The pedagogic training of graduate students at the Faculty of Medicine of Ribeirão Preto. In a discussion of the objectives of the Post-Graduate Course of the Faculty of Medicine of Ribeirão Preto, i.e., the training of future medical educators as teachers, the author describes the major effort of the School to achieve this objective during the last twenty years. Special emphasis is placed on the disciplines "Pedagogy in the Biomedical Area-Seminars I and II" which have presented persistent administrative difficulties. The author comments about the attitudes that seem to prevail in Brazilian Universities, which attribute a very high value to scientific productivity at the expense of teaching, and therefore represent an obstacle to achieving the objective in question. Some procedures that may result in changes in attitude towards medical education, with an improvement in the area, are also discussed.


[Kidney failure as a complication of the treatment of pulmonary paracoccidioidomycosis]. Sulfadiazine is one of the drugs of choice in the treatment of paracoccidioidomycosis. Side effects are uncommon. Cutaneous and gastrointestinal reactions and rarely leucopenia and jaundice may occur. A patient on sulfadiazine 6 g daily exhibiting a good response is reported. On the 12th day, he presented an intense abdominal pain and reduced urinary output. An ultrasonography revealed diffuse calculosis in urinary tract. Peritoneal dialysis, urinary alkalinization and rehydration were instituted with complete restoration of renal function. This case illustrates the importance of an adequate hydratation of patients receiving sulfadiazine, a simple measure in the prevention of this uncommon complication which carries a high morbidity.


[Effects of previous distribution of documents containing educational objectives on the teaching of pediatrics]. The authors discuss the usefulness of giving to the students of the Department of Pediatrics of the University of São Paulo instructions regarding the educational objectives of the graduation course. An inquiry was carried out with the collaboration of: 1) members of the Departmental Council and of the Commission for Graduation; 2) teachers responsible for the course, and 3) the students. Only 32.9% of the consulted students affirmed that the knowledge of educational objectives had a positive influence on their study.


[Effect of 9-alpha-fluorhydrocortisone (Florinef) in extrarenal regulation of potassium]. There are evidences that adrenal hormones regulate extrarenal potassium homeostasis. The present study evaluated the effect of Florinef modulation on extrarenal mechanisms in potassium adaptation of adrenalectomized rats. The results demonstrated that the rats treated with Florinef had serum potassium levels at normal range probably due to an increase in cellular potassium uptake consequently to en enhanced activity of Na-K-ATPase.


[Morphological study of the inguinal hernial sac]. The pathological changes observed in 76 hernial sacs removed at operations for inguinal hernias are reported. The shape and size of hernial sacs varied from case to case, containing from small to large amounts of adipose tissue and, sometimes, also of smooth muscle. The role played by this muscular tissue is not yet clear; it is possible that it acts as supportive tissue against hernial sac enlargement. In one case schistosomotic granuloma and in the other a metastasis of ovarian carcinoma was found. For better understanding of the evolution of hernias and for the diagnosis of associated diseases there is the need for routine pathological examination of hernial sacs.


[Barrett esophagus in the esophageal stump after subtotal esophagectomy with cervical esophagogastroplasty]. In 48 patients that suffered subtotal esophagectomy and were submitted to esophagogastroplasty a long term follow-up study was carried out. In four of these patients (8.3%) pathological changes that characterize Barrett's esophagus were found in the esophageal stump. As far as we could verify, such changes were never reported at this localization before. The Barrett's esophagus seems to be intimately linked to reflux esophagitis resulting from the action of acid-peptic and biliary secretions.


[Transport of electrolytes in the respiratory epithelium in patients with cystic fibrosis]. In the conducting airway, the epithelial electrolyte transport processes play an important role in determining the composition of the respiratory tract fluid. Apical membrane Cl- and Na+ channels control the secretion and absorption of epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis.


[Epidemiological study of Lyme disease in Brazil]. Lyme disease is a tick-born infection first reported in United States of America in 1977 by Allen C. Steere. It occurred in the state of Connecticut; other cases have been discovered in others states of USA and also in other countries (Canada, Soviet Union, Japan, China, Australia). This disease has not been reported in South America yet. In order to investigate this disease in Brasil, a multi-disciplinary group including microbiologists, entomologists and clinicians was created at the University of São Paulo. The aim of this report is to describe the elaboration of this research in our center and also to present the preliminary results.


[Gastric liposarcoma: report of a case]. The authors present a case of a well differentiated gastric liposarcoma. The tumor was situated in the submucosa of the antrum, had a diameter of 9 cm, and showed well defined borders. A subtotal gastrectomy with BI reconstruction was performed. No abdominal metastases were observed. The patient is in good condition, without recurrence of the tumor 19 months after its removal.


[Heart transplantation: donor with Chagas' disease and clinical course of the receptor]. Because of an operational misshape, a female patient received a heart transplant whose donor was chronically infected by Trypanosoma cruzi. After 3 months, transmission of the parasite has not been detected, what deserves communication, considering the clinical and scientific implications of the case.


[Recurrence in post-splenectomy spherocytosis anemia due to presence of accessory spleens]. A young female patient had a recurrence of severe spherocytic anemia there years after having been submitted to splenectomy. The presence of two accessory spleens with a size of 3.5 cm and 4.0 cm, was demonstrated by technetium scanning and ultrasound images. Surgical removal of these noduli resulted again in prompt recovery. The development of the spleen beings in the fifth week of intrauterine life at the dorsal mesogastrium. The lack of fusion of splenic lobuli is responsible for the occurrence of accessory spleens. A surgeon performing splenectomy for the treatment of hemolytic anemia should always search for possible existences of accessory spleens.


[Persistence of the right valve of the sinus venosus with partial division of the right atrium]. A case of persistent right valve of the sinus venosus (the so-called cor triatriatum dexter) is described. There were associated severe pulmonary valve stenosis and probe patent foramen ovale. The patient survived to the age of 25, without complaints. His death was due to complications of pulmonary thromboembolism. The anomaly was detected prior to death by echo-doppler-cardiogram. The postmortem findings of the heart are described in detail.


[Primary disorders of neutrophils: principal clinical and laboratory aspects]. This article presents a review of different categories of inherited neutrophil dysfunctions, such as chronic granulomatous disease, disorders of chemotaxis, defects of degranulation and the leukocyte adherence deficiency. The clinical manifestations and laboratory findings of these phagocyte abnormalities, with particular reference to various tests of neutrophil functions used for diagnostic elucidation are described.


[Sarasinula marginata (Semper, 1885) (Mollusca, Soleolifera) from Belo Horizonte (MG, Brasil) as a potential intermediate host of Angiostrongylus costaricensis Morera, Cespedes, 1971]. Specimens of Sarasinula marginata were collected in kitchen and house gardens of Belo Horizonte, Minas Gerais. The susceptibility of these molluscs for Angiostrongylus costaricensis was tested by infecting 15 laboratory--reared slugs (F1). The positivity demonstrated was of 80.0%.


[Laboratory diagnosis and symptoms of dengue, during an outbreak in the Ribeirão Preto region, SP, Brazil]. A dengue type 1 outbreak started in the Ribeirao Preto Region, North of Sao Paulo State, Brazil, in November of 1990. About 3500 dengue cases were confirmed by blood tests until February of 1991. The Virus Research Unit of The Faculty of Medicine of Ribeirao Preto-Sao Paulo State University, studied 502 dengue suspect cases. The serologic diagnosis of dengue type 1 was confirmed by haemagglutination inhibition test (HAI) in 19% of the cases. Diagnosis was done later by using an enzyme immuno assay on infected cultured cells (EIA-ICC) which discriminated IgG and IgM dengue, antibodies. EIA-ICC was less sensitive (89%) but more effective than HAI. EIA-ICC is a simple technique. It dispenses a second serum sample for diagnosis and it can be completed in about 5 hours. Dengue virus was isolated from the blood of 21 patients by inoculation in culture of mosquito C6/36 cells. The isolated virus were identified by indirect immunofluorescent test, by using an antisera pool to the flavivirus family and dengue type specific monoclonal antibodies. The dengue most frequent symptoms in 71 patients were observed: fever (90%), myalgias (57%) and arthralgias (41%).


[Thoracic radiologic aspects in paracoccidioidomycosis]. In this paper the authors analyse 159 radiographs from paracoccidioidomycosis patients seen at the Evandro Chagas Hospital/Fiocruz in the period between January 1960 to December 1988. Twenty four cases (15.09%) of association with tuberculosis were observed; one with pneumoconiosis; one with aspergillosis, and two with carcinoma. Twenty cases were excluded from the radiologic analysis: in 8 of these the diagnosis of tuberculosis occurred concomitantly, and in 12 patients, lung fibrosis due to previous treatment for tuberculosis or paracoccidioidomycosis was present in the 139 remaining cases, the radiographic abnormalities encountered were grouped according to the predominance of lesions at the various lung sites, if alveolar or interstitial, according to Magalhães' (1982) classification modified by the authors: infiltrate 55 cases (39.6%); mist 28 (20.1%); pneumonic 23 (16.6%); nodular 16 (11.5%); micronodular 10 (7.2%), and fibrotic 7 (5.0%). In 113 cases it was possible to follow the regression of the pulmonary process radiologically. In 85 (75.2%) patients, regression took place within 6 months; in 17 (15.0%) cases between 7 and 12 months; in 4 (3.5%) between 13 and 24 months, and in 7 (6.1%) cases no changes in the radiographic pattern were noted.


Hepatitis B vaccine--proposal for a standardized assessment of immune response. The authors developed a comparative study of the various methods of assessment of immune response to Hepatitis B vaccine. Eighty-six health care professionals underwent a vaccination programme with three doses of plasma-derived vaccine against Hepatitis B (H-B-Vax, Merck, Sharp & Dohme) given intramuscularly. Assessment of immune response was carried out three months after the end of the programme, by radioimmunoassay (RIA) and enzyme immunoassay (EIA). The results showed that the semi-quantitative assessment of Anti-HBs antibodies by RIA or EIA was perfectly comparable to the reference method (quantitative determination of antibodies by RIA). In view of these findings, the authors suggest a standardization of assessment of immune response to the vaccine, thus permitting correct planning of booster doses and easier comparison between different studies.


[Evaluation of an ELISA technique for detection of antigens and circulating immune complexes of Trypanosoma cruzi by a field study in an endemic zone of Argentina]. The ELISA technique for detection of T. cruzi circulating antigens (cAg) and immune complexes (CIC) in the sera of chronic chagasic patients was field-tested in a well-known endemic area of Argentina (San Luis province). Of 215 individuals screened, 51 were positive for ELISA-CIC and 45 for ELISA-cAg. Seventy four subjects were considered por T. cruzi-infected, as they showed serologic reactivity at least by two different techniques. In this group, 49 (66.21%) were ELISA-CIC positive, whereas in 43 (58.11%) of them cAg was found by ELISA. Unspecific reactions were observed in only 2 cases with reactive serology for Chagas disease. Within the group considered as noninfected, a false-positive outcome was obtained at low dilution by one of the serologic tests in 16 (11.35%) of 141 individuals. These sera yielded consistently negative results by ELISA-CIC and cAg, showing the utility of antigen detection in situations of conflictive serology. While antibody determination merely provides an indirect proof of infection, our ELISA tests for demonstration of T. cruzi-specific antigenic fractions in the host's circulation allow a parasitologic diagnosis in chronic patients, with higher sensitivity than that exhibited by traditional methods for detection of the whole parasite.


[Non-cholera vibrios in enterobacteriologic routine]. Of 3250 diarrheal stools received for microbiologic diagnosis at a private clinical laboratory in Recife, Brazil, strains of Vibrio were isolated from 55 (1.7%). The study was carried out from May 1989 through May 1991. For recovering Vibrio, fecal samples were enriched in alkaline peptone water supplemented with 2% NaCl and subcultured on thiosulfate-citrate-bile salts-sucrose agar (TCBS). Of the recovered species, V. parahaemolyticus was most commonly found (24 strains), followed by V. furnissii (15 strains), V. cholerae non-01 (6 strains), V. alginolyticus (4 strains), V. fluvialis (2 strains), and Vibrio sp. (1 strain). The low isolation rate of Vibrio raises doubts about the cost-effectiveness of the use of TCBS in the routine enterobacteriologic workup of clinical laboratories.


[Ecological aspects of American cutaneous leishmaniasis. 9. Prevalence/incidence of the human infection in the municipality of Pedro de Toledo and Miracatu, São Paulo, Brazil]. The epidemiologic study was conducted during the 1973-1984 period. The clinical prospective exam and Montenegro skin, immunofluorescent and passive hemagglutination tests have been carried out in three small localities between Pedro de Toledo e Miracatu municipalities, São Paulo, Brazil. The retrospective study of human-cases involved 108 and 65 cases registered in Pedro de Toledo e Miracatu, respectively. In the three communities studied, 273 people were examined clinically and serologically. Twenty two individuals had had signals of cutaneous leishmaniasis; 10.2 and 12.8% were seropositive to IF and HA. Leishmanin skin testing of a sample of 154 people residents in Pedra do Largo showed prevalence of Leishmania infection in 25.5%. This result involved individual of all ages and sex. However, 5.8% of them were from 0 to 9 years old. The data confirmed that active parasite transmission didn't occur every year, either. The human infection seems not to depend on man contact with a forest. The incidence relatively low suggests low endemic area for cutaneous leishmaniasis and an explosive behavior of the cases. The temporal distribution of disease was irregular and the epidemiological pattern seen was different from the other endemic area of South America.


[Preservation of fungi and actinomycetes of medical importance in distilled water]. Several methods have been used for the preservation of fungi, all of them presenting advantages and disadvantages. The choice of the methods depends upon the laboratory availabilities, time of preservation, genetic stability of the cultures and other factors. In this work the results obtained through the utilization of Castellani's method (preservation in distilled water) for the maintenance of 174 strains belonging to the "Micoteca do Instituto de Medicina Tropical de São Paulo" are presented. These strains were analyzed after 6, 12, 18 and 24 months, with regard to the percentage of viability taking into consideration the rates of growth and contamination. The smallest percentage of viability occurred in the group of the actinomycetes (50 to 100%) and the largest one in the group of the yeasts (near 100%). According to other authors, the Castellani's method, besides being simple and economically feasible for small size laboratories, yields good results.


A low cost method to produce a gaseous environment for the isolation of Helicobacter pylori. A low cost method (LCM) to produce a gaseous environment for the isolation of Helicobacter pylori, was compared with the standard Gas Park system. The LCM uses a carbonated antacid tablet, a plastic bag with tap water, a candle, and a wide-mouthed glass jar provided with a tight-fitting metallic screw cap and a rubber gasket. Antral gastric biopsies from 153 cases were incubated by duplicate on blood agar plates and treated with the two methods. In 95 cases the agent was isolated from both, and only from the standard method in 10 cases; the opposite condition was found in five cases, and 43 were negative. That difference is not significant (Pearson's chi 2 = 93.25 p > 0.05).


Activity of two different triazoles in a murine model of paracoccidioidomycosis. A new orally absorbable triazole (Schering 39304) with a long serum half-life in man (60 hours), was tried in a murine model of progressive paracoccidioidomycosis and compared with itraconazole, another triazole which has proven effective in this mycosis. Only 15% of the infected, untreated mice survived while 53 to 75% of the animals receiving itraconazole survived. Mice treated with Schering 39304 exhibited higher (86-100%) survival rates. Statistically, the 5 mg/kg Sch 39304 was superior to the 50 mg/kg itraconazole dose. Lung cultures showed that 20 mg/kg/day of Sch achieved sterilization of the infectious foci. These results indicate that the new triazole will have a place in the treatment of paracoccidioidomycosis.


[Evaluation of the therapeutic activity of itraconazole in chronic infections, experimental and human, by Trypanosoma cruzi]. Other authors have demonstrated that itraconazole has antiparasitic activity against Trypanosoma cruzi both in vitro and in animal acute infection. Because of these observations, we decided to evaluate the chronic phase of this protozoal disease, since it is the most important form under a clinical and assistential point of view. We studied an infected mouse model as well as human cases of Chagas' disease. One hundred mg/kg/day by gastric tube, and 100 or 200 mg/day orally were given, respectively, during three months, without showing any beneficial effect, at least with the adopted methods.


[The efficacy of the bothropic-crotalic antivenom in the neutralization of the main Bothrops jararacussu venom effects]. Myonecrosis is one of the effects of Bothrops jararacussu venom, from which a myotoxin was isolated showing structural homology to phospholipase A2 (PLA2), but without enzymatic activity. Such myotoxic activity is also present in the Crotalus durissus terrifucus venom, and is attributed to crotoxin and to PLA2 (crotoxin B), the basic component of the crotoxin complex. The Bothrops jararacussu venom showed three proteins with immunologic identity to PLA2 from crotoxin. The bothropic (AB) and the bothropic/crotalic (AB/C) anti-venoms, two commercial polyvalent anti-venoms produced at Instituto Butantan, were compared in order to assess their capacity for neutralization of the lethal, hemorrhagic, coagulant and myotoxic activities of Bothrops jararacussu venom. Both anti-venoms showed the same level of hemorrhagic activity neutralization. However, AB/C was about three times more efficient than AB in neutralizing the myotoxic activity, and two times more potent for neutralization of lethality and coagulant activity of Bothrops jararacussu venom. These data suggest that the use of AB/C could be of value in the treatment of patients bitten by snakes of this species.


[Bone marrow involvement and eosinophilia in paracoccidioidomycosis]. The authors described three acute paracoccidioidomycosis patients with bone marrow involvement. P. brasiliensis yeast forms were observed in bone marrow smears of all them, and in one case, culture also revealed fungus growth. The mononuclear phagocytic system involvement, the blood eosinophilia and the negative skin hypersensibility responses were emphasized in all of them, as well as the severity of the disease in one case, with disseminated bone lesions and 20.260 eosinophils/mm3 in peripheral blood. The authors discuss the possible role of eosinophil in the host-parasite interaction in paracoccidioidomycosis, suggesting that TH 2 subpopulation activation and increased IL 5 and GM-CSF secretions may be responsible by eosinophilia in the most severe case.


[Epidemiologic markers of Salmonella typhimurium and Salmonella agona]. Among S. typhimurium and S. agona strains isolated during the period from 1971 to 1987, the biotypes, colicine types and resistance patterns were determined for 734 S. typhimurium and 631 S. agona strains. Among 734 S. typhimurium strains 65 biotypes were disclosed with prevalence of biotypes 1a (28.34%), 1b (29.84%) and 9 bi (18.28). Concerning S. agona, the biotype 1a represented by 87.16%, was the commonest clone among our strains. Although colicine typing added little information to characterize these serotypes, it should be usefull when applied in epidemiological study of outbreaks. It was observed multiply antimicrobial resistance mainly among human strains, particularly from nosocomial origins.


[Detection of cytotoxicity of biocompatible materials in MRC-5, HeLa, and RC-IAL cell lines]. The sensitivity of diploid and heteroploid cell lines for detection of cytotoxicity using the agar diffusion method on cell culture, was tested with ascorbic acid solution of different concentrations. A total of 562 samples of 21 various materials were tested. The heteroploid cell line, RC-IAL, showed in relation to the MRC-5 and HeLa cell lines, greater sensitivity because it showed the presence of cytotoxic effect with the lowest concentration used (10 and 25 micrograms/ml) of ascorbic acid and showed greater diameter of cytotoxic halo in 15 samples and equal diameter in 16 of the 43 positive samples (7.6%). Out of 43 positive samples, the MRC-5 line did not show cytotoxicity in 3 sponge samples and 1 of acrylic resin. The PVC (polyvinylchloride) and polyethylene rarely showed positivity, while, the plastic, cotton and acrylic resin demonstrated cytotoxicity in about 5% of samples. We thus suggest the use of the RC-IAL and HeLa cell lines for continuation of this type of analysis at Adolfo Lutz Institute.


Determinants and effects of waiting time to coitus. Nonuse of contraception at first intercourse among adolescents is well documented in the adolescent sexuality research literature. This study provides a formal test of the hypothesis that an increase in the courtship period (i.e., waiting time to intercourse) increases the likelihood that a couple will discuss contraception and use it at first intercourse. The data analyzed are from personal interviews with 1,314 women aged 20-29 in the 1983 National Survey of Unmarried Women. The results are mixed about the effect of waiting time on contraceptive behavior, providing weak support for the hypothesis, but also elucidating individual and relative characteristics of the couples, such as age and education of the respective partners, that affected contraceptive behavior at first intercourse. Support for the hypothesis may prove more robust in data with different characteristics from the survey used here.


Discrepancies between men and women in reporting number of sexual partners: a summary from four countries. Men and women in national surveys from four countries, the United States, Canada, Great Britain, and Norway, give mutually inconsistent reports of numbers of opposite-gender sexual partners. In all cases the number of female partners reported by men exceeds the number of male partners reported by women. Gender difference in reporting bias seems to be the most plausible explanation for the discrepancies.


Household crowding and reproductive behavior. Ethological studies suggest that animal populations that live in crowded conditions display a number of behaviors that tend to limit the size of the population, such as aberrant forms of sexual behavior, small litter sizes, a higher incidence of spontaneous abortion, ineffectual maternal care, and even cannibalism of their young. Studies of household crowding in North America cities have produced only modest and selective evidence that crowding has similar effects among humans. In this paper, we examine the effect of household crowding on marital sexual relations, on desire for additional children, and on fetal and child loss in Bangkok, Thailand, a city with a much wider range of household crowding than is typically found in North America. In spite of the wider range, and higher mean level of crowding, we find that both the objective and subjective dimensions of crowding have only modest selective effects on sexual and reproductive behavior.


Effects of interpregnancy intervals on preterm birth, intrauterine growth retardation, and fetal loss. This study examines the magnitude and shape of the interpregnancy interval (IPI) effect on three pregnancy outcomes: preterm low birthweight (PRETERM-LBW), intrauterine growth-retardation low birthweight (IUGR-LBW), and fetal loss (LOSS). A multinomial logistic regression model is estimated, based on data from the 1988 National Survey of Family Growth which contains pregnancy histories. The results indicate that both short and long intervals raise the risk of IUGR-LBW and LOSS, net of sociodemographic and behavioral variables, but IPI effects on PRETERM-LBW are not clear.


Phosphoglucomutase genetic polymorphism and human fertility. We studied the phosphoglucomutase phenotype in relation to fertility parameters in a consecutive series of 204 women who had delivered a normal live-born child in Rome. A highly significant association was found between age of the women and phosphoglucomutase phenotype, suggesting a reduced rate of reproduction among women of phosphoglucomutase Type 1. Previous spontaneous abortion appears related to both age and phosphoglucomutase enzymatic type. An increased incidence of abortion in women of older ages was observed only in phosphoglucomutase Type 1. Gestational duration and fetal intrauterine growth rate are also significantly associated with maternal phosphoglucomutase phenotype. The pattern is complex, but also in this instance the influence of maternal age was evident. Considered altogether, the data suggest that phosphoglucomutase may have an important role in zygote development and survival through the whole span of intrauterine life.


Ecological determinants of divorce: a structural approach to the explanation of Japanese divorce. This paper examines the ecological determinants of contemporary Japanese divorce rates on the prefectural level. LISREL and computer-generated graphics are the analytic methods used. The aggregate level of analysis demands the use of the ecological model which posits that demographic changes, economic activities, migration patterns, and the level of urbanization are significant predictors of divorce rate. Our analysis demonstrates that sex ratio, female labor force participation, female in-migration patterns, population increase, and net household income all play a significant role in affecting the divorce rate. Our findings also confirm the well-supported hypothesis that both population density and modernization positively influence modern Japan's divorce rates. The residual analysis also points out that in order to account for the large proportion of the unexplained variance of Japanese divorce, behavioral-related variables and island- or prefecture-specific dimensions need to be included in the ecological model of divorce.


Maternal nativity status and pregnancy outcome among U.S.-born Filipinos. Using 1979-87 Hawaii vital record data on single live births, this study compares by nativity status of the mother the maternal characteristics and pregnancy outcomes of resident Filipino women. Among ethnic minorities in the United States, the pregnancy outcomes of U.S.-born mothers have been reported to compare unfavorably to their foreign-born counterparts. In this study, unequivocally preferential pregnancy outcome indicators were not observed for foreign-born women. Contrary to expectations, a significant, but modest, increase in the risk of preterm delivery was found for infants of Philippines-born mothers, along with a less favorable mean birth weight and gestational age. As a growing minority population in the United States, the atypical determinants and patterns of pregnancy outcome in this population warrants further investigation.


Human sex ratio as it relates to caloric availability. The relationship between human sex ratios at birth and caloric availability per capita was examined across different countries. Significant positive correlations were obtained between the amount of food a country had available and the percentage of male births. Furthermore, increases or decreases in a country's caloric availability were related to corresponding changes in that country's sex ratio. These results provide evidence of adaptive sex ratio biasing in humans. The physiological mechanism by which this effect operates is probably higher mortality rates for male embryos and fetuses as a result of nutritional deficiencies and associated stressors.


Age at return marriage and timing of first birth in India's Uttar Pradesh and Kerala states. The study investigates the relationship between age at marriage and the length of first birth interval in two states of India: Uttar Pradesh and Kerala. Life tables of first-birth intervals and median first-birth intervals are computed for several subgroups of the study population. Multivariate hazards modelling technique is used to study the net effect of age at marriage, controlling for a multiple of socioeconomic factors. The result shows that the average first-birth interval varies by age at marriage and is much longer in Uttar Pradesh than in Kerala.


Unemployment and marital status in Great Britain. During the past decade, the marriage rate has declined and the divorce rate has increased in Great Britain. Becker (1981) attributes such changes to improvements in the economic status of women, in that high-wage women gain less from marriage relative to other women. However, an empirical analysis of data from the General Household Survey 1985 in Great Britain shows that male unemployment is another important determinant of changes in marital status. High rates of male unemployment reduce the incidence of marriage and increase the likelihood of divorce.


Assortative versus selective mating: is the distinction worthwhile? The study examines the distinction between assortative and selective mating made by Lewontin, Kirk, and Crow in 1968 and finds it unproductive. Not only has the difference been ignored on many occasions even as it was invoked, but maintaining it obscures several useful properties of both nonrandom mating schemes and some formally equivalent systems such as fertility selection. The elucidation of these similarities could have accelerated the work of population biologists.


Pattern of menstrual cycle length in south Indian women: a prospective study. Data on 8,308 menstrual cycles from 1,740 South Indian women prospectively recorded were analyzed to identify the effect of age on menstrual cycle length. The distribution was skewed to the right with the mean (SD) cycle length of 31.8 (6.7) days. The range of 25-40 days constituted 10-82 per cent of menstrual cycle lengths. In no age group did 28-day cycles occur in more than 9 per cent of women. Variability as measured by the standard deviation was high among those below 19 years of age, stabilized during 25-39 years, and then increased in women aged 40 years or more. The findings are discussed in the light of other studies and possible implications in fertility control programs.


Selection intensities and consanguineity in the Yadava and Vadabalija of Visakhapatnam, Andhra Pradesh, India. The opportunity for intensity of selection is studied in two backward caste groups with different occupations, namely Yadava (pastoral) and Vadabalija (fishing) of Bheemunipatnam Taluk of Visakhapatnam District, Andhra Pradesh. The amount of selection intensity is found to be higher in Vadabalija (0.8583) than Yadava (0.5827). The contribution of mortality to the total index of selection is greater in Vadabalija (0.6095) than in Yadava (0.3326). When consanguineity is considered, the fertility components of selection intensity (0.1885) are lower in the consanguineous group of Yadava than in the nonconsanguineous group (0.2845), whereas in Vadabalija they are higher in the consanguineous group (0.5261) than in the nonconsanguineous group (0.2223).


Pathobiology of experimental acute pancreatitis. Pancreatic duct obstruction, even in the absence of biliary obstruction and/or bile reflux into the pancreatic duct, can trigger acute hemorrhagic necrotizing pancreatitis. The earliest changes are seen within acinar cells. Early derangements in acinar cell biology include inhibition of digestive enzyme secretion and the co-localization of lysosomal hydrolases with digestive enzyme zymogens. Under appropriate conditions, this co-localization could lead to digestive enzyme activation within acinar cells.


Intracellular proteolysis of pancreatic zymogens. Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.


Quantal calcium release and calcium entry in the pancreatic acinar cell. In the past decade, there have been remarkable advances in our understanding of the calcium messenger system that mediates the effects of various agonists. The purpose of the present article is to describe two areas of current interest in the calcium signaling field--quantal calcium release and calcium entry into the cell--using the pancreatic acinar cell as a model. Proposed mechanisms describing these phenomena and the role they play in the kinetics of calcium movements in the cell are discussed.


The biochemical characterization of the native pancreatic cholecystokinin receptor using affinity labeling approaches. Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using "intrinsic" probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a "topographical mapping" probe, which incorporates a flexible spacer of variable length between a CCK-like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other.(ABSTRACT TRUNCATED AT 250 WORDS)


Glucocorticoids have opposite effects on ornithine decarboxylase and cell growth in pancreatic acinar AR42J cells. This paper reviews the relationships between the effects of glucocorticoids on rat pancreatic acinar AR42J cell polyamine levels and cellular growth and differentiation. Glucocorticoids inhibit the growth of AR42J cells. Glucocorticoids either stimulate or inhibit the formation of polyamines in a variety of cell types. Cells require polyamines for normal growth. Therefore, we tested the hypothesis that polyamines mediate the effects of glucocorticoids on AR42J cells. First, to confirm that AR42J cells required polyamines for growth we examined the effects of inhibiting ornithine decarboxylase (ODC). ODC is the most important and generally rate-limiting enzyme in the synthesis of the polyamines. As expected, the ODC inhibitor difluoromethylornithine (DFMO) inhibited AR42J cell DNA synthesis, and the addition of exogenous putrescine reversed this effect. The levels of growth inhibition by glucocorticoids and DFMO treatment were similar. Second, we examined the effects of glucocorticoids on ODC. Surprisingly, glucocorticoids increased levels of AR42J cell ODC mRNA, ODC activity, and putrescine. Glucocorticoids increased these parameters over a similar time-course as they decreased DNA synthesis. Analog specificity studies indicated that a glucocorticoid receptor mediated both the growth inhibitory and ODC stimulatory effects. Dose-response studies indicated, however, that growth inhibition was more sensitive to dexamethasone (DEX) than were ODC levels. Therefore, polyamines do not account for the effects of glucocorticoids on AR42J cell growth. In these cells, glucocorticoids have opposite and independent effects on ODC and growth.


Recent results in animal models of pancreatic carcinoma: histogenesis of tumors. Animal models of carcinoma of the pancreas provide new information regarding the pathways for histogenesis of the tumors. Four models, induced by chemical carcinogens or transgenic methods, are reviewed briefly from this perspective. Recent reports indicate that carcinomas with a ductal phenotype can arise from transformed acinar cells in rodents. A transgenic mouse model provides evidence that anaplastic carcinomas and islet cell tumors may arise from primitive cells that express the elastase gene, yet retain the potential to differentiate as islet cells. In a nitrosamine-induced hamster model, ductal carcinomas appear to arise directly from ductal cells. Carcinomas in this model contained mutations in the c-K-ras oncogene that are similar to those reported in about 75 percent of human pancreatic carcinomas, whereas acinar cell carcinomas of rats lacked this mutation. The histologic type of a carcinoma may reflect the cell of origin, but this statement is not always true. Therefore, classification of tumors on the basis of phenotype rather than on the presumed cell of origin is recommended. Among the animal models, the carcinomas in hamster pancreas rank as most similar to human pancreatic ductal adenocarcinomas in regard to the phenotype of the tumors and the prevalence of the c-K-ras mutation.


Receptor strategies in pancreatitis.

Receptor strategies in pancreatitis. A variety of receptors on pancreatic acinar and duct cells regulate both pancreatic exocrine secretion and intracellular processes. These receptors are potential sites of action for therapeutic agents in the treatment of pancreatitis. Cholecystokinin (CCK) receptor antagonists, which may reduce the level of metabolic "stress" on acinar cells, have been shown to mitigate the severity of acute pancreatitis in a number of models. Not all studies have shown a benefit, however, and differences may exist between different structural classes of antagonists. Because increased pancreatic stimulation due to loss of feedback inhibition of CCK has been proposed to contribute to the pain of some patients with chronic pancreatitis, CCK receptor antagonists could also be of benefit in this setting. Somatostatin and its analogs diminish pancreatic secretion of water and electrolytes and have been effective in treating pancreatic fistulas and pseudocysts. These agents are also being evaluated for their ability to reduce pain in chronic pancreatitis (perhaps by reducing ductal pressure by diminishing secretory volume) and mitigating the severity of acute pancreatitis (possibly by reducing the metabolic load on acinar cells). Recently described secretin receptor antagonists may also have therapeutic value as a means of selectively inhibiting pancreatic secretion of water and electrolytes.


Interferons in the management of neuroendocrine tumors and their possible mechanism of action. Alpha interferons at doses of 3-9 MU subcutaneously, three to seven times/week, have been administered to 32 patients with malignant endocrine pancreatic tumors. The objective biochemical response rate was 63 percent with a median duration of 20.5 months. Significant reduction of tumor size was only noticed in 20 percent of the patients. Alpha interferon administered to 111 patients with malignant carcinoid tumors showed objective biochemical responses in 42 percent of the patients with a median duration of 32 months. Another 39 percent of the patients showed stabilization of disease without any further tumor growth. Subjective improvement was noticed in 70 percent of the patients. When survival data are analyzed in patients with malignant carcinoid tumors, the median survival from start of treatment was 80+ months in the group of patients treated with alpha interferon, which should be compared with only eight months in a historical group treated with chemotherapy (streptozotocin plus 5-fluorouracil). The adverse reactions to alpha-interferon treatment are dose-dependent and include, mainly, flu-like symptoms, fatigue, and low-grade weight loss. Autoimmune reactions are noted in about 20 percent of the patients. Patients treated with recombinant alpha interferons might develop neutralizing interferon antibodies (6-27 percent), which abrogate the anti-tumor response. The anti-tumor effect in neuroendocrine tumors includes anti-proliferation, apoptosis, differentiations, and cytotoxic/cytostatic effects. Furthermore, immunomodulation is obtained by increased expression of class I antigens on tumor cells. Four patients also developed antibodies directed against carcinoid tumor cells. Alpha interferons induce several nuclear enzymes such as 2'-5'-A synthetase, p-68 kinase, and Mx-A proteins, which are involved in a downregulation of expression of growth factors, oncogenes, and peptide hormones, leading to anti-proliferation and/or apoptosis. The response to alpha-interferon treatment might be predicted by analysis of the induction of 2'-5'-A synthetase in samples from neuroendocrine tumors. Stimulatory tests of hormone secretion, such as meal stimulation of pancreatic polypeptide secretion or secretin test, clearly demonstrate a normalization during alpha-interferon treatment, which might depend on reduced peptide production and/or secretion but also on eradication of malignant cell clones. In summary, alpha interferons have demonstrated significant anti-tumor effects in patients with malignant neuroendocrine gut and pancreatic tumors. The adverse reactions are dose-dependent and manageable. The anti-tumor effects of alpha interferons are pleiotropic and include several direct effects on tumor cells but also immunomodulation.


Somatostatin receptors in the gastrointestinal tract in health and disease. The multiple actions of somatostatin are mediated by specific membrane-bound receptors present in all somatostatin target tissues, such as brain, pituitary, pancreas, and gastrointestinal tract. Three different types of tissues in the human gastrointestinal tract express somatostatin receptors: (1) the gastrointestinal mucosa, (2) the peripheral nervous system, and (3) the gut-associated lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogs. Somatostatin receptors are also expressed in pathological states, particularly in neuroendocrine tumors of the gastrointestinal tract. Ninety percent of the carcinoids and a majority of islet-cell carcinomas, including their metastases, usually have a high density of somatostatin receptors. Only 10 percent of the colorectal carcinomas and none of the exocrine pancreatic carcinomas, however, contain somatostatin receptors. The somatostatin receptors in tumors are identified with in vitro binding methods or with in vivo imaging techniques; the latter allow the precise localization of the tumors and their metastases in the patients. Since somatostatin receptors in gastroenteropancreatic tumors are functional, their identification can be used to assess the therapeutic efficacy of octreotide to inhibit excessive hormone release in the patients.


The influence of peripheral nerve graft's predegeneration stage on the regrowth of hippocampal injured neurites and concomitant changes in submicrosomal fraction proteins of grafts. The present work has a twofold aim: 1. To ascertain whether the stimulative influence of peripheral nerve grafts on injured hippocampal neurons depends on the time lapse after transection and; 2. To examine whether the mentioned effect runs parallel to the time-dependent changes of proteins contents and composition in the submicrosomal fraction from transected rat sciatic nerves. Fluorescence microscope examination revealed that FITC-HRP labeled cells extending their neurites into the implanted peripheral nerve segments were particularly numerous among the hippocampal neurons when 7- and 35-day-old predegenegated distal stumps were used as grafts. Discontinuous SDS-slab polyacrylamide gel electrophoresis of submicrosomal fraction proteins obtained from distal stumps of rat sciatic nerves was performed at the 7, 14, 21 and 35 days after transection. Among the obtained protein fractions the most interesting seem to be the ones of 47 and 54 kDa, which reached maximal levels at the 7th day and the 50 kDa fraction with a maximum at the 35th experimental day. It is possible that the growth promoting power of the employed grafts depends on the presence of proper proteins.


The effect of different prostaglandins on gastric mucosal intracellular second messenger system in the rat. After an oral administration of 100 micrograms/kg dose, the investigated prostaglandins: PGF2 alpha, PGE2 and a synthetic PGE2 derivative: FCE-20700, exerted a significant effect on cAMP and cGMP content of both parts (antral and fundic) of gastric mucosa, resulting in an elevated cAMP/cGMP ratio, while 6-keto-PGF1 alpha, the stable break-down product of prostacyclin, was inactive. Since the above-mentioned phenomenon seems to be proportionate to the cytoprotective (anti-ulcerogenic) property of the investigated prostaglandins, this cAMP/cGMP ratio "shift" is interpreted as a probable (molecular) sign of the reparative, (anti-ulcerogenic) processes.


Effect of intravenous cimetidine, ranitidine and pentagastrin and intragastric prostaglandin E1 treatments on gastric transmucosal potential difference in the rat. Effects of intravenous cimetidine, ranitidine and intragastric prostaglandin E1 (alprostadil) treatments on the transmucosal potential difference (PD) of the stomach were compared. It was also investigated whether the above-mentioned drugs influenced the decrease of PD which followed both intragastric administration of 30% alcohol or Ca++ solution in 5 Mm final concentration and intravenous administration of pentagastrin. Both cimetidine and ranitidine treatments led to significant (p < 0.05) increase of PD, the effect of ranitidine was dose dependent. Prostaglandin E1 in a dose of 40 micrograms/kg led to significant decrease of PD (< 0.05). Both intragastric administration of prostaglandin E1 in a dose of 40 micrograms/kg and intravenous administration of ranitidine in a dose of 10 mg/kg significantly diminish the effect of Ca++ and alcohol to decrease PD. Neither prostaglandin E1, nor ranitidine pretreatment had any effect on the rapid and highly significant (p < 0.01) decrease of PD following i.v. pentagastrin administration. It is hypothesized that transmucosal PD of the stomach provides information not only on the actual condition of the mucosal barrier but on the electrophysiology of gastric secretion as well.


Polymerase chain reaction and other gene techniques in pharmacogenetics: an introduction and review. The author gives a short description of the polymerase chain reaction technique and restriction fragment length polymorphism which revolutionized molecular biology and have entered pharmacogenetics, too. Examples of the use of these techniques are given in the polymorphism of oxidative (phase I) and conjugative (phase II) reactions.


Electrophysiological changes induced by lysophosphatidylcholine, an ischaemic phospholipid catabolite, in rabbit atrial and ventricular cardiac cells. The effects of palmitoyl-lysophosphatidylcholine (LPC) were studied on the cellular electrical activity of rabbit heart preparations. LPC (100 mumol/l) caused a considerable enhancement of the automaticity of the SA nodal and Purkinje fibers and frequently induced irregular firing in both supraventricular (SA node, atrium, AV junction) and ventricular (Purkinje fibers, papillary muscle) myocardial regions. The 'automatotropic' and arrhythmogenic effects of LPC were accompanied by a lengthening of the atrioventricular conduction time. In ventricular muscle fibers LPC (100 mumol/l) decreased the resting potential (RP), the maximum rate of depolarization (Vmax) and the amplitude (APA) and duration (APD) of the action potential, and often evoked action potentials of 'slow response' type. In atrial muscle cells, 100 mumol/l LPC was capable of inducing hyperpolarization, with concomitant increases in RP, Vmax, APA and APD; higher concentrations (300 and 600 mumol/l) of LPC resulted in decreases in RP, Vmax, APA and APD, i.e. phenomena similar to those observed with 100 mumol/l LPC in the ventricular myocardium. The results seem to support the assumption that lysolipids accumulating in the ischaemic myocardium may play a pathogenetic role in the development of both supraventricular and ventricular dysrhythmias accompanying coronary artery occlusion.


Intact kidney function during contralateral renal artery clamping in dogs. Experiments were carried out on 32 Nembutal anaesthetized mongrel dogs from both sexes. After 45 min control period unilateral renal ischemia was achieved by clamping the left renal artery for 90 min. In part of the experiments (n = 8) after clamp removal 3 consecutive 45 min periods were performed. The function of the intact right kidney was investigated. Mean arterial pressure (MAP), heart rate (HR), glomerular filtration rate (GFR), urine flow rate (V), fractional excretions of sodium (FENa), potassium (FEK) and chloride (FECl) and plasma levels of atrial natriuretic peptide, dopamine and antidiuretic hormone were evaluated. During ischemia MAP was elevated from 122.5 +/- 3.1 to 140.2 +/- 2.7 mmHg (p < 0.001), HR decreased from 119 +/- 4 to 102.5 +/- 3.9 beats/min (p < 0.01) as compared to the control period. GFR did not change significantly, while all excretory parameters increased: V from 8.7 +/- 1.2 to 14.5 +/- 1.7 microliters/min/gr kidney tissue (p < 0.05); FENa from 2.3 +/- 0.2 to 3.6 +/- 0.3% (p < 0.01); FEK from 40.0 < 3.5 to 51.2 < 2.8% (p < 0.05); FECl from 1.8 < 0.3 to 2.6 < 0.3% (p < 0.05). MAP remained elevated in the first and the second postischemic periods and was paralleled by the sustained increase in FENa and FECl, while FEK remained higher to the end of the experiment. ANP was significantly elevated during ischemia: on 75 min--p < 0.01 and on 105 min.--p < 0.05. AVP and dopamine showed no statistically significant changes during the investigated periods.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of small changes in extracellular magnesium concentration on the tone of feline mesenteric arteries: involvement of endothelium. The aim of this study was to investigate the effect of small alterations in extracellular magnesium concentration on the tone of feline mesenteric arteries and to examine the role of endothelium in these responses. We measured isometrical tension of isolated arterial rings, placed between two stainless steel wires in a tissue chamber containing Krebs-Henseleit solution, aerated with a gas mixture containing 95% O2 and 5% CO2 at 37 degrees C. After precontraction with noradrenaline, a decrease of extracellular magnesium concentration from 1.2 mM to 1.0 and 0.8 mM resulted in sustained relaxations, whereas the elevation of extracellular magnesium from 0.8 mM to 1.2 mM caused an increase in vascular tone when endothelium was intact. The magnesium-withdrawal related dilations were absent in endothelium-denuded vessels and were inhibited by oxyhemoglobin (5 x 10(-6) M) and methylene blue (10(-5) M), suggesting the involvement of endothelium-derived relaxing factor in this vascular response. Nifedipine (5 x 10(-7) M) or dichlorobenzamil (3 x 10(-5) M), however, did not affect the magnesium-deficiency related relaxations. Therefore, in this vascular action of magnesium, nifedipine-sensitive calcium channels or the sodium- calcium antiport system are not involved. We conclude that small alterations in extracellular magnesium concentration, possibly within the physiological range, are able to modify the basal formation and release of EDRF, and thus alter arterial smooth muscle tone in this vascular bed. This endothelium- and magnesium-dependent system appears to be more sensitive than the direct smooth muscle actions of magnesium. The possible physiological and pathophysiological consequences of these observations are discussed.


Calcium as a counteractive agent to streptomycin induced respiratory depression: an in vivo electrophysiological observation. Effect of streptomycin on respiratory function in cats was studied. It was observed that streptomycin at a dose of 40 mg/kg body weight intravenously (i.v.) caused respiratory failure or streptomycin induced respiratory depression (SIRD). This respiratory failure is not linked with Herring-Breuer stretch receptors because the effect remained unaltered in artificially ventilated cats. The involvement of central structures in SIRD can be discarded since intracarotid and intraventricular administration of streptomycin failed to produce any change in respiration. Studies on monosynaptic reflex, dorsal and ventral root activities of spinal phrenic and intercostal nerves, and on fusimotor and alpha-motor neuron activities of spinal intercostal and phrenic nerves in decerebrated cats indicated clearly that respiratory depression is not only due to blockade at neuromuscular junction but due to functional depression at the level of muscle receptors and spinal cord motor neurons. The respiratory depression induced by streptomycin was more or less completely reversed when calcium was administered intravenously from external source. It is speculated that streptomycin induced respiratory depression may be mediated through calcium inhibition which can be treated with external calcium in conjunction with artificial respiration.


Pathology of wave III of brainstem auditory evoked potentials (BAEPs). In a group of 195 consecutively examined patients with brainstem neurological symptomatology parameters of the wave III BAEPs were pathological in 20 patients. In 70% of them the clinical symptomatology and/or the cranial computed tomography examination results pointed to a lesion at the level of pontomedullary junction and lower pons Varoli where according to the recent opinion the possible generators of this BAEP wave are situated (superior olivary complex, nuclei cochleares, corpus trapezoideum). These results support the supposed localization of possible generators of the wave III BAEPs.


Impact of a single insulin treatment (imprinting) applied during liver regeneration on hepatic insulin receptor development, blood glucose level and liver function parameters in adult rats. Rats subjected to partial hepatectomy (surgical removal of two thirds of the liver) showed no appreciable change in serum cholesterol, bilirubin, albumin, total protein and A/G values at 2, 5, 12 and 21 days after the intervention. The enzyme activities characteristic of liver damage (GOT, GPT, LDH, AP) were high in the control group and low in the insulin-imprinted group at 2 days, tended to normalize in both groups at 5 days and changed slightly at 12 days. The blood glucose level was markedly decreased in the control group and to a lesser degree also in the experimental group at 2 and 5 days of sampling. Insulin treatment (loading) performed at 2 and 5 days accounted for a drop of blood glucose which was followed by normalization within 2 h. Starving value and response to insulin loading uniformly fell into the physiological range at 21 days, whereas at 12 days no normalization occurred in either group within 2 h of insulin loading, although the starving value was physiological. The binding capacity of the insulin receptor was markedly low in the control group as long as 12 days, and tended to normalize by 21 days. In the insulin-imprinted group the binding capacity increased over the control at 2 and 5 days and normalized by 12 days.


[Opportunistic toxoplasmosis in a case of heart transplantation]. Infection with toxoplasma gondii is a serious complication in the immunocompromised heart transplant recipient. We reported a case of toxoplasmosis in a seronegative heart transplant recipient detected on endomyocardial biopsy. This patient was treated with oral pyrimethamine and sulphadiazine. Because primary toxoplasmosis occurred in seronegative patients receiving hearts from sero-positive donors, it is necessary to screen both recipients and donors for T. gondii. Prophylaxis with pyrimethamine in sero-negative patients transplanted with a heart from sero-positive donors confers considerable benefit.


[Sterilization by the plasma procedure]. Plasma sterilisation of heat sensitive materials appears as an attractive substitute for ethylene oxide processing which leaves adsorbed toxic residues. In oxygen-based plasmas, the de-activation of pathogenic organisms is assumed to be due to their slow combustion with the active species which produces CO2 and H2O. In the absence of ion bombardment, the concentration of oxygen atoms is an important parameter entering the plasma sterilisation efficiency. Means for achieving high production of oxygen atoms are reviewed and discussed. In particular, an initial improvement would be to generate the plasma in the sterilisation volume itself. Uniform plasma excitation at electron cyclotron resonance (ECR) which allows resonant coupling in the whole treatment volume (no shadowing) and requires reduced electric fields to sustain the discharge (no heating of biomaterials) is particularly adapted to this purpose.


[Infectious complications of heart-lung transplantation]. This study describes the infectious complications in 68 heart-lung transplant patients. We focused interest on early post-transplant infections so called nosocomial pulmonary infections, their exacerbating cofactors their clinical expression, and elements of diagnosis and treatment. Furthermore we describe the principal infections seen during long-term followup, which are opportunist infections caused most often by immunosuppression: Cytomegalovirus infections have a high incidence with serious clinical consequences in heart-lung transplant patients. As well as pneumocystis carinii infections and fungal infections, such as aspergillosis.


[General rules of infection prevention in organ transplantation, bone marrow transplantation and prosthetic surgery]. General rules to prevent are categorized into four classes. The first one concern patient before operation: well-balanced nourishment latent infections treatment, digestive tract micro flora elimination, immunizations, antiseptic gargles, skin antisepsis. The second one concern operation: previous fumigation, very high efficiency filtration and laminar flow air, transplant care, antibioprophylaxis, operating theatre discipline. The third one concern post-operation days: cubicle initial fumigation, high efficiency filtration and positive pressure air, strict protective insulation, single-use things, controlled food, specific anti viral prevention, catheters and tubes removal or replacement. The fourth one concern going home patient: well-balanced nourishment, body hygiene and hands washing, infected people shunning, gardening and cleaning proscribing, medical follow-up and latent infections systematic detection.


[Rare opportunistic fungal diseases in patients with organ or bone marrow transplantation]. Candidiasis, aspergillosis and cryptococcosis are the most common fungal infections in transplant recipients. However other fungal infections have been reported. Mucormycosis, Scedosporium infections, fusariosis and trichosporonosis represent the largest part of these rare mycosis. The clinical and mycological features are described here. In addition, cases of very uncommon mycosis, most of them only once reported, have been reviewed. Overall the diagnosis is difficult as mycological examinations are often negative till the disease is disseminated. Amphotericin B remains the reference treatment except in Scedosporium infections which respond more likely to azole antifungal agents. Despite the treatment the outcome is usually fatal.


[Invasive aspergillosis in immunocompromised patients. An analysis of 57 patients]. Fifty seven cases of invasive aspergillosis have been analyzed. Most patients were severely neutropenic. The main underlying diseases were acute leukemia, malignant lymphoma and bone marrow transplantation. The clinical and radiological manifestations and the mycological data rare presented. A dramatic increase of the annual incidence has been observed since 1986. The main causes appear to be the increase in intensity of chemotherapy regimen for acute leukemia and the progressive colonisation of a part the department. Overall mortality is especially high (74%) but the mortality rate appears to decrease since 1989.


[Do water mycobacteria present any infectious risk in immunocompromised patients?]. Atypical Mycobacteria were demonstrated in tap water. Mycobacteria are generally more resistant to chemical disinfection than other bacteria and grow and survive in water. In an effort to clarify the role of water in the transmission of Mycobacteria, water from various sites in the hospital was analysed. Atypical Mycobacteria were isolated from 58 of 60 samples of cold water distributor. Species more frequent are M.Kansasii, M.Gordonae, M.Fortuitum. 3 of 10 samples of hot water were positives M.Xenopi was isolated once. 1 of 10 samples of mineral waters was contaminated with M.Gordonae. The atypical Mycobacteria in normal patient are relatively less virulent, in a host with an impaired cellular immunity they caused active diseases. The number of published cases is low. The incidence in transplant patients ranges from 0.5 to 1%. Infections with atypical Mycobacteria differ in several clinical features. Person to person does not occur. Water is a source of infection induce direct inoculation inhalation and ingestion.


[Bacteriological control of food for immunocompromised host]. This study concerning bacteriological controls of food for immuno-suppressive patients have shown that food sterilized in sterilizer or with pressure-cooker contains sporulated bacteria after treatment. Unit packaging or freeze-drying commercial foods offer no bacteriological quality guarantees. Clinicians have to define acceptable contamination rate according to their patients' condition.


[Does automatized disinfection of traps by chlorine reduce water related contamination?]. Sanitary U bends are very contaminated places from a microbiological point of view. They may even be dangerous for immunocompromised patients. Although daily chloride disinfection of U-bends is ineffective, it seems to work when performed after each use of sanitary devices. On line disinfection reduces not only U-bend bacteriological contamination but also all surrounding surfaces.


[Practice of oral feeding in immunocompromised patients isolated in hematologic units]. 41 french clinical hematologic departments were questioned about their oral feeding practice for compromised patients. In conclusion, an agreement seems necessary to define the goals of the hematological patients' feeding, to set going the measures and to assess the food proceeding treatments.


[Trichosporon capitatum septicemia. Apropos of 5 cases]. Invasive Trichosporon capitatum infections are seldom reported. We present here five cases of septicemia. All patients had an acute myeloblastic leukemia and were severely neutropenic. They have also been treated before the onset of the fungal infection with broad-spectrum antibiotherapy and also with an oral azole antifungal agent. The role of this antifungal therapy in the development of T. capitatum infection is discussed. The prognosis of T. capitatum infections is severe. Eight of the 10 published cases had a fatal outcome and one of our patients died of the fungal infection in spite of the treatment.


[Incidence of hepatitis C virus in kidney transplantation recipients]. The investigation was carried on 122 waiting renal transplantation hospitalized patients. Detection of HCV antibodies was done before transplantation and after renal transplantation. HCV antibodies were detected by immunosorbent assay (ELISA) for C 100-3 protein of HC virus (Lab. ORTHO). Positive results were checked by a second test (immunoblot RIBA II) to detect antibodies against C100-3, 5-1-1, C33, C22 proteins of HCV genome. Before transplantation, 112 patients were negative and 10 positive. After transplantation, 104 were checked: 103 had identical serology (93- and 10+); only one patient has shown a seroconversion six months after the transplantation, demonstrating the late apparition of HCV antibodies, but immunological status of donor was unknown. Renal transplantation does not seem a risk factor of HCV contamination: only 1 seroconversion on 122 patients or 0.8%: near percentage of French blood donors (0.68%). The percentage of positivity HCV before transplantation (9%) answered with that of European hemophils (5 to 20%). Second generation tests demonstrate a better sensibility and specificity than the first.


[Sterilization of biocompatible materials: which method to choose?]. Sterilization of biomaterials, in hospitals, must be considered as the re-sterilization (and not reuse) of devices: prostheses, implants, catheters... This practice is not allowed, according to the circular of may 14 th, 1986; however it is a necessity in various cases. It must be realised with an extreme care, after evaluation of the different methods of sterilization: steam, dry-heat, ethylene oxide, formaldehyde or ionizing radiations and of their effects on the behaviour of the biomaterial.


[Preparation of a polymer named Porimid, and its sterilization]. From a concrete example, the authors remind the interest to envisage the possibilities of sterilization as early as the initial period of a research about biomaterials. Indeed certain kinds of sterilization may be too much aggressive for biomaterials, especially the polymers, whom the structure is so impaired. These biomaterials may become ineffective even dangerous and blight a long time of research. The authors also expose the necessity to respect the good manufacturing practices of laboratory and the rules of hospital hygienics which is indispensable for all the members of the teams which participate to biomedical or clinical researches. A condition for the respect of these rules is the utilization of a common and well understood language between the medical and no medical teams.


[Biomaterials and infection in dental and maxillofacial surgery]. Based on metals, polymers, ceramics, carbons or natural products, biomaterials represent an essential contribution to the repair or reconstruction of the hard or soft tissues of the dental and maxillofacial area. In order to avoid unsuccessful results these materials should realize a satisfactory tissue integration without bacterial colonization able to compromise the tissue-biomaterial finalized cooperation. The evolution of the science and technology of biomaterials should allow to get tissue toxicity-free materials which also inhibit microorganisms adherence, the infectability becoming a criteria as important as the biocompatibility itself.


[Risk of infection related to the use of cryopreserved bone grafts from domestic banks]. The authors report their experience of a home bone bank of cryopreserved femoral heads on a period of 3 years. They remind the rules to observe for the harvesting, the storage and the utilization of these allografts, and also the indispensable biological tests (at the moment when the article was written). About their series, the authors remind the risks of infection in relation with the utilization of femoral head from bone bank, which justify a great rigour in the organization and the managing of these home bone bank.


[Risk of infection after cataract surgery with intraocular implant]. A personal series of 10 patients with post surgical bacterial endophthalmitis is reviewed. Clinical signs, risk factors and diagnostic modalities are analysed and compared to other reports in literature. The role of intraocular lens is discussed. While it does not seem to be a risk factor in acute endophthalmitis, it plays a major role in chronic bacterial endophthalmitis. Endophthalmitis remains a rare but serious complication after cataract surgery and prevention modalities, especially using prophylactic antibiotherapy, have still to be evaluated.


[Comparison of polyurethane and polyethylene for central venous catheter in intensive care units]. Polyurethane (PU) and polyethylene (PE) central venous catheters were compared for their respective responsabilities in catheter related sepsis (CRS). From may 1988 to may 1989, 300 central venous catheters were inserted. Insertion sites were freely chosen by physicians. The polymer type was randomized. Catheters were removed after 10 days in place. Microbial loads were assessed on insertion sites, catheter hubs and tips, and blood drawn through the catheters lumen. One hundred eighty three catheters were available for complete evaluation (101 PE, 82 PE). Eleven were responsible for CRS, 4 were colonized (BB3 according to Brun-Buisson's classification), 19 were contaminated (BB2), and 149 were sterile (BB1). When comparing the "infected" group (CRS+BB3) and the "noninfected" (BB2 + BB1), no difference appeared between the tested polymers. CRS were significantly associated with insertion into the internal jugular vein. It seems useless to exclude from clinical practice any of the biomaterials tested.


[Bone substitutes and infection in maxillofacial surgery]. Bony substitutes are in fashion in maxillofacial surgery. They are used to fill bony cavities, in bony reconstruction to give shape-lines and re-create area of support or to fill bony defects. Then their use is frequently crowned by success in general surgery, it is not the same thing in maxillofacial surgery because of the usual impossibility to fulfil a requirement to biomaterial utilisation: the watertight. Furthermore, they don't have yet, for the most part, an essential quality: malleability. In our experience, their use are frequently disappointing because of postoperative infections and we stay faithful to the autograft bone. However it is highly probable that these biomaterials will take an importance more and more considerable when these problems will be overcame.


[Urinary tract infections during the 1st month after kidney transplantation]. The incidence and the predisposal factors of urinary tract infections (UTI) in the first month post-transplant were studied in 255 kidney transplantations (252 patients). UTI episodes were demonstrated in 73.7% of the grafts. The most common organisms were: Escherichia coli (35.8%), Staphylococcus (33.6%), Streptococcus D (11.2%), Klebsiella (5.3%). The infectious episodes were recurrent in 39% of the cases. The majority of the UTIs were asymptomatic but 7% of the infections led to septicaemia. Etiology of end-stage renal disease, pre-graft binephrectomy, asymptomatic vesicoureteral reflux into the patient's own kidneys, type of immunosuppressive treatment, acute tubular necrosis, rejection episodes, urological complications, coexistent other infections were not predisposal factors. Bacteriuria was more frequent in female than in male patients. The incidence of UTI was found to be statistically increased with history of UTI preoperatively (p = 0.039) and the use of ureteral catheter (p = 0.018). Occurrence of UTI was less common when the donor was treated by antibiotics before brain death (p-0.025). These results provide additional support for regular monitoring of urine cultures in the first month post-transplant. They should help to identify means of reduction of this infectious risk.


[Herpes simplex virus pneumonia following transplantation]. The HSV (1 or 2) is the cause of serious pulmonary infections among patients who have had a transplantation. This study in retrospect is based on the analysis of 145 patients who underwent a cardiothoracic transplant at the CHU. in Nancy. Confronted with clinical signs calling to mind breathing difficulties, the analysis of the broncho alveolar lavage (or of the bronchial brushing) revealed the viral aetiological agent. The answer from the laboratory is quickly available by immunofluorescence or by immunoperoxidase with viral anti-protein monoclonal antibodies and by the multiplication in vitro of the virus into cell cultures. The HSV 1 was responsible for 8 herpetic lung infections. The specific Acyclovir treatment was used 6 times successfully. When such a direction of treatment was impossible (in 2 cases) the outcome was fatal. The carry HSV is highly frequent and recurrences under immuno-suppressor treatment require an Acyclovir prophylaxis among patients admittedly carrying the virus in a pre-transplanted serum assessment.


[Bacteriological relationship between organ donor and recipient. A survey apropos of 100 brain dead patients]. The aim of the study was to evaluate the relationship between germs found in samples systematically taken in the organ donors and germs found in the corresponding recipients in the 8 post-operative days. The 100 brain-dead patients received oxacillin (2g every 4 hours) when the bacteriological samples were taken. 41% of the donors were germ carriers. The germs were mainly located in the respiratory track (Staphylococcus aureus meti-S (34%) and Hemophilus (29%)). Among the 86 patients harvested, 40 donors were germ carriers and gave 132 organs, 46 donors were not germ carrier and gave 151 organs. The comparison between these two groups showed no difference. Assessment of infection occurring in organ recipient in the 8 post-operative days (germ, location, evolution) showed no difference, whether organs were removed from germ carrier donors or no. Comparison between the germs found in germ carrier donors and those found in recipients with sepsis showed a similitude in three cases (2.2%). In the other cases, there is no relationship between the germs found in the donors and post-operative sepsis in the recipient.


[Organ, tissue transplantation, prosthetic surgery and dental focal infections: attitude of the dental surgeon]. The rational use of oral surgery therapy and techniques allows the preservation of total, or part of the infected dental organ. With patients who are about be grafted or undergo prosthetic surgery, the oral state is of prime importance and requires special attention. Getting such patients ready for operation must take into consideration the pathology for which they have been admitted to hospital. Therefore the length of oral care fully justifies its priority in the patient's preparation previously to surgery.


[Sterno-mediastinitis after heart transplantation. An easy treatment: irrigation-lavage]. After cardiac transplantation bacterial mediastinitis is a severe early complication. Between 1986 and 1990, we performed 49 cardiac transplantations. Six patients developed purulent mediastinitis. Treatment consisted in surgical debridement, local irrigation, drainage and systemic antibiotics. No patient died of this bacterial mediastinitis. Low cardiac output, re-sternotomy for bleeding, prolonged artificial ventilation were significantly higher in the group with sternal infection. Closed tube irrigation is a simple and efficient treatment of mediastinitis.


Epstein-Barr viral latency and cell immortalization as targets for antisense oligomers. The approach of using an antisense oligonucleotide to oppose the synthesis of a single selected viral gene product needed to maintain the EBV episome in non-virus-producing cells appears to be promising not only for possible cure of latent viral infection, but also for reversal of EBV-driven cell proliferation. It is also possible that targeting multiple latent viral genes might mount a synergistic effect that would prove to be more efficient at curing latent infection. However, further work is needed to identify the reasons for the variable results that are observed as well as to prove specificity of the inhibitory effects. Finally, we are also working on other assays and methods to screen compounds rapidly.


Reversal of transformed phenotypes by antisense fos. The therapeutic use of antisense DNA has started a revolution in pharmacology. As a model system for demonstrating the therapeutic power of the antisense concept, we sought to interrupt signal transduction in H-ras transformed cells to attempt to down-regulate their oncogenic phenotype. We hypothesized that down-regulation of c-fos translation by antisense-fos expression would decrease oncogenic signal transduction through the fos pathway and thus reverse the tumorigenic phenotype of these cells. To test this hypothesis, we transfected H-ras cells with a plasmid containing an 84-base sequence antisense to the 5' end of the mouse c-fos gene. The antisense-fos was under the transcriptional control of the MMTV promoter and inducible by dexamethasone. Two of the antisense-fos clones grew in a density-dependent manner, exhibiting both a flat morphology and a quiescence in low serum medium unlike the sense-fos controls. Antisense-fos also inhibited soft agar growth to 1% of control values and dramatically reduced tumor growth in nude mice. Antisense-fos had no effect on ras expression but greatly reduced c-fos protein levels as assayed by immunofluorescence. These findings suggest that down-regulation of signal transduction pathways by antisense therapeutic compounds might have major therapeutic benefits against malignant cells transformed by ras or other oncogenes.


Antisense and differentiation.

Antisense and differentiation. The use of antisense technology in animals has great potential for studying individual genes and for generating animal models of human disorders. Further control can be obtained by the use of inducible promoters to regulate the expression of antisense constructs, so that the timing and degree of antisense inhibition can be manipulated. In addition, tissue-specific promoters and enhancers provide the potential for targeting antisense inhibition to specific organs. Transgene expression in mice directed by the Cyp1a-1 promoter and enhancer elements can be increased in the liver up to 10,000-fold by administration to the animals of the inducer, 3-methylcholanthrene. Transfected antisense constructs containing splice site regions of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene can reduce HPRT activity to less than 1% of levels in parental NIH-3T3, COS, or HeLa cells. Antisense constructs including splice site regions and driven by the inducible Cyp1a-1 promoter should prove to be powerful tools in generating mouse models of human disorders.


Antisense oligodeoxynucleotides as probes of T-lymphocyte gene function. Conventional and thiophosphonate-derivatized oligonucleotides were employed to specifically regulate functional gene expression in murine T-cell hybridomas. For example, induction of apoptotic cell death following activation of T-cell hybridomas was examined using antisense oligonucleotides corresponding to several protooncogenes. We found that antisense oligodeoxynucleotides corresponding to c-myc inhibited both the characteristic DNA fragmentation and the loss of cell viability following activation without affecting production of lymphokines. Functional antisense oligonucleotides corresponding to c-fos had no effect in this system. These results demonstrate the use of antisense oligonucleotides to regulate function in T-cell hybridomas and provide valuable insights into the molecular bases of this biological phenomenon. Antisense oligonucleotides were also used to study another problem, the relation of T-cell-derived antigen-specific immunoregulatory factors to the T-cell receptor (TCR). Because the translation start of each TCR gene usually varies from one T cell to another, antisense oligonucleotides corresponding to the TCR V alpha or V beta of different cells were shown to act in a cell-specific manner. Furthermore, this method was used to demonstrate that a soluble antigen-specific regulatory activity produced by one of the T-cell lines depends on expression of the specific TCRa, an observation that has since been confirmed by gene transfer experiments. Expression of the CD3-TCR complex on the cell surface was also blocked by antisense oligonucleotides corresponding to CD3 gamma and CD3 zeta; however, neither these nor TCR V beta antisense oligonucleotides had any effect on production of the soluble regulatory activity.


Cell cycle effects of microinjected antisense oligodeoxynucleotides to p34cdc2 kinase. In this study the effect of antisense oligomers targeted against the mRNA transcripts of p34cdc2 kinase on G1 progression into S-phase was examined. For this purpose, antisense, sense, or nonsense oligomers were introduced directly into the cytoplasm of T98G cells grown in monolayer cultures by glass-capillary microinjection. The microinjection of antisense oligomers (but not sense or nonsense oligomers) into growth-arrested cells before serum stimulation inhibited G1 progression into S-phase. This inhibition was correlated with a reduction in the steady-state levels of nuclear p34cdc2 protein. Microinjection of antisense oligomers into cells at 2 and 6 hours after serum stimulation also resulted in a marked inhibition in the ability of cells to enter S-phase. The inhibitory effect decreased when cells were microinjected at 12 hours after serum stimulation. When cells were microinjected at 18 and 24 hours after serum stimulation, only a slight inhibition was observed. As the antisense oligomers were introduced directly into the cytoplasm of cells at each of the time points examined, the observed differences in the inhibitory effects of the antisense oligomers at later times after serum stimulation cannot be explained by differences in uptake. An alternative explanation is that after a certain threshold level of nuclear p34cdc2 protein is reached in late G1 phase; no further increase is necessary, because the cells become committed to enter S-phase. In yeast, p34cdc2 appears to play an important role in the G1/S-phase transition at a control point in late G1 phase called START (reviewed by Lewin). In mammalian cells a control point that could be equivalent to START is the "restriction point" which is defined as the time after which inhibition of protein synthesis fails to block entry into S-phase (reviewed by Pardee). The effects observed with antisense oligomers to p34cdc2 kinase are strikingly similar to what is observed when low concentrations of the drug cycloheximide are added to these cells at different times after serum stimulation; entry into S-phase is significantly inhibited when cycloheximide is added up to 12 hours postimulation. Thus, the results reported in this study are in agreement with the idea that p34cdc2 kinase plays a role in the G1/S phase transition in mammalian cells. Finally, introduction of antisense oligomers directly into the cytoplasm of cells grown in monolayer cultures by glass-capillary microinjection appears to be a viable alternative to simply adding the oligomers to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)


Use of antisense oligomers to study the role of c-jun in G1 progression. In actively proliferating Swiss 3T3 fibroblasts, expression of the protooncogene c-jun is maximally induced early in G1, immediately after completion of mitosis. Within 2 hours, c-jun mRNA levels drop to a basal amount that is approximately 30% of the maximum. This is maintained throughout the remainder of G1. To access the functional implications of this pattern of c-jun expression, antisense oligomers specific to c-jun were added to either actively proliferating or synchronized Swiss 3T3 cells, and their ability to inhibit DNA synthesis and division was determined. Our results show that if Swiss 3T3 cells are treated with anti-c-jun while actively growing or at any time during G1 after completion of mitosis, they exhibit a reduced ability to enter S-phase and subsequently divide. These results demonstrate that the regulation of G1 progression following mitosis depends on the expression and function of the protooncogene c-jun.


Modulation of double-stranded RNAs in vivo by RNA duplex unwindase. The double-stranded RNA (dsRNA) unwinding/modifying activity is a recently discovered cellular activity capable of unwinding or denaturing dsRNAs by modifying multiple adenosine residues to inosines and creating I-U mismatched base-pairings. The biological functions of this activity, which can potentially mutate the coding capacity of messenger RNAs (mRNAs), are presently not known. However, this unwinding/modifying activity is likely to affect the secondary structures, processing, and turn-over of various eukaryotic as well as viral transcripts. Although the activity was originally found and proposed as a cellular factor that interfered with the use of antisense RNA, it now appears more likely that the activity in fact may participate in antisense RNA suppression of target genes, either by altering the coding capacity of the sense mRNAs or by accelerating the degradation of duplex RNAs. Further understanding of this novel enzymatic activity, and thus, in turn, of the metabolism of dsRNAs in vivo, should allow us to derive a better strategy for designing antisense RNA.


Antisense RNA. History and perspective.

Antisense RNA. History and perspective. Antisense RNA was first an in vitro curiosity that was found to shut off protein synthesis in cell-free extracts. It was later shown to function in prokaryotic cells as a natural modulator of the synthesis of some proteins. Artificial antisense constructs can inhibit protein synthesis in prokaryotic and eukaryotic cells. To inhibit synthesis of proteins effectively, high ratios of antisense to sense RNAs are required. Thus, the challenge is to develop strategies to locate suitable targets and provide for amplification of the antisense RNA. This report provides a summary of our original work on antisense RNA.


Variables affecting antisense RNA inhibition of gene expression. We have sought to determine what variables affect the extent of inhibition of gene expression by stable nuclear-derived antisense RNAs. Myosin heavy chain gene II (MHCII) expression in Dictyostelium was used as a model system, because previous results have shown that nearly complete inhibition of expression can be achieved under appropriate conditions. Various fragments of the myosin gene were inserted into several transformation vectors in both sense and antisense orientation, and the effects on expression of protein and RNA from the endogenous MHC II gene were assayed. The results indicate that the critical factor was the particular fragment of the gene used to produce the antisense RNA. Some fragments produced complete inhibition of expression, whereas others gave only slight inhibition. The fragments that produced the greatest inhibition were from the tail region of the gene. In addition, cells were capable of overcoming the inhibition while still expressing the antisense RNA. We hypothesize that the three-dimensional topology of the antisense and sense RNAs determines their accessibility for interstrand hybridization. Transformation with sense fragments of the myosin gene also caused inhibition of the endogenous myosin gene. The inhibition seen with sense expression is not as dramatic as that for antisense, but it had the same phenotypic consequences for the cells. This phenomenon, which has now been documented in other systems, may be mechanistically similar to inhibition by antisense RNA in our system.


Modulation of c-myc transcription by triple helix formation. The human c-myc oncogene promoter was used as a model with which to study the mechanism of action of oligodeoxyribonucleotides targeted to a gene regulatory region. The nuclease-hypersensitive element, NHE, lying -115 bp from the P1 promoter of the human c-myc gene, is known to be required in cis for transcription of the gene from both P1 and P2 promoters (Fig. 1). Inhibition of c-myc transcription by an oligonucleotide designed to bind to NHE by triplex formation has been observed in a cell-free transcription assay. Using a reconstituted transcription system with the semipurified PuF transcription factor whose site of interaction resides within the NHE, it is shown here that the oligonucleotide inhibits PuF-mediated transcription. These findings, together with data presented elsewhere showing that: (1) PU1 binds to cloned DNA fragments to form a colinear triplex; (2) PU1 inhibits transcription in nuclear extracts; (3) triple helix formation inhibits the binding of PuF to its target NHE element in an in vitro binding competition assay (E. Postel, R. Durland, and M. Hogan, submitted); (4) triplex formation at the NHE target site can occur in living HeLa cells treated with the triplex-forming PU1 oligomer, and (5) c-myc mRNA synthesis in these treated cells is repressed, clearly support the proposed model in which the oligonucleotide targeted against the c-myc NHE promoter region binds to form a triplex, thereby blocking access to the regulatory protein PuF. This results in promoter-sensitive repression of transcriptional activation of the c-myc gene. The potential for manipulation of gene expression by oligonucleotides targeted to a DNA sequence of the c-myc oncogene promoter and other gene promoters is clear.


Inhibition of cell cycle progression by antisense oligodeoxynucleotides. We have used the antisense strategy to study the role of certain genes in cell cycle progression. In particular, we used antisense oligodeoxynucleotides to study: (1) the role of the IGF-1 receptor in the control of cell proliferation; and (2) the sequence of gene expression during the cell cycle. Our results can be summarized as follows: (1) the activation of the IGF-1 receptor by its ligand, IGF-1, is an obligatory step in the proliferation of fibroblasts and hemopoietic cells; and (2) the expression of DNA synthesis genes, such as PCNA, DNA polymerase alpha, and cdc2, is dependent on the expression of previous genes. A tentative temporal order is: c-myc > c-myb > IGF-1 receptor > DNA synthesis genes.


Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation. Anyone considering the use of AS RNA, generated endogenously, to inhibit gene expression should plan to generate several independent transfectants with nonoverlapping sequences; strategies that maximize both the transcription rate and the stability of the AS RNA are obviously desirable. Reasons why different results are obtained in different systems or with different constructs likely include the specific nucleotide sequence under investigation, the location of the AS gene in the nucleus relative to the endogenous gene, and the rate-limiting step in the expression of the target gene. Splicing may not be necessary, but an efficient polyadenylation signal likely is. Employment of a ribozyme-mediated strategy, discussed by various investigators in this volume, may be beneficial. There is no reason at present to conclude that any gene, however abundant its transcript might be, is inherently recalcitrant to AS-mediated down-regulation of expression.


Antisense RNA and p53 regulation in induced murine cell differentiation. p53 expression is strongly modulated during the process of induced differentiation, at the same time as both cell cycle and genetic expression become modulated, giving rise to a commitment to terminal differentiation. We took advantage of two murine cell lines inducible for differentiation, an erythroleukemia and a melanoma cell line, to outline common features of the regulation of p53 expression during the differentiation process. We found that p53 mRNA decreased early after induced differentiation and that regulation was controlled at a posttranscriptional level. Our data showed that this regulation affects p53 pre-mRNA maturation. Because, in both systems used, actinomycin D treatment abolished the inducer-mediated decrease of p53 mRNA, we looked for induced RNAs potentially involved in this process. Using different parts of the p53 gene and flanking regions as probes, we identified three RNA species whose expression is modulated during induced differentiation. A first species is made of high molecular weight RNAs that accumulate in the nuclear compartment and seem to represent antisense transcripts of the p53 gene. A second species, 1.3-kb long, was found to accumulate in the nucleus of induced MEL cells and was homologous to a restricted part of the first intron of the p53 gene due to the presence of a B1 repetitive element in an antisense orientation with respect to the p53 pre-messenger RNA. Finally, a family of B2-containing RNAs was observed in both cytoplasmic and nuclear compartments. The variation in the amounts of sense and antisense RNAs, respectively, suggested an interesting speculative model for the maturation of B2-containing pre-messenger RNAs.


Antisense promoter mapping. Inhibitory methods of transcriptional analysis. We have employed antisense methods to study the transcriptional functions of c-fos protein (Fos). Clones expressing inducible anti-fos RNA have been employed to inhibit c-fos expression, resulting in activation of c-fos transcription by inhibiting its normal repressor function. The sites of negative regulation by Fos have been mapped using this antisense mapping method which demonstrates that the serum response element represents the major site of repression by endogenous c-fos protein. A similar strategy (antisense cloning) has been employed to clone four target genes that are Fos dependent. These cDNAs encode mRNAs that are rapidly induced by serum (although this induction is blocked by cycloheximide) but are blocked by induction of anti-fos RNA. These inhibitory methods of studying transcription factor function are extremely useful for transcription factors (like Fos) that require cooperation with other factors to modulate gene transcription.