the following agents which destroy extracellular blood dna were used : bovine pancreatic dnase ( sigma , specific activity 2 400 kunitz units / mg ; samson - med , specific activity 1 500 kunitz units / mg ), recombinant human dnase 1 ( gentech , specific activity 1000 u / mg ). extracellular dna from blood plasma was isolated as follows : fresh plasma ( no more than 3 - 4 hours after sampling ) with anticoagulant ( sodium citrate ) was centrifuged on ficoll - plaque plus ( amersham - pharmacia ) during 20 minutes at 1500 g . at room temperature . ½ of plasma was detached , not affecting the rest of cells on the ficoll pillow , and further centrifuged at 10000 g . during 30 min for separation from cell fragments and debris . supernatant was detached , without affecting of the sediment , and was toped up to 1 % of sarkosil , 50 m m tris - hcl , ph 7 . 6 , 20 m m edta , 400 m m nacl , and than mixed with equal volume of phenol - chloroform ( 1 : 1 ) mixture . the prepared emulsion was incubated during 2 hours at t = 65 ° c ., then phenol - chloroform mixture was separated by centrifuging ( 500 g during 20 minutes , room temperature ). the procedure of deproteinization with phenol - chlorophorm mixture was repeated 3 times , and then the water phase was processed with chloroform and diethyl ether . separation from organic solvents was made by centrifugation at 5000 g during 15 minutes . then equal volume of izopropanol was added to resulting aqueous phase and the mixture was incubated overnight at 0 ° c . after sedimentation the nucleic acids were separated by centrifugation at 10000 g during 30 minutes . the sediment of nucleic acids was dissolved in of 10 m m tris - hcl buffer , ph 7 , 6 with 5 m m edta , and inflicted to the cscl gradient ( 1m , 2 . 5m , 5 . 7m ) in test - tube for rotor sw60ti . the volume of dna solution was 2 ml , volume of each step of cscl was 1 ml . ultracentrifugation was conducted in l80 - 80 ( beckman ) centrifuge during 3 hours at 250000 g . dna was collected from the surface of each gradient step into fractions . these fractions were dialyzed during 12 hours ( t = 4 ° c .) and pooled . presence of dna in fractions was determined by agar electrophoresis and dna was visualized by ethidium bromide staining . the amount of dna was determined with spectrophotometer ( beckman du70 ) in cuvet ( 100 mkl ) at wavelength of 220 - 230 nm . escherichia coli k1 , escherichia coli atcc 25922 , staphylococcus aureus vt38 , staphylococcus aureus vt - 2003r , candida albicans vt2002s , p . bergehi , a fumigatus vt2002a were used in this study . the strains designated vt were isolated from patients in the clinics of the st . petersburg state pavlov medical university . shr and c57bl mice were obtained from “ rappolovo ” animal breeding house . sequencing of extracellular blood dna from the patient suffering from bacillary dysentery , shigelosis caused by shigella flexneri the probes of patient &# 39 ; s extracellular blood dna were taken before initiation of antibacterial chemotherapy and week after completion of the therapy . the extracellular dna was cloned by the method which allows to get non amplified plasmid libraries of blood extracellular dna with representativeness up to one million of clones with the average size of 300 - 500 base pairs . the dna which has been isolated using the protocol specified above was treated with proteinase k ( sigma ) at 65 ° c . and subjected to additional phenol - chloroform extraction step with further overnight precipitation by ethanol . the dna fraction was than treated by eco ri restrictase or by pfu polymerase ( stratagene ) in presence of 300 mkm of all desoxynucleotidetriphosphates for sticky - ends elimination . the completed dna was phosphorylated by polynucleotidkinase t4 and ligated to pbluescript plasmid ( stratagene ), which had been digested with ecori or pvuii and dephosphorylated by phosphatase cip ( fermentas ). the process of ligation was conducted with rapid legation kit ( roche ). the ligated library was transformed into dh12s cells ( life technologies ) by electroporation ( e . coli porator ; biorad ). 12 - 20 electroporation covets were used for the transformation of one library . the library serial dilutions were cloned on 1 . 5 % agar and lb media supplemented with ampicilline . in both cases the libraries represented 2 - 3 × 10 6 clones . analysis of 50 randomly selected clones with the size 300 - 1000 base pairs from the “ before treatment ” library revealed 12 clones containing shigella flexneri dna specified in the table below : number of gene clones role in microorganism physiology s . flexneri 3 clones glycinedecarboxylase , p - protein of glycine gcvp gene cleavage system ; known as the part of conserved metabolic pathways involved in the chronic persistence of microorganism in hosts . s . flexneri 5 clones conserved membrane protein involved into yigm gene biofilm formation s . flexneri 4 clones coding for ferrisiderophor reductase ; ubib ubib gene e . coli mutants have alternate , as - yet unidentified hydroxylation mechanisms used in the anaerobic synthesis of ubiquinone . analysis of 50 randomly selected clones with the size 300 - 1000 base pairs from the “ after treatment ” library does not revealed any dna of bacterial origin . thus , extracellular blood dna from patient having systemic bacterial infection contains significant non - random presence of pathogen - specific dna coding for proteins involved in pathogen metabolic pathways and biofilm formation . the extracellular blood plasma dna were obtained as follows : s . aureus vt38 and escherichia coli k 1 was injected intravenously at ld50 dose to 10 shr mice ( 5 mice for each pathogen ) to induce sepsis . on day 3 the diseased mice were sacrificed and extrcellular blood dna were collected as specified above . biofilm formation assay was performed as follows : escherichia coli k1 and staphylococcus aureus vt38 pathogenic strains were grown at 378 c , and liquid cultures were incubated without shaking . before use in biofilm formation assay , cells were harvested and washed twice with 0 . 15m isotonic phosphate - buffered saline ( ph7 . 2 ), and the cell suspensions were standardized to an optical density ( od ) of 0 . 8 at 520 nm . the inoculums containing 7 . 53 ±□ 0 . 22 log 10 cfu / ml , was added to the wells of 96 - well plates ( 200 mkl / well ) and plates were incubated for 24 h at 378 c . different amounts of extracellular blood plasma dna were added to the wells 2 hours following seed . quantification of bacterial biomass in biofilms was performed by spectrophotometric method . liquid medium with bacteria was aspirated from the wells , and the wells were then washed with isotonic phosphate buffer ( 0 . 15 m , ph 7 . 2 ) without disturbing the adherent film . biofilm cells were scraped off the well walls and resuspended in 100 ml of phosphate buffer ( 0 . 15 m , ph 7 . 2 ). the well contents were then aspirated and bacteria were harvested by 10 min of centrifugation at 5000 rpm ( eppendorf 5415 c centrifuge ; eppendorf geratgebau gmbh , germany ). the pelleted bacteria were diluted in 2 ml pbs , and absorption was determined at 570 nm in a lkb biochrom novaspec 4049 spectrophotometer ( cambridge , united kingdom ). forming biofilm of s . aureus vt38 ( od at 570 nm ) effect of extracellular blood dna derived from blood of k1 infected animals ( ebdna k1 ) on biomass of forming biofilm of e . coli k1 ( od at 570 nm ) ebdna k1 ebdna k1 ebdna k1 control ( pbs ) 0 . 5 mkg / well 5 mkg / well 50 mkg / well 0 . 405 0 . 505 0 . 875 1 . 015 extracellular blood dna promotes spread of antibiotic resistance and clonogenic potential of microorganisms the extracellular blood plasma dna were obtained as follows : escherichia coli ki was injected intravenously at ld50 dose to 10 shr mice to induce sepsis . on day 3 animals were treated with subtherapeutic doses of ampicillin and kanamycin for 3 consecutive days . on day 6 the diseased mice were sacrificed and extrcellular blood dna were collected as specified above . biofilms of e . coli atcc 25922 were grown as described in example 2 with or without extracellular blood dna from e . coli k1 infected animals treated with antibiotics ( 50 mkg / well ) on selection media containing ampicillin or kanamycin or both . colony - forming assay was performed as follows : the term “ the number of cfu ” means the number of viable bacteria that grow on nutrient media in the current conditions . biofilms were grown in 96 - well plates for 24 or 48 h . at 37 ° c . liquid medium with bacteria was aspirated from the wells , which were then washed with isotonic phosphate buffer ( 0 . 15m , ph 7 . 2 ). biofilms were scraped thoroughly , with particular attention to well edges . the well contents were aspirated again , then placed in 1 . 0 ml of isotonic phosphate buffer ( 0 . 15m , ph 7 . 2 ), and dispersed by drawing up and down through a fine - tip pipette , and the total cfu number was determined by serial dilution method and plating on appropriate media . antibiotic in colony - forming units per 1 ml selection biofilms in ebdna containing media biofilms in regular media media 24 h . growth 48 h . growth 24 h . growth 48 h . growth control 3 . 0 - 5 . 0 × 10 9 6 . 0 - 8 . 0 × 10 9 1 . 0 - 3 . 0 × 10 9 2 . 0 - 4 . 0 × 10 9 km 2 . 0 - 5 . 0 × 10 9 2 . 0 - 5 . 0 × 10 9 1 . 0 - 3 . 0 × 10 9 1 . 0 - 3 . 0 × 10 9 amp 7 . 0 - 9 . 0 × 10 8 8 . 0 - 9 . 0 × 10 8 2 . 0 - 4 . 0 × 10 8 3 . 0 - 5 . 0 × 10 8 km + amp 1 . 0 × 10 2 - 5 . 0 × 10 3 1 . 0 × 10 2 - 5 . 0 × 10 3 0 . 0 - 1 . 0 × 10 2 0 . 0 - 1 . 0 × 10 2 thus , pathogen - specific extracellular blood dna promotes spread of antibiotic resistance and clonogenic potential of microorganism . abrogation of pathogenic potential of circulating extracellular blood dna by different doses of dnase the extracellular blood plasma dna were obtained as follows : s . aureus vt38 and escherichia coli ki was injected intravenously at ld50 dose to 10 shr mice ( 5 mice for each pathogen ) to induce sepsis . on day 3 the diseased mice were sacrificed and blood were collected into heparinized 1 ml tubes . 1 ml aliquots of blood from both s . aureus vt38 and escherichia coli ki infected animals were incubated within 30 min at 37 ° c . with different amounts of bovine pancreatic dnase , having specific activity of 1500 kunitz units per milligram . after incubation the reaction was stopped by placing tubes on ice and extracellular blood dna fraction were collected from each 1 ml sample as described in “ methods ” section . the biofilm formation assay was performed as specified in example 2 with presence of extracellular blood dna extracted from blood samples incubated with dnase ( extracellular dna originating from 1 ml blood aliquot was assayed in 3 parallel wells ). the electrophoresis assay of extracellular blood dna was performed before and after dnase treatment . the results of experiment are presented on fig1 . doses of dnase less than 1 mkg / ml ( 1 . 5 kunitz units / ml ) do not eliminate the pathogenic influence of extracellular blood dna ( promotion of biofilm growth ) and cant reduce the number average molecular weight of circulating extracellular blood dna . thus , unusually high doses of dnase required for realization of inventive treatment . treatment of the experimental sepsis caused by candida albicans and s . aureus group 1 — 30 shr mice were intravenously inoculated with 1 × 10 10 bacteria of pathogenic s . aureus vt - 2003r strain . recombinant human dnase 1 ( genentech ) was intraperitoneally administered at dose of 500 mkg / kg at 2 , 6 , 10 and 14 hours after the infection . group 2 — 10 shr mice were intravenously inoculated with 1 × 10 10 bacteria of pathogenic s . aureus vt - 2003r strain . phosphate buffer was intraperitoneally administrated at 2 , 6 , 10 and 14 hours after the infection . after last injection of recombinant human dnase 1 , 20 mice from group 1 were divided into two subgroups ( 1a and 1b ). subgroup 1a ( 10 mice )— 2 hours following last injection of recombinant human dnase 1 got intravenous injection of blood extracellular dna isolated from blood plasma of not treated s . aureus vt - 2003r infected animals at 15 hours post infection . subgroup 1b ( 10 mice )— 2 hours following last injection of recombinant human dnase 1 got intravenous injection of blood extracellular dna isolated from blood plasma of not treated c . albicans vt2002s infected animals at 72 hours post infection . the efficacies of dnase treatment and influence of extracellular blood dna on disease course were assessed based on the survival rate during 32 hours after infection . the results of challenge experiments are presented at the table below group 3 — 10 shr mice were intravenously inoculated with ld50 dose of pathogenic c . albicans vt2002s strain . recombinant human dnase 1 ( genentech ) was intraperitoneally administered at 1 mg / kg dose twice daily on day 2 , 3 and 4 after infection . group 4 — 10 shr mice were intravenously inoculated with ld50 dose of pathogenic c . albicans vt2002s strain . amphotericin b was intraperitoneally administered at 20 mg / kg dose twice a day on day 2 , day 3 and day 4 after infection . group 5 — 10 shr mice were intravenously inoculated with ld50 dose of pathogenic c . albicans vt2002s strain . phosphate buffer was intraperitoneally administered as negative control twice a day on day 2 , day 3 and day 4 after infection . the efficacies of treatments were assessed based on the survival rate during 7 days after infection . the results of challenge experiments are presented at the table below : thus the inventive method and is effective against systemic bacterial and fungal infection . blood extracellular dna from the infected animals possesses strong negative influence on the development of the infectious process . forty c57bl mice c57bl got intraperitoneal injection of erythrocytes , obtained from balb / c mice previously infected with p . bergehi ( 10 6 erythrocytes per mice ). mice were divided into 4 groups as follows : group 1 — 10 mice were treated with recombinant human dnase 1 ( genentech ) at 500 mkg / kg intramuscularly , 4 times per day , starting 24 h . following infection for three consecutive days . group 2 — 10 mice were treated with negative control ( phosphate buffer ) at the same schedule . group 3 — 10 mice were treated with recombinant human dnase 1 ( genentech ) at 50 mkg / kg intramuscularly , 4 times per day , starting 24 h . following infection for three consecutive days the efficacies of treatments were assessed based on the survival rate during 7 days after infection . the results of challenge experiments are presented at the table below : group 1 group 2 group 3 survival rate , % 90 10 20 thus the inventive method and is effective against systemic protozoan infection an isolate of a . fumigatus was obtained from the diagnostic microbiology laboratory at the st . petersburg medical academy and cultured on sabouraud dextrose agar at 25 ° c . for 3 days . 70 shr mice were used in challenge experiments . cyclophosphamide ( biochimic , saransk ) was administered intraperitoneally at 200 mg / kg of body weight 4 days prior to a . fumigatus challenge and 1 day after infection to induce a immunodeficient condition in mice . the pml counts were & lt ; 100 / μl for 6 days after infection . the inoculums , a 0 . 05 - ml droplet containing 7 . 0 × 10 5 cfu / mouse , were given via intranasal route . the recombinant human dnase 1 ( gentech ) ( at 5 mkg / kg , 50 mkg / kg , 100 mkg / kg , 500 mkg / kg , or 1000 mkg / kg ) was administered intravenously twice daily for 4 days , starting at 1 . 5 h after infection . 2 mice from each group were sacrificed at day 5 to perform quantification of extracellular blood plasma dna . the efficacies of dnase were assessed based on the survival rate at 10 days after infection . the results of challenge experiments are presented at the table below : thus , high doses of dnase according to inventive treatment are able to decrease the quantities of circulating extracellular blood plasma dna and are effective against systemic fungal infection in order to explore pharmacodynamics and perform exposure - response analysis of action of dnase administered into a circulating blood system of a patient diagnosed with systemic infection on extracellular blood dna we perform the clinical trial involving 25 patients suffering from odontogenic abscesses and phlegmons who has a 50 % probability of disease progression despite initiation of treatment . ( scores between 91 - 159 points according to m . m . solovev topical indicative prognosis scale ). scores between 91 - 159 points according to m . m . solovev topical indicative prognosis scale surgical drainage was performed at admittance day ( day 1 ); infusion therapy and antihistamine therapy were performed as supportive therapy . antibacterial therapy comprises cefazoline 1 . 0 g × 3 times daily . patients were randomly assigned into four groups to receive 50 , 150 , 300 and 600 mg daily doses of bovine pancreatic dnase ( samson med ). daily dose of dnase were applied as 6 daily im injections with 4 hours interval at day 1 , day 2 and day 3 . the patients were sampled for electrophoresis analysis of extracellular blood dna before first dnase injection and 24 h . later . at day 3 all patients were qualified by responsible physician either as “ progressing ” or “ stable ”. number of patients with decreased number average molecular number of total weight of patients with number of daily dose daily dose extracellular disease patients ( mg ) ( kunitz units ) blood dna . progression 9 50 85 000 0 5 8 150 255 000 0 3 8 300 510 000 5 1 8 600 1 020 000 8 0 thus , we surprisingly found that unusually high doses of dnase enzyme ( almost × 10 times higher than those reported in the literature ) required to achieve abrogation of disease progression and to achieve visible decrease of number average molecular weight of extracellular blood dna ( which in fact reflects enzymatic depolymerisation of dna ) ( fig2 ) kidney filtration system works as natural “ concentrator ” for circulating extracellular blood dna . concentrated “ transrenal dna ” further is excreted through lower urinary tract out from organism . transrenal dna is a extracellular urinary dna that originates from circulating blood extracellular dna . transrenal dna containing fetal sequences has been isolated from the urine of pregnant women , tumor - specific mutations have been detected in transrenal dna from patients with colon and pancreatic tumors , and donor dna has been found in transrenal dna isolated from recipient urine . furthermore , proviral hiv dna , bacterial and parasite dna sequences have been detected in transrenal dna from infected patients in very large quantities . ( umansky s r , tomei l d , expert rev mol diagn . 2006 march ; 6 ( 2 ): 153 - 63 . transrenal dna testing : progress and perspectives ). in fact , that results in exposition of infective microorganisms spreading in kidneys and lower urinary tract to extremely large quantities of disease - stimulating extracellular blood dna . thus , systemic infection developing in kidneys and lower urinary tract might be especially sensitive to the treatment according present invention . autosomal dominant polycystic kidney disease ( adpkd ) is a late - onset disorder characterized by progressive cyst development and bilaterally enlarged kidneys with multiple cysts . it is a genetic disorder resulting from mutations in either the pkd - 1 or pkd - 2 gene . cyst formation begins in utero from any point along the nephron , although & lt ; 5 % of total nephrons are thought to be involved . as the cysts accumulate fluid , they enlarge , separate entirely from the nephron , compress the neighboring renal parenchyma , and progressively compromise renal function . urinary tract infection occurs with increased frequency in adpkd . infection in a kidney or liver cyst is a particularly serious complication . it is most often due to gram - negative bacteria and presents with pain , fever , and chills . blood cultures are frequently positive , but urine culture may be negative because infected kidney cysts do not communicate directly with the collecting duct system . a 43 - year - old woman ( height : 165 cm weight : 72 kg ), with established history of adpkd , was admitted to nephrology center of st . petersburg medical academy with 25 day history of malaise , severe bilateral flank pain , dysuria , frequency , chills , and fever . on examination patient was febrile and ill . pretibial edema and tenderness over the costovertebral angles were noted . laboratory studies showed anemia , elevated level of blood urea nitrogen ( bun ), serum creatinine , and serum glucose . urinalysis showed pyuria , and urine culture yielded growth of escherichia coli and pseudomonas aeroginosa ( more than 10 5 colonies per high power field ). abdominal ultrasonography demonstrated both enlarged kidneys measuring 20 cm on the right side and 23 cm on the left contained multiple cysts . the largest cysts in the right and left kidney were 7 cm and 12 cm respectively , with most of the remaining cysts measuring 3 to 5 cm . no hepatic cyst was noted . intravenous piperacillin / tazobactam / ciprofloxacin therapy was initiated . further ultrsonography investigation on the 7th day of treatment showed numerous cystic space - occupying lesions with low level internal echoes , and increased wall thickness of the cysts that representing infected cysts . additional intravenous trimethoprim - sulfamethoxazole scheme was added to the assigned antibiotic therapy . this 2 - week intensive antibacterial therapy had no beneficial effect and no ultrasonographic or symptomatic relief was achieved . after informing the patient signed consent , the continuous intravenous infusion of bovine pancreatic dnase in daily dose of 1 000 mg ( 1 700 000 kunitz units ) was initiated . after 3 days , fever disappeared and clinical state and laboratory tests showed improvement . electrophoresis examination of circulating extracellular blood dna showed significant decrease of number average molecular weight ( fig3 ). repeated ultrasonography showed decreasing amount of internal echoes and thick septa of the drained cysts after one week of dnase therapy . then , dnase infusion was stopped and antibiotic therapy was appropriately changed to oral trimethoprim - sulfamethoxazole . after 2 months of hospitalization , patient completely recovered and was discharged from clinics . a 38 - years - old man has been admitted to the department of internal medicine of st . petersburg medical academy in grave condition . twelve days before he was diagnosed with acute respiratory viral infection . because of febrile temperature , severe asthenia and chest pain he was further diagnosed with acute pneumonia 5 days prior hospital admittance . intramuscular cefazoline injection and oral roxitromycine were prescribed , but no improvement was noted . two days before admittance to the hospital the patient has developed fever ( 39 . 5 - 40 ° c . ), sickness , headache . numerous hemorrhagic rush on the skin , muscle pain , jaundice and diarrhea appeared in the last day before hospitalization . on admittance the temperature was 39 . 3 ° c ., arterial pressure was 100 / 60 ; tachycardia of 120 beats per minute , negative meningeal symptoms , cold and cyanotic limbs were present . data of laboratory examination : moderate leucocytosis and left shift of haemogram ( 18 % of young forms ), appearance of neutrophils with toxic granulation , increased bilirubin , ast and alt . numerous small zones of heterogeneity were found at liver ′ ultrasound examination . the patient was diagnosed as having sepsis . bacteriological control of blood was performed . intravenous gentamycin / ampicillin / metronidazole / heparin therapy were initiated . despite intensive antimicrobial polychemotherapy , hemasorbtion and plasma transfusion patient ′ condition has become worse . vancomycin ′ infusions were prescribed as s . pneumonia was isolated from the patient ′ blood . during next 48 hours despite of vancomycin &# 39 ; s infusions condition of patient continued to become worse . symptoms of multiple organ failure have appeared . continuous intravenous infusions of bovine pancreatic dnase at 800 mg / day ( 1 600 000 kunitz units ) dose were initiated following consent from relatives . symptoms of condition ′ stabilization has appeared twelve hours after initiation of dnase infusion : improvement of peripheral blood circulation and of systemic hemodynamic indexes , appearance of urination . dnase infusions continued during next 5 days . at that time patient laboratory indexes , hemodynamics , renal function become normal and patient was transferred on self - breathing . for the realization the methods there were used well - known materials and equipment manufactured in plant conditions and according to aforesaid the invention conformances to requirements of “ industrial applicability ” criteria ( ia )
a treatment for the diseases that are accompanied by quantitative and qualitative changes of blood extracellular dna and , more particularly , to a treatment of systemic bacterial , fungal and protozoan infections . the inventive method consist from introducing a treatment agent into a circulating blood system of a patient diagnosed with systemic infection caused by bacteria , fungi or protozoa when said treatment agent destroys extracellular dna in said blood of said patient and wherein said treatment agent used to destroy said extracellular dna is a dnase enzyme : said agent must be administered in doses and regimens which sufficient to decrease number average molecular weight of circulating extracellular blood dna in the blood of said patient ; such decrease of number average molecular weight might be measured by gel electrophoresis of extracellular blood dna fraction from the blood of said patient . a dnase enzyme may be further applied in a dose and regime that provide a dna hydrolytic activity measured in blood plasma exceeding 1 . 5 kunitz units per 1 ml of blood plasma for more than 12 hours within a period of 24 hours .