of using physiological concentrations within in vitro experimental models . in contrast to a previous study , we observed a decreased gene expression of the tlr modulatory factors socs1 and tollip when pbmcs were stimulated in the presence of c4 , which thus can not explain the inhibitory effects on cytokine production . this , together with our data showing that c4 does not affect map kinase activity , suggests that c4 does not act at the level of tlr signalling , as shown previously . as a third possible mechanism , we assessed whether c4 exerts its effects through eicosanoid metabolism . the eicosanoid pathway is under influence of scfas [ 30 , 56 ] and may modulate the host response to mtb [ 5155 ] . c4 did not affect expression of cox-2 , a key enzyme in the eicosanoid pathway , in contrast to previous reports that used supraphysiological c4 concentrations [ 30 , 56 ] . in addition , inhibition of the eicosanoid pathway using aspirin did not counteract the effects of c4 . therefore , the eicosanoid pathway is unlikely to be involved in mediating the effects of c4 . the effect of diabetes on the host immune response to mtb might also be explained by altered cellular metabolism , with a possible role for scfa . one previous study noted that c4 influences metabolic processes in colonocytes , which use butyrate as their primary energy source . however , we did not observe any effect of c4 on lactate production , the redox status , tca cycle gene expression , or -oxidation in pbmcs . we therefore conclude that cellular metabolism does not mediate the effect of c4 on mtb - induced cytokine production . finally , we further examined the effect of c4 on the anti - inflammatory cytokine il-10 . il-10 is detrimental to tb outcome , while it may improve dm symptoms [ 6877 ] . in line with previous studies [ 33 , 81 , 82 ] , we report an upregulation in il-10 production induced by c4 . removal of all intermediary protein , including il-10 , from pbmcs stimulated with h37rv and c4 led to a significant increase in tnf- transcript , thereby counteracting the decrease in tnf- production induced by c4 . moreover , blocking il-10 specifically fully restored il-6 responses in pbmcs stimulated with h37rv and c4 and partly restored tnf- and il-1 responses . these data suggest that the anti - inflammatory cytokine il-10 may play a role in the inhibitory effects of c4 on mtb - induced inflammatory responses . currently , much research focuses on modulation of the gut microbiota in order to treat obesity and type 2 dm [ 8386 ] . administration of sodium butyrate or butyrate - inducing probiotics in mice significantly increased plasma insulin levels and insulin sensitivity and suppressed body weight gain [ 8789 ] . the anti - inflammatory effects of c4 may attenuate the chronic inflammatory state associated with type 2 dm , thereby improving dm symptoms . if chronic inflammation is a causal factor of the impaired host response to mtb in type 2 dm patients , attenuation of this hyperinflammatory state may improve not only dm but also tb outcome in patients with coincident dm and tb disease . some limitations of our study need to be addressed . firstly , we studied the effects of c4 on mtb - induced inflammation in pbmcs in vitro . scfa levels have been shown to be altered in dm patients [ 1822 ] , but this in vitro model does not include other aspects of the pathophysiology of dm such as hyperglycemia , hyperinsulinemia , or dyslipidemia , phenomena which have also been reported to affect immunity [ 9094 ] . furthermore , dm medications possibly interfere with the intestinal microbiota and immune responses in patients [ 9597 ] . it is therefore unclear how accurately our in vitro model reflects the in vivo situation in dm patients . in conclusion , we show an anti - inflammatory effect of low , physiological doses of c4 on mtb - induced inflammatory responses . the anti - inflammatory cytokine il-10 may play a role in mediating the inhibitory effects of c4 on the host immune response to mtb . further studies are needed to precisely explore the pathways by which physiological concentrations of c4 exert their anti - inflammatory effects and to define the mechanism of increased tb sensitivity in type 2 dm patients . moreover , current research on modulating gut microbiota in dm should include its possible effects on tb .	type 2 diabetes mellitus confers a threefold increased risk for tuberculosis , but the underlying immunological mechanisms are still largely unknown . possible mediators of this increased susceptibility are short - chain fatty acids , levels of which have been shown to be altered in individuals with diabetes . we examined the influence of physiological concentrations of butyrate on cytokine responses to mycobacterium tuberculosis ( mtb ) in human peripheral blood mononuclear cells ( pbmcs ) . butyrate decreased mtb - induced proinflammatory cytokine responses , while it increased production of il-10 . this anti - inflammatory effect was independent of butyrate 's well - characterised inhibition of hdac activity and was not accompanied by changes in toll - like receptor signalling pathways , the eicosanoid pathway , or cellular metabolism . in contrast blocking il-10 activity reversed the effects of butyrate on mtb - induced inflammation . alteration of the gut microbiota , thereby increasing butyrate concentrations , can reduce insulin resistance and obesity , but further studies are needed to determine how this affects susceptibility to tuberculosis .
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Proceed to summarize the following text: he did not show any constitutional symptoms such as fever , malaise , or fatigue . inflammatory markers were suggestive of mild disease ; the erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) were 13 mm / hr ( normal range , 0 to 10 mm / hr ) and 1.1 mg / l ( normal range , 0 to 5 mg / l ) , respectively . coronary angiography ( cag ) showed 80% concentric stenosis at the left main coronary artery , chronic total occlusion of the left anterior descending ( lad ) artery , and 95% stenosis of the proximal right coronary artery ( fig . transthoracic echocardiography showed normal left ventricular systolic function with severe hypokinesia at the mid - inferior and inferolateral wall . computed tomography ( ct ) of the aorta revealed a diffuse severe tapered narrowing of the descending thoracic artery through the abdominal aorta with the narrowest dimension of 6 mm at the level of the renal arteries . total occlusion of the celiac trunk and the superior mesenteric artery ( sma ) was identified with major visceral collateral circulation from both the inferior mesenteric artery ( i m a ) and the bilateral internal thoracic arteries ( itas ) ( fig . the ankle - brachial index had decreased to 0.7 on both sides . in order to enlarge the narrowing aortic lumen and to increase the forward flow to the i m a and the lower extremities , a vascular stent for the abdominal aorta ( 12 mm100 mm , hercules vascular ; s&g biotech inc . , after this procedure , the patient received steroid therapy under the impression of takayasu arteritis ( ta ) . during a 5-month follow - up period , the ankle - brachial index also recovered to the normal range , and inflammatory markers such as esr and crp likewise dropped to the normal range . considering the patient s young age and the absence of possible arterial grafts first , drug - eluting balloon angioplasty ( sequent please ; b. braun , melsungen , germany ) for the proximal right coronary artery ( rca ) lesion was done during the follow - up cag ( fig . two weeks later , off - pump coronary artery bypass grafting ( opcab ) for the lad and the large ramus intermedius ( ri ) was carried out using the right ita in situ and its large side branch . aspirin was continued on the day of surgery , and clopidogrel was stopped 1 week before surgery . the right ita was selected because it provided relatively less collateral circulation to the visceral organs than the left ita . in the operating field ( fig . 2c , d ) , the right ita was very large ( diameter : 5 mm ) , and the lumen was high - quality and did not show any calcifications . a large side branch was also identified and used for lengthening the graft by connecting the free side branch to the end of the in situ right ita ( i - compo - site graft ) . the right ita was anastomosed in situ to the lad , and the i -composite graft ( side branch ) was anastomosed to the ri branch . the peak postoperative creatine kinase - myocardial band troponin - t levels were 4.8 ng / ml and 0.15 ng / ml , respectively . the postoperative course was uneventful , and the patient was discharged from the hospital on postoperative day 6 without any complications . the postoperative crp level was 6.4 mg / l before discharge . at the most recent follow - up at the outpatient clinic ta can be classified according to the involved vessels as follows : type i ( branches of the aortic arch ) , type iia ( ascending aorta and aortic arch ) , type iib ( ascending aorta , aortic arch , and descending thoracic aorta ) , type iii ( descending thoracic aorta and abdominal aorta ) , type iv ( abdominal aorta and/or renal arteries ) , and type v ( combined features of types iib and iv ) . the distribution of the types varies among countries , but a recent study reported that type v ( 54.5% ) and type ii ( 22.3% ) were the most common types of ta in korea . coronary arterial involvement is not uncommon in ta , and occurs in 10% to 30% of cases . typical lesions involve stenosis at the coronary ostium or proximal segments of the coronary arteries . this patient was relatively old , considering that ta is typically seen in young patients with an age of onset of less than 40 years . other possible large vessel vasculitis conditions can occur at this age , such as infectious aortitis , giant cell arteritis ( gca ) , or behcet disease . symptoms of cranial aortitis , which is characteristic of gca , such as new - onset headache , jaw claudication , or visual problems , were also absent . a neurysms involving large branches of the pulmonary artery , which are typically seen in behcet disease , were not present either . as this patient was considered to be in the active stage of ta with elevated inflammatory markers and a thickened aortic wall at the first presentation , he underwent opcab after stabilization of the disease activity using oral steroid treatment following aortic stenting . surgery is strongly recommended during the inactive phase of vasculitis because subsequent steroid treatment can lead to a significant regression of the coronary artery stenosis , resulting in the occlusion of bypass grafts from flow competition . we performed total arterial revascularization considering the patient s young age . according to the american college of cardiology foundation / american heart association guidelines for coronary artery bypass surgery , complete arterial revascularization may be reasonable in patients less than 60 years of age . in this patient , although the i m a provided most of the collateral circulation in the sma territory , the left ita provided a significant amount of the collateral circulation in the celiac trunk territory . thus , we used the right ita as a blood source for coronary artery bypass grafting . fortunately , there was a large side branch of the right ita that was used for lengthening the in situ right ita graft for the sequential anastomosis of the lad and ri branch . side branches of the ita are rarely used for grafts because of their small caliber and histologic differences . however , in the present case , the size and the quality of the side branch were considered excellent for a graft , and a good mid - term patency rate has been reported when the side branch of the ita was used . we adopted an i - composite graft considering the location of the target coronary artery and the length of the free side branch . this graft made the total graft longer than if a y - composite graft , which anastomoses the second graft in an end - to - side fashion , were used . some controversy exists regarding the in situ use of the ita in ta because of subclavian artery involvement . however , in the present case , both subclavian arteries were normal and the quality of the right ita was excellent . long - term patency is considered acceptable in coronary artery bypass grafting using an appropriate - quality ita even in ta patients [ 2 , 4 ] . as the available arterial grafts were limited in patients with ta , we adopted a hybrid approach ( percutaneous intervention for the rca with a drug - eluting balloon and opcab ) . we first applied the intervention for the rca lesion before surgery because doing so reduced the risk of perioperative myocardial infarction in the rca territory . drug - eluting balloon angioplasty can suppress intimal hyperplasia and has been proven to be safe and effective in terms of mid - term outcomes . moreover , opcab can allow aortic manipulation to be avoided in cases with systemic vasculitis and irregular aortic wall thickening . although there have been some reports of coronary artery bypass grafting using a dilated ita in cases with coarctation of the aorta , to the best of our knowledge , this is the first report of hybrid opcab using a dilated ita and its branch to treat a patient with ta . although the quality of the dilated ita and its branch was good , the long - term clinical outcome and patency need to be confirmed over the course of follow - up .	coronary arterial involvement in takayasu arteritis ( ta ) is not uncommon . herein , we describe a case of ta with celiac trunk and superior mesenteric artery occlusion combined with coronary artery disease . bilateral huge internal thoracic arteries ( itas ) and the inferior mesenteric artery provided the major visceral collateral circulation . after percutaneous intervention to the right coronary artery , off - pump coronary artery bypass grafting for the left coronary territory was done using a right ita graft and its large side branch because of its relatively minor contribution to the visceral collateral circulation .