Patent Application: US-67899503-A

Abstract:
the invention relates to the field of immunology . specifically , the invention relates to the field of immune - mediated disorders such as allergies , auto - immune disease , transplantation - related disease or inflammatory disease . the invention provides for an immunoregulator , use of an ir in preparing a pharmaceutical composition for treating an immune - mediated disorder and a method for treating an immune - mediated disorder .

Description:
first trimester pregnancy urine ( 2 liters ) was collected in a bottle from a healthy volunteer and was refrigerated until delivered at the laboratory within 2 days . upon delivery , 1 gram per liter of sodium azide was added , and the ph was adjusted to 7 . 2 - 7 . 4 with sodium hydroxide and allowed to sediment for 1 hour ( h ) at room temperature ( rt ). approximately , 75 % of the supernatant was decanted and the remainder close to the precipitate was centrifuged ( 10 min at 25000 rpm at 40c ) to remove sediment and added to the rest of the supernatants . the supernatants were filtered through 0 . 45 ( m in a minitan ) ( millipore ) transversal filtration set - up . subsequently , the filtrate ( 2 liter ) was concentrated in an amicon ultrafiltration set - up equipped with a ym diopore membrane with a 10 kda cut - off . the final volume ( 250 ml ) was dialyzed against 2 changes of 10 liters of milli q water . next , the sample was further concentrated by 10 kda cut - off in an amicon ultrafiltration to a final volume of 3 ml . gel permeation : a pharmacia fplc system equipped with a superdex 75 gel permeation column was used to analyze the treated urine sample ( ir - u ) and commercial hcg preparation ( ir - p ) ( pregnyl ; organon ; oss , nl ). the running conditions are shown elsewhere in this document : in order to purify lower molecular weight fractions from first trimester pregnancy urine , 50 ml of urine was directly desalted with an fplc system equipped with an fdc ® g25 in 50 mm ammonium bicarbonate . the running conditions used are shown below : to analyze the ir - u ( first trimester urine ) obtained from method 1 and 2 , we also used a shimadzu hplc system equipped with alltech macrosphere size exclusion ( gpc ) column 60 å or 300 å ( 250 × 4 . 6 mm ) in 50 mm ammonium bicarbonate . the separation range for both columns was 28 , 000 - 250 and 1 , 200 , 000 - 7 , 500 dalton , respectively . sample load volume was 10 - 50 ml . the flow rate was 0 . 3 ml / min for 25 minutes . external molecular weight standards were also employed to calibrate the column elution positions . the markers used were : aprotinin ( 6 , 500 da ), cytochrome c ( 12 , 400 ), carbonic anhydrase ( 29 , 000 ), albumin ( 66 , 000 ) and blue dextran ( 2 , 000 , 000 ). to analyze ir further , two different hcg preparations , ir - p ( pregnyl ; organon ; oss , the netherlands ) and ir - a ( apl ; wyeth ayerst ; philadelphia , usa ), were used . ir - p was further separated by two methods . a pharmacia fplc system equipped with a superdex 75 gel permeation column ( hr 5 / 30 ) ( pharmacia , sweden ) was used to analyze the ir - p . for the running buffer 50 mm ammonium bicarbonate was used . the separation range of this column was 100 , 000 - 3 , 000 da for globular proteins . sample load volume was 1 ml and the flow rate was 0 . 5 ml / min for 45 min . in addition , macrosphere gpc 60 å ( 250 × 4 . 6 mm ) was also used . this column separates proteins , peptides , and other water - soluble macromolecules by size exclusion chromatography . the separation range of this column was 28 , 000 - 250 dalton . three selected areas were fractionated , ir - p1 which elutes apparently with molecular weight of & gt ; 10 kda , ir - p2 which elutes apparently with molecular weight between the 10 kda - 1 kda , and ir - p3 which elutes apparently with molecular weight & lt ; 1 kda . purification of ir from lower molecular fraction first trimester pregnancy urine ( ir - u / lmdf ) and commercial hcg preparations ( pregnyl , apl ): method 4 : procedure : the lyophilized low molecular mass fraction (& lt ; 2 kda ) obtained from first trimester pregnancy urine and from commercial hcg preparations ( pregnyl , apl ) by method 3 were further analyzed by gel filtration chromatography on a bio - gel p - 2 column ( 96 × 1 . 5 cm ). fraction ( 13 - 17 mg ) was suspended in bi - distilled water ( 8 - 12 ml ). the material was not completely dissolved . the sediment ( 8 - 11 mg ) was separated from the supernatant by centrifugation ( sigma 201 , 10 min , 3000 rpm ). the supernatant ( 6 - 8 ml ) was fractionated by gel filtration chromatography on a bio - gel p - 2 column . the column was eluted with water at a flow rate of 15 ml / min . the elution was monitored with an lkb 2142 differential refractometer and an lkb 2238 uvicord sii ( 206 nm ). fractions ( 20 min ) were collected by a pharmacia frac 100 fraction collecter . definite fractions were pooled and lyophilized . these fractions were further tested for anti - shock activity . gel permeation : a pharmacia fplc system equipped with a superdex 75 gel permeation column was used to analyze the treated urine sample ( ir - u ) and commercial hcg preparation ( ir - p ) ( pregnyl ; organon ; oss , nl ). the running conditions used are shown below : anion exchange chromatography : in order to further separate the overlapping fractions , 1 ml mono q hr 5 / 5 fplc anion exchange column was used . the running conditions are shown below and the buffer combination consisted of 10 mm pbs , ph 7 . 3 as buffer a and pbs containing 1 m nacl as buffer b : further treatment of the ir - u and ir - p : to reduce covalent binding between protein species present in the urine sample , we treated the urine ( ir - u ) and hcg preparation ( ir - p ) sample with 60 mm 2 - mercaptoethanol for 3 min at 100 ° c . subsequently , the treated ir - u and ir - p sample were applied to the superdex 75 column under identical running conditions . activity determination of fplc fractions of ir - u : the protein concentration of urine fractions was determined by od280 nm divided by 1 . 4 . from this value , the amount of hcg units was calculated using 5000 iu / ml pregnyl preparation of hcg corresponded to 100 μg . alternative methods for purifying and / or isolating ir comprise gel filtration on , for example , a superdex 75 column in an fplc system using pbs with or without ethanol to increase resolution and disrupt hydrophobic interactions , optionally followed by cationic exchange . samples can be submitted in reduced or unreduced form . another method comprises lectin affinity chromatography to better separate carbohydrate containing components from other components , whereby the effluent is further subjected to gel filtration . it is , of course , possible to derive at synthetic or recombinant ( poly ) peptide sequences with methods known in the art , and to select ( synthetic ) antibodies , i . e ., phage - derived , to further select ir . the non - obese diabetic ( nod ) mouse is a model for auto - immune disease , in this case insulin - dependent diabetes mellitus ( iddm ), which main clinical feature is elevated blood glucose levels ( hyperglycemia ). the elevated blood glucose levels are caused by the immune - mediated destruction of insulin - producing β cells in the islets of langerhans of the pancreas ( bach et al . 1991 , atkinson et al . 1994 ). this destruction is accompanied by a massive cellular infiltration surrounding and penetrating the islets ( insulitis ) by a heterogeneous mixture composed of a cd4 + and cd8 + t - lymphocytes , b - lymphocytes , macrophages and dendritic cells ( o &# 39 ; reilly et al . 1991 ). the easiest and most reliable way to detect the onset of diabetes in these mice is to test for glucose levels in the blood . the nod mouse represents a model in which auto - immunity against beta - cells is the primary event in the development of iddm . in general , t - lymphocytes play a pivotal role in initiating the disease process ( sempe et al . 1991 , miyazaki et al . 1985 , harada et al . 1986 , makino et al . 1986 ). diabetogenesis is mediated through a multifactorial interaction between a unique mhc class ii gene and multiple , unlinked , genetic loci as in the human disease . moreover , the nod mouse demonstrates beautifully the critical interaction between heredity and environment . differences between the cleanliness of the housing conditions illustrates how environmental factors can affect the action of diabetes - mediated genes ( elias et al . 1994 ). as for the auto - immunity recorded in nod mice , most antigen - specific antibodies and t - cell responses have been studied after these antigens were detected as self - antigens in diabetic patients . understanding the role that these auto - antigens play in nod diabetes may allow to distinguish between primary pathogenic auto - antigens and auto - immunity that is an epiphenomenon . moreover , one should bear in mind that iddm patients are genetically and pathogenically heterogeneous . a typical longitudinal histological examination of the nod pancreas demonstrates infiltrating cells surrounding the blood vessels at 3 - 4 weeks of age , but the islets are typically still clear at 6 - 7 weeks . infiltrating cells than reach the islets , either surrounding them or accumulating at one pole . between 10 and 12 weeks , the infiltrating cells penetrate into the islets and the islets become swollen with lymphocytes . as mentioned above , differences between the housing conditions and microbiological and environmental factors can affect the penetration of diabetes - susceptible genes . in our hands , typically between 14 - 17 weeks nod mice become diabetic . however , this varies from lab to lab ( average 14 - 19 weeks ) ( elias et al . 1994 ). cd4 + t - cells can be separated into at least two major subsets th1 and th2 . activated th1 cells secrete ifn - γ and tnf - α , while th2 cells produce il - 4 , il - 5 and il - 10 . th1 cells are critically involved in the generation of effective cellular immunity , whereas th2 cells are instrumental in the generation of humoral and mucosal immunity and allergy , including the activation of eosinophils and mast - cells and the production of ige ( abbas et al . 1996 ). a number of studies have now correlated diabetes in mice and humans with th1 phenotype development ( liblau et al . 1995 , katz et al . 1995 ). th2 t - cells are shown to be relatively innocuous . some have even speculated that th2 t - cells , in fact , may be protective . but katz et al have shown the ability of cd4 + t - cells to transfer diabetes to naive recipients resided not with the antigen specificity recognized by the tcr , per se , but with the phenotypic nature of the t - cell response . strongly polarized th1 t - cells transferred disease into nod neonatal mice , while th2 t - cells did not , despite being activated and bearing the same tcr as the diabetogenic th1 t - cell population . moreover , upon co - transfer , th2 t - cells could not ameliorate th1 - induced diabetes , even when th2 cells were co - transferred in 10 - fold excess ( pakala et al . 1997 ). th1 - polarized t - cells can transfer disease in neonatal nod mice , something th2 - polarized t - cells fail to do . both th1 - and th2 - polarized t - cells can transfer disease in nod . scid mice and other immune - compromised recipients . th2 - mediated diabetes in nod . scid recipients exhibited a longer pre - diabetic phase and a lowered overall - incidence . moreover , the diabetic lesion created by th2 cells is unique and quite unlike the lesion found in spontaneously diabetic or th1 t - cell - induced diabetes in either neonates or nod . scid mice ( pakala et al . 1997 ). in addition , ifn - γ correlates with diabetes ( in nod as well as in humans ) and anti - ifn - γ prevents disease ; under disease , ifn - γ + cells are present in islets and antigen - specific th1 clones accelerate the onset of diabetes ( pakala et al . 1997 , o &# 39 ; garra et al . 1997 ). furthermore , th2 cells only induce insulitis in neonatal nod , but have the capacity to induce diabetes in immuno - compromised nod . scid ; also , disease is inhibitable by anti - il - 10 , but not by anti - il - 4 ( pakala et al . 1997 ). this suggests that non - th2 type regulator t - cells are present in normal mice , but these are absent in immunodeficient mice . these results stress the existence of cells regulating the balance between activated th - sub - populations . possible disturbances in this balance induced by altered reactivity of such regulatory t - cell populations can cause immune - mediated diseases , which results in absence or over - production of certain critically important cytokines ( o &# 39 ; garra et al . 1997 ). some auto - immune diseases , in particular th1 mediated diseases , like rheumatoid arthritis ( ra ) ( grossman et al . 1997 , russel et al . 1997 , buyon et al . 1998 , hintzen et al . 1997 ) can remit during pregnancy . furthermore , successful pregnancy is a th2 type phenomenon ( raghupath et al . 1997 ). we tested hcg preparation and its fractions from pregnyl organon , oss on the development of diabetes in nod mice and in an in vitro model . surprisingly , we found that intraperitoneal treatment of nod mice of age 15 weeks , with an hcg preparation for three times a week for a month can delay or inhibit the onset of diabetes . in addition , transfer of total spleen cells from these treated nod mice into nod . scid mice can delay or prevent diabetes in nod . scid whereas transfer of non - treated spleen cells cannot . this anti - diabetic effect resides in a fraction obtainable from a pregnant woman but not in hcg . mice . nod mice were bred in our facilities under specific pathogen - free conditions . the spontaneous incidence of diabetes in our colony is 85 % in females at 15 weeks of age . nod . scid mice were also bred in our facilities under specific pathogen - free conditions . transfer of diabetogenic cells from nod to nod . scid at the age of 8 weeks induces diabetes after 22 days . diabetes . diabetes was assessed by measurement of venous blood using an abbott medisense precision q . i . d . glucometer and also monitored for glucosuria ( gluketur test ; boehringer mannheim , mannheim , germany ). animals were considered diabetic after two consecutive glucose measurements of higher than 13 . 75 mmol / l ( 250 mg / dl ) onset of diabetes was dated from the first consecutive reading . in instances of sustained hyperglycemia of & gt ; 33 mmol / l animals were killed to avoid prolonged discomfort . immunohistochemistry . mice were killed by co2 asphyxiation . the entire pancreata were removed and snap frozen in oct compound ( tissue - tek ) for cry - sectioning . 5 - μm cryo - sections were obtained , air dried , and stored at − 20 ° c . until used . formalin - fixed sections were deparaffinised in xylene and alcohol , and stained with hematoxylin and eosin for general morphology . immunohistochemistry for insulin was then performed using a two - step protocol . endogenous peroxidase activity was blocked , and slides were incubated with a rabbit antiserum to insulin ( dako corp ., carpenteria , calif . ; 1 : 500 in 5 % normal mouse serum for 30 min ). after washing steps , staining was revealed with horseradish peroxidase conjugated anti - rabbit ig ( dako ; 1 : 500 in 5 % nms for 30 min ), developed with amino - ethyl - carbazole ( aec ; pierce ) for 10 min and mounted in crystal mount . in vivo anti - diabetic effect : nod mice at the age of 15 weeks were treated with pbs ( n = 4 ), 300 iu pregnyl ( n = 4 ), or 600 iu pregnyl ( n = 4 ) i . p ., 3 times a week for four weeks and diabetes was assessed as mentioned above . after four weeks , the treatment was stopped and the pbs and the 600 iu pregnyl group were killed after one week . the 300 iu pregnyl group was left alive until the age of 28 weeks . spleen cell transfer . the spleen was removed from 600 iu pregnyl treated nod and pbs control treated nod mice , and total spleen cells were recovered . these cells were washed twice with pbs and 20 × 10 6 cells were i . p . transferred into an 8 - wk - old nod . scid mouse . transfer experiments total spleen cells were recovered from 9 - wk - old nod mice and stimulated in vitro in rpmi supplemented with 10 % fbs with coated anti - cd3 ( 145 - 2c11 ; 25 mg / ml ) and il - 2 ( 50 u / ml ) along with 300 iu / ml ir - p , 100 mg / ml ir - u3 - 5 or ir - u / lmdf . plates were then incubated at 37 ° c . in 5 % of co 2 in air for 48 hrs . after 48 hrs , cells were twice washed with pbs and 20 × 10 6 cells were i . p . transferred into an 8 - wk - old nod . scid mouse . in vitro restimulation . total spleen cells ( 1 × 10 6 cells / ml ) from 20 - wk - old nod were stimulated in rpmi + supplemented with 10 % fbs with lps ( ecoli ; 10 μg / ml ) or coated anti - cd3 ( 145 - 2c11 ; 25 μg / ml ) with different doses of hcg - pregnyl ( 50 , 100 , 300 , 600 , 800 iu / ml ), fraction 1 - 2 ( 200 ) μg / ml ), fraction 3 - 5 ( 200 ( g / ml ), human recombinant hcg , α - hcg , and β - hcg ( each at 200 μg / ml ) in flat bottom 96 - well plates . wells with anti - cd3 coating were implemented with il - 2 ( 40 iu / ml ). plates were incubated at 37 ° c . in 5 % co2 in air for 48 hrs . after 48 hrs of incubation the supernatants were collected for cytokine analyzes . cd4 + t - cells were isolated from total spleen cells of 20 - wk - old nod and stimulated as mentioned above with anti - cd3 at different conditions . these wells were implemented with il - 2 ( 40 μg / ml ) and anti - cd28 ( 10 μg / ml ). after 48 hrs of incubation the supernatants were also collected for cytokine analyzes . to determine the effect of ir on the potential of cd4 cells to differentiate into th1 or th2 cytokine - producing effector cells , th polarization assay was performed in the presence or absence of ir . total spleen cells from 8 - wk - old female nod were used as a source to purify cd4 + cells . purified cd4 + t - cells from the spleen were obtained by negative selection due to complement depletion with antibodies specific for b - cells , nk - cells , monocytes / macrophages and granulocytes . cells were further purified using magnetic activated cell sorting with a cocktail of biotinylated mabs against cd11b , b220 , cd8 and cd40 , followed by incubation with streptavidin conjugated microbeads ( milteny biotech , bergisch gladbach , germany ). cd4 + cells used for experiments were always 90 - 95 % purified as determined by flow cytometry . for primary stimulation , purified cd4 + t - cells were cultured at 1 × 10 5 cells / well in flat bottom 96 - well plates ( nalge nunc int ., naperville , ill ., usa ), and stimulated with plate - bound anti - cd3 mab ( 145 - 2c11 , 25 mg / ml ), anti - cd28 , and il - 2 ( 50 u / ml ). for differentiation of th1 cells , anti - il - 4 mab ( 11b11 ; 10 mg / ml ) and il - 12 ( 10 ng / ml ) were added to the cultures . priming for th2 cells was with il - 4 ( 35 ng / ml ) and antiifn - g mab ( xmg 1 . 2 ; 5 mg / ml ). furthermore , in th1 and th2 priming conditions , also 300 iu / ml ir - p and 100 mg / ml ir - u / lmndf in the presence or absence of blocking anti - il - 10 ( 10 mg / ml ), anti - tgf - b ( 10 mg / ml ), and vitd3 ( 10 mg / ml ). unprimed cultures contained only anti - cd3 , anti - cd28 and il - 2 . all doses were optimized in preliminary experiments . after 4 days of culture , the cells were washed 3 times and transferred to new anti - cd3 - coated 96 - well plates and restimulated in the presence of il - 2 ( 50 u / ml ) and anti - cd28 ( 10 mg / ml ). forty - eight hours later , supernatants were collected and assayed for il - 4 , ifn - g and il - 10 production by elisa as a readout for th1 versus th2 polarization . in rodents , the switch in the production of antibodies from igm to igg and other classes appears to be largely under t - cell control mediated by cytokines . dominant th1 polarization mediate switching b - cells from igm production to igg2a under the influence of massive production of ifn - gamma , while th2 polarization induces isotype switching in b - cells to igg1 production . we treated nod mice at the age of 8 - 10 weeks with pbs ( n = 5 ) or ir - p and its fractions ir - p1 , ir - p2 , ir - p3 , or recombinant hcg ( rhcg ) and rhcg in combination with ir - p3 , each with 200 mg i . p . for three days . total spleen cells were isolated from all groups and stimulated with lps or coated anti - cd3 as mentioned before . at different time points , cytokines and proliferation was measured as follows : anti - cd3 stimulated proliferation ( t = 12 , 24 , 48 h ), anti - cd3 stimulated ifn - gamma ( t = 24 , 30 , 48 h ), lps stimulated igg2a production ( t = 7 days ). in order to determine the effect of ir treatment on th1 polarization , we isolated cd4 + cells and performed th1 polarization assays as mentioned before . to separate the immune - modulating activity of ir from its beneficial clinical effects , we treated healthy balb / c mice i . p . with 300 iu ir - p or 100 mg / ml of iru / lmdf ( n = 5 ). this strain is generally considered to react upon stimulation with a th2 driven immune response . after four days of treatment with ir , purified cd4 + spleen cells from control and ir - p treated mice were analyzed for th polarization as mentioned above . in order to determine the effect of ir - p on cytokine levels produced by splenic apcs , spleen cells from control and ir - p treated balb / c mice were stimulated in vitro with lps ( e . coli 026 : b6 ; 10 mg / ml , difco laboratories , detroit mich ., usa ). after 48 hours of incubation , supernatants were collected for cytokine analysis ( il - 12p70 , il - 6 ). to determine the in vivo effect of ir - p in il - 10 gene targeted ( il - 10ko ) mice , we treated such mice ( n = 2 ) i . p . with 300 iu ir - p / day for 4 consecutive days . after 4 days of treatment spleen and lymph nodes cells were recovered and tested for their ability to proliferate in response to lps and anti - cd3 . in addition , cd4 + cells were purified from control and ir - p treated mice and analyzed for th polarization potential as mentioned above . in order to determine ir - induced effects on dendritic cells ( dc ) derived from bone marrow ( bm ), bm of 9 - wk - old female nod mice ( n = 2 ) were isolated and incubated with 20 ng / ml gm - csf ( 2 . 0 × 10 5 cells / ml ) for 6 days and at day 7 co - cultured with 300 iu / ml ir - p or 100 mg / ml ir - u ( ir - u , ir - u - f3 - 5 [ superdex 75 - derived ], or ir - u / lmdf [ fdc - derived ]) for an additional 24 hrs . briefly , femora and tibiae were cleaned of muscles and tendons and ground in a mortar using dbss - fcs . single cell suspensions were obtained by aspiration through a 22 gauge needle into a 2 ml syringe , followed by sieving the cell suspension twice over nylon filters ( mesh size 100 and 30 mm respectively ; polymon pes , kabel , amsterdam , the netherlands ). furthermore , in order to know whether ir also has an effect on the maturation of dc , bm from nod mice were also directly co - cultured with gm - csf and ir for 7 days . at day 8 , all cells were analyzed by a flow cytometer for expression of the following markers : cd1d , cd11c , cd14 , cd31 , cd40 , cd43 , cd80 , cd86 , cd95 , er - mp20 , er - mp58 , f4 / 80 , e - cad , mhc ii , mhc i , rb6 8c5 . a similar experiment was performed with bm cells from a 9 - wk - old female balb / c mice ( n = 3 ). in order to test the immunosuppressive activity of ir on transplantation rejection , we performed allo - mlr . bm cells from 9 - wk - old female balb / c ( n = 3 ) were isolated as mentioned above and treated with ( recombinant mouse ) rmgm - csf ( 20 ng / ml ) and ir ( ir - p ; 300 iu / ml , ir - u ; 300 mg / ml , ir - u3 - 5 ; 300 mg / ml , ir - u / lmdf ; 300 mg / ml ) for 7 days . after 7 days , the dc generated were irradiated ( 2 , 000 rad ) and co - cultured with splenic cd3 + cells isolated from 9 - wk - old female c57bl6 / ly . these cd3 + and dc cells were cultured at various ratios and t - cell proliferation was measured via [ 3 h ] tdr incorporation ( 0 . 5 mci / well during the last 16 hrs in culture ). cytokine elisa . il - 4 was detected using monoclonal anti - il - 4 antibody ( 11b11 ) as the capture antibody and revealed with biotinated - conjugated rat anti - mouse il - 4 monoclonal antibody ( bvd6 24g2 . 3 ). ifn - γ was detected using monoclonal anti - ifn - γ antibody ( xmg1 . 2 ) as the capture antibody and revealed with biotinylatedconjugated rat anti - mouse ifn - γ monoclonal antibody ( r46a2 ). in both cases , abts substrate was used for detection . flat bottom microplates ( 96 - wells , falcon 3912 , microtest ii flexible assay plate , becton dickinson , oxnard , usa ) were coated with cytokine - specific capture antibodies for il - 6 , il - 10 , il - 4 and ifn - g diluted in pbs ( 1 mg / ml 20f3 and sxc - 1 ; 5 mg / ml 11b11 and xmg1 . 2 , respectively ) at 4 ° c . for 18 hrs . after coating , plates were washed ( pbs , 0 . 1 % bsa , 0 . 05 % tween - 20 ) and blocked with pbs supplemented with 1 % bsa at room temperature for 1 hr . after washing , samples and standards were added and incubation was continued for at least 4 hrs at room temperature . thereafter , plates were washed and biotinylated detection antibodies were added ( 1 mg / ml 32c11 ( il - 6 ) and r46a2 ( ifn - g ); 0 . 1 mg / ml 2a5 . 1 ( il - 10 ) and bvd6 . 24g2 ( il - 4 )) and incubated overnight at 4 ° c . after washing , streptavidin - peroxidase ( 1 / 1500 diluted , jackson lmmunoresearch , west grove , pa ., usa ) was added . after 1 hr , plates were washed and the reaction was visualized using 2 , 2 ′- azino - bis - 3 - ethylbenz - thiazoline - 6 - sulfonic acid ( abts , 1 mg / ml , sigrna , st . louis , mo ., usa ). optical density was measured at 414 nm , using a titertek multiscan ( flow labs , redwood city , usa ). the amounts of il - 12p70 , tnf - a and tgf - b were measured with commercially available elisa kits ( genzyme corp , cambridge , mass .) according to the protocols provided by the manufacturer . there are three common mouse models used to investigate sepsis or septic shock : high dose lps , low dose lps with d - galactosamine sensitization and low dose superantigen with d - galactosamine . one of the first models used for investigating sepsis or septic shock involved treatments with rather large doses of lps in the inter - peritoneal cavity ( between 300 - 1200 μg ). mice are quite resistant to bacterial toxins , yet succumb to this high dose . it has been suggested that a high dose of lps in mice might correlate with a lower dose in humans ( mietheke et al .). approximately 70 % of sepsis or septic shocks in humans are caused by gram - negative bacterial endotoxin and up to 30 % are created by exotoxins released from gram - positive bacteria . the traditional endotoxin , the distinctive lipopolysaccharide ( lps ), is associated with the cell membrane of the gram - negative organism and represents the most common initiator of the sepsis or septic shock pathogenetic cascade . the endotoxin molecule consists or an outer core with a series of oligosaccharides that are antigenically and structurally diverse , an inner oligosaccharide core that has similarities among common gram - negative bacteria , and a core lipid a that is highly conserved across bacterial species . the lipid a is responsible for many of the toxic properties of endotoxin . the systemic effects of endotoxins , such as lps , seem to be largely mediated by macrophages , since adoptive transfer of endotoxin - sensitive macrophages renders previously endotoxin - resistant mice sensitive to the toxin ( freudenberg et al . 1986 ). the more commonly used model of endotoxin sepsis or septic shock takes advantage of the increased susceptibility of balb / c mice to low doses of lps after being simultaneously treated with galactosamine ( d - gal sensitized ). this d - gal treatment dramatically sensitizes animals to the toxic effect of lps , so that nanogram amounts induce a liver toxicity that is lethal for wild - type animals in a period of 6 - 7 h . this systemic effect of endotoxin seems to be largely mediated by macrophages . ( gutierrez - ramos et al . 1997 ). although certain mediators are undoubtedly more important than others in producing sepsis , probably dozens of organism - and host - derived mediators interacting , accelerating , and inhibiting one another , are responsible for the pathogenesis of sepsis or septic shock . on response to lps , tnf , and other mediators , endothelial cells and macrophages can release a potent vasodilator agent , endothelial - derived relaxing factor ( edrf ), which has recently been identified as nitric oxide . this molecule causes smooth muscle cell relaxation and potent vasodilatation . inhibiting nitric oxide production with competitive inhibitors of nitric oxide synthase results in increased blood pressure in animals with endotoxin shock . this suggests that nitric oxide may be partially responsible for the hypotension associated with sepsis . although inhibition of nitric oxide restores blood pressure , such inhibition may reduce tissue blood flow . ( bennett et al .) endotoxin can also activate the complement cascade , usually via the alternative pathway . this results in the release of the anaphylotoxins c3a and c5a , which can induce vasodilatation , increase vascular permeability , platelet aggregation , activation and aggregation of neutrophils . these complement - derived mediators may be responsible in part for the microvascular abnormalities associated with sepsis or septic shock . further , endotoxin can result in the release of bradykinin via the activation of factor xii ( hageman factor ), kallikrein , and kiniogen . bradykinin is also a potent vasodilator and hypotensive agent . lps activation of factor xii also leads to intrinsic and ( through macrophage and endothelial cell release of tissue factor ) extrinsic coagulation pathway activation . this results in consumption of coagulation factors and dic . tnf also activates the extrinsic pathway and may contribute to these coagulation abnormalities . different metabolism of the arachidonic acid cascade are also known to cause vasodilatation ( prostacyclines ), vasoconstriction ( thromboxanes ), platelet aggregation , or neutrophil activation . in experimental animals , inhibiting cyclo - oxygenase or thromboxane synthase has protected against endotoxin shock . elevated levels of thromboxane b2 ( tbx2 ) and 6 - ketoprostaglandin f1 ( the end product of prostacylin metabolism ) are present in patients with sepsis . a number of cytokines can cause release of these arachidonic acid metabolites from endothelial cells or leukocytes . in a similar fashion , exotoxin shock model d - gal sensitized balb / c mice are treated with low doses of tsst - 1 or seb . these superantigens stimulate the proliferation and activation of a large proportion of t - cells . in fact , the t - cell activation induced by these super - antigens can almost be viewed as a polyclonal t cell activation in that t - cells expressing a specific vbeta family are all activated through non - antigen - specific binding of the tcr / mhcii / and superantigen ( fig1 ). d - galactosamine has been shown to be a transcription inhibitor which targets the liver , interfering with the synthesis of acute phase proteins . it is believed that these acute phase proteins , in fact , help the liver detoxify or deactivate tnfα . in fact d - galactosamine treatment in the low dose endotoxin or exotoxin models is accompanied by tnfα mediated hepatic apoptosis . d - galactosamine treatment alone does not result in hepatic apoptosis , and these organ damaging effects can be neutralized in both low dose models by neutralizing anti - tnfα antibodies ( gutierrez - ramos et al . 1997 ). mice used in sepsis or septic shock experiments : female balb / c and sjl mice between 8 - 12 weeks of age were used for all experiments . the animals were bred in our facility under specific pathogen - free conditions according to the protocols described in the report of european laboratory animal science associations ( felasa ) working group on animal health ( laboratory animals 28 : 1 - 24 , 1994 ). for the exotoxin model , balb / c mice were injected with 20 mg d - galactosamine dissolved in 100 μl sterile saline solution ( 9 %) intraperitoneally . they were then given 4 μg of tsst - 1 dissolved in 100 μl sterile saline solution ( 9 %) injected subcutaneously in two sites approximately 0 . 5 cm below each shoulder blade . control groups were injected with either 4 μg tsst - 1 subcutaneously without d - galactosamine , or treated with d - galactosamine alone . a group of d - galactosamine sensitized balb / c mice were also pre - treated i . p . with 700 iu ir - p for 3 days before the treatment of tsst - 1 . lps model ( n = 6 ) for the endotoxin model , balb / c and sjl mice were treated i . p . with 600 μg lps . control groups were treated only with pbs i . p . to test the effect of ir - p , we also pre - treated balb / c and sjl mice with 700 iu for 3 days and then injected with 600 μg of lps . moreover , a group of balb / c mice was also pre - treated with ir - u fractions ( iru1 , ir - u2 , ir - u3 - 5 ), each with the same doses of 200 μg i . p . for 3 days and then injected with 600 μg of lps . in order to test low molecular weight fraction , we tested ir - u / lmdf ( which also contains ir - u5 [& lt ; 10 kda ] fraction ), ir - p3 ( obtained by method 3 ), ir - a and ir - a3 ( obtained by method 3 ), and their fractions obtained by method 4 for anti - shock activity . in addition , we also tested three fractions from peptide column ( f1 - 3 ) for anti - shock activity ( methods are shown elsewhere in this document ). we also treated balb / c mice with 700 iu ir - p twice i . p . after 1 and 2 hours of injection with lps , respectively . semi - quantitative sickness measurements : mice were scored for sickness levels using the following measurement scheme : 1 percolated fur , but no detectable behavior differences from normal mice . 2 percolated fur , huddle reflex , responds to stimuli ( such as tap on cage ), just as active during handling as healthy mouse . 3 slower response to tap on cage , passive or docile when handled , but still curious when alone in a new setting . 4 lack of curiosity , little or no response to stimuli , quite immobile . 5 labored breathing , inability or slow to self - right after being rolled onto back ( moribund , sacrificed ). wbc and platelets counts : 100 μl of blood was obtained from 2 randomly selected mice per group utilizing a tail bleed method at the 24 hour time - point from tsst - 1 model . whole blood was collected in edta tubes and analyzed in an automated blood hematology analyzer . animals and treatments : 8 - 10 - wk - old female balb / c mice obtained from harlan were used in this study . animals were killed and livers and spleens were excised for further study as indicated below . mouse handling and experimental procedures were conducted in accordance with the american association of accreditation of laboratory animal care guidelines for animal care and use . injection protocols : lps from escherichia coli ( sigma chemical co ) was administered intraperitoneally at 150 mg / kg for the high - dose lps shock model . to test the effect of ir , mice were pre - treated with ir - p ( pregnyl ; organon ; oss , the netherlands ) and its fractions , ir - pi , ir - p2 , ir - p3 and with ir - a3 ( apl ; wyeth ayerst , philadelphia , pa ., usa ) for 3 days ( t =− 3 , t =− 2 , t =− 1 ) each with the same dose of 200 mg i . p . and then lps was injected at t = 0 h . a group of mice was also treated with ir - p or dexamethasone twice i . p . after 1 and 2 hours of injection with lps , respectively . blood test : from each group blood was withdrawn by a tail bleed of 3 mice at each time point ( t =− 72 h , − 1 h and 48 h ) and pooled for routine measurement of leukocytes , platelets , plasma enzymes ldh , alat and asat . mice were then sacrificed and liver and spleens were excised and studied as indicated below . animals and treatment : in order to determine whether ir - p is able to protect allograft , we treated balb / c mice ( n = 5 ) with 600 i . u . ir - p / day i . p . or pbs for two days . on day 3 , tail skin of c57bl / 6 donors was grafted to the dorsal thorax of ir - p or pbs treated balb / c recipients using a modification of the method of billingham and medawar . grafts were considered rejected when no viable donor skin / hair was detectable . after transplantation , ir - p pre - treated balb / c recipients were treated for an additional two days . induction of eae . 8 - 12 week - old female sjl mice ( n = 5 ) were immunized s . c . with 50 ml ( 0 . 5 mg / ml ) of plp - peptide at four different places ( t = 0 ). after 24 hours , 10 10 bordetella pertussis was injected i . v . in tail . subsequently , after 72 ( t = 3 ) hours mice were again immunized with bordetella pertussis . from day 7 , mice were weighted and clinical signs of eae were graded daily on a scale of 0 to 5 as follows : ir treatment : a group of mice were also treated from day 8 with 600 i . u . ir - p / day i . p . three times a week for two weeks , while control group was treated with same volume of pbs . streptozotocin model : streptozotocin injections . for multiple dose streptozotocin ( md - stz ) model , 25 mg / kg of stz ( sigma ) were dissolved in citrate buffer ( ph 4 . 2 ) and injected intraperitoneally within 5 min of solubilization as described previously . male mice were injected on 5 consecutive days ( experiment day 1 through day 5 ) at 6 - 9 weeks of age . after 5 consecutive days of stz , mice were treated with ir - p ( 600 i . u . i . p .) ( n = 5 ) or citrate buffer ( n = 5 ) four times a week for three weeks . for high dose streptozotocin ( hd - stz ) model , hyperglycemia was induced in mice by a single intraperitoneal injection of streptozotocin ( 160 mg / kg ). mice in the control group received a corresponding volume of citrate buffer alone . hcg fraction preparation and characterization . gel filtration of the solution of 1 or 2 vials of commercial grade hcg - pregnyl ( 5 , 000 iu / vial ) was performed on a pharmacia fplc system equipped with a superdex 75 column ( hr 5 / 30 ) ( pharmacia , sweden ) in pbs . sample load volume was 1 ml . the flow rate was 0 . 5 ml / min for 45 min followed . the 1 minute flow rate of 0 . 2 ml / min was implemented because of the viscosity of the commercial grade hcg solution which has a high lactose content . hcg and a very low amount hcg core fragment were present in the relatively purified pregnyl preparation of hcg and their positions were used as internal size markers . hcg eluted as 78 kda molecules and the hcg β - core eluted as 19 kda molecules on gel filtration . there were 1 - 5 fractions collected whereby fraction 1 - 2 contained hcg and fraction 5 contained the hcg (− core fragments ). fraction 1 - 2 and fraction 3 - 5 were tested for anti - diabetic effect by treating in vitro total spleen cells of 20 - wk - old nod and transferring them into nod . scid . in this way , human recombinant hcg , α - hcg , and β - hcg ( sigma , st . louis , mo . usa ) were also tested . gel permeation of ir - u and ir - p : fig1 represents a fplc chromatogram of 50 μl of undiluted ir - u sample . the running buffer was pbs . the chromatogram indicates 4 major peaks at 70 , 37 , 15 and 10 kda . to identify these peaks , a sample of 500 μl ( containing 5000 iu ) of ir - p ( pregnyl ) was applied on the same column under similar running conditions . the profile obtained ( fig1 ) displayed also these 4 peaks although the ratios were different . peak fraction 2 represents ( alpha / beta ) heterodimer hcg ( 37 kda ) while fraction 3 represents individual chains , homodimers of these chains or beta - core residual chains and other molecules ( 15 - 30 kda ). from these results we concluded that first trimester urine contains the same 4 major protein fractions that are also present in commercial hcg preparation , as could be expected . we named them as ( ir - p1 , ir - p2 , 1 r3 - 5 [ pooled ]), ( ir - u1 , ir - u2 , ir - u3 - 5 [ pooled ]). fraction 5 contains no protein or protein less than 10 kda weight . in addition , overlapping fractions 2 and 3 were seen in ir - p as well as in ir - u which suggested covalent binding of protein species present in these fractions . further separation of the overlapping fractions 2 and 3 , was done on a 1 ml mono q hr 5 / 5 anion exchange column . fig1 represents a chromatogram of 50 μl of ir - u sample diluted 1 : 20 in pbs . two major protein peaks eluted at 43 % and 55 % buffer b , but were not separated suggesting covalent binding between these protein species . even using a discontinuous elution gradient with a 50 % buffer b hold did not result in separation of these peaks ( data not shown ). therefore , we concluded that ion exchange chromatography could not be used for further purification due to covalent binding of protein species present in the urine sample . to reduce the presumed covalent binding between the important protein species present in the ir - u sample , we treated the sample with 60 mm 2 - mercaptoethanol for 3 min at 100 ° c . and the sample was then applied to the superdex 75 column under identical conditions . fig1 represents the elution profile showing that peak 1 ( 70 kda ) remains present ( see also fig1 - 17 ), fraction 2 ( representing hcg , 37 kda ) nearly disappeared and resulted in two new peaks of a low molecular weight (& lt ; 10 kda ). peak 3 remained present and , therefore , is likely to contain isolated beta - core and monomeric proteins is excess . peak 4 ( 10 kda ) also disappeared due to the reducing treatment . a similar reducing treatment was applied to a sample of ir - p ( pregnyl ). like the profile of the ir - u sample also treated , hcg ( fig1 ) displayed the decrease in peak 2 , increase in peak 3 , while a new protein peak appeared between peaks 1 and 2 . moreover , an increase in the breakdown product peak (& lt ; 10 kda ) was apparent . total spleen cells were recovered from 9 - wk - old nod and stimulated in vitro in rpmi + supplemented with 10 % fbs with coated anti - cd3 ( 145 - 2c11 ; 25 mg / ml ) and il - 2 ( 50 u / ml ) along with 300 iu / ml ir - p , 100 mg / ml ir - u3 - 5 or iru / lmdf . plates were then incubated at 37 ° c . in 5 % of co 2 in air for 48 hrs . after 48 hrs , cells were twice washed with pbs and 20 × 10 6 cells were i . p . transferred into an 8 - wk - old nod . scid mouse . in vivo anti - diabetic effect of ir : four 15 - wk - old nod female mice ( n = 4 ) were treated with pbs , 300 iu pregnyl , or 600 iu pregnyl intraperitoneally , 3 times a week for four weeks . after the treatment , all mice in the pbs group were diabetic ( blood glucose & gt ; 33 mmol / l ), they lost weight and looked uncomfortable , while the 300 iu pregnyl and 600 iu pregnyl groups remained free of disease . their blood glucose levels never exceeded 6 mmol / l and they looked very healthy ( fig1 and 3 ). in order to assess possible infiltrations and intact insulin - producing cells in the pancreas , mice from the pbs and the 600 iu pregnyl groups were killed after treatment and entire pancreata were removed for immunohistochemistry for insulin . pancreas sections from the pbs group showed many infiltrating cells in the pancreas and these cells penetrated the islets . there were also a large number of b - lymphocytes and t - lymphocytes present in the pancreata of the pbs - group . this finding was consistent with our other finding of an elevated ratio of splenic cd8 / cd4 cells due to a selective reduction in the number of cd4 + cells and a decrease in the number of b lymphocytes in the spleen of these mice ( data not shown ). in the 600 iu pregnyl group , pancreata were free of infiltration and , surprisingly , a number of new insulin - producing islets were seen . there was also a decrease in the number of b - lymphocytes and t - lymphocytes in the pancreas , which was consistent with normal levels of the cd8 / cd4 ratio and the number of b - lymphocytes in the spleens of these mice . mice from the 300 iu pregnyl group were kept alive until the age of 28 weeks . they appeared healthy , did not lose their weight and never had blood glucose levels above 8 mmol / l ( fig1 and 3 ). immunohistochemistry for the presence of insulin was also performed . there were still infiltrating cells present and some insulin - producing islets in the pancreas . these mice were treated for four weeks with pregnyl along with the 600 iu pregnyl group and from wk 20 until 28 they were left untreated . in order to determine whether the spleen cells of treated and untreated nod mice still had the potential to induce diabetes in nod . scid , we transferred spleen cells from the pbs and the 600 iu pregnyl group into nod . scid mice . 22 days after transferring , the pbs nod . scid group were positive for diabetes and within a week they reached a blood glucose level above 33 mmol / l , while nod . scid mice receiving spleen cells from the 600 iu pregnyl group remained normal ( blood glucose & lt ; 7 mmol / l ). 7 weeks after transferring , the pbs group looked very uncomfortable ( fig2 ), while the 600 iu pregnyl nod . scid group still had blood glucose levels less than 9 mmol / l and remained healthy . mice from both groups were killed at this time . in vitro restimulation . since high levels of ifn - γ , il - 1 , and tnf - alpha were reported during the course of disease in nod and this cytokine profile fits in a selective activation of the th1 subset , we tested in vitro the effect of pregnyl on cytokine production by total spleen cells and purified cd4 + cells from 20 - wk - old nod female mice . in order to assess whether the anti - diabetic effect resides in hcg or in one of its subunits or in other factors contained in the preparation used , we also tested the effect of different fractions obtained by gel permeation chromatography from pregnyl ( fig1 ) and human recombinant hcg and its subunits on cytokine production . the effect of these fractions were also tested in vivo on blood glucose levels in reconstituted nod . scid mice . we observed a strong inhibition of ifn - γ production by spleen cells obtained from mice treated with 50 - 600 iu / ml of pregnyl , f3 - 5 ( 58 - 15 kda ) and to a lesser extent with human recombinant - βcg ( fig4 - 6 ). there was only a moderate increase in ifn - γ production splenocytes from mice treated with 800 iu / ml pregnyl . a similar pattern was observed when analyzing il - 4 production ( fig5 ). in addition , a marked inhibition of il - i and tnf - alpha production was observed in stimulated splenocytes from mice treated with 300 - 600 iu / ml pregnyl , with a concomitant stimulation of il - 6 and il - 10 production ( data not shown ). furthermore , transfer experiments showed that total spleen cells of 20 - wk - old nod mice treated with f3 - 5 or 600 iu pregnyl can delay or even prevent the onset of diabetes in nod . scid as compared to reconstitution with pbs treated nod cells ( fig7 ). however , no significant effect was observed with f1 - 2 ( 80 - 70 kda ) on the onset of diabetes in nod . scid mice . in order to test whether pregnyl also has an effect on th2 type mice , we treated balb / c mice ( n = 5 ) with 300 iu pregnyl i . p . for four days and with pbs ( n = 5 ). after isolating cd4 + cells from spleens , we stimulated them with anti - cd3 / il - 2 for 48 hours and the supernatants were collected for the determination of ifn - γ and il - 4 cytokines . we also treated cd4 + cells with different doses of pregnyl . subsequently , the supernatants were collected for cytokine analyses . there was a marked inhibition of ifn - γ and a concomitant stimulation of il - 4 found in cd4 + cells stimulated with anti - cd3 / il - 2 only ( th1 -& gt ; th2 ), while the inverse was seen in cd4 + cells treated in vitro with different doses of pregnyl ( th2 -& gt ; th1 ). in order to test the anti - diabetic activity of ir - u / lmdf (& lt ; 5 kda ), we treated diabetogenic cells in vitro with this fraction and with pbs ( control ). transferring of these cells into nod . scid mice revealed that reconstituted nod . scid mice with ir - u / lmdf - treated cells had delayed onset of diabetes as compared to the control group ( n = 3 ). to determine the effect of ir on the potential of cd4 + cells to differentiate into th1 cytokine - producing effector cells , the th polarization assay was performed in the presence or absence of ir . we also tested recombinant hcg ( rhcg ) and beta - hcg in this th polarization assay . a strong inhibition of ifn - gamma was found with ir - p and ir - u / lmdf on cd4 + cells polarizing towards the th1 phenotype ( fig2 ). there was only a moderate inhibition of ifn - gamma production observed with recombinant beta - hcg and no effect was seen with recombinant hcg ( fig2 ). to determine whether ir - p3 needed an additional factor , such as hcg , to exert its full activity , we also treated nod mice with ir - p , its fraction ir - p3 , rhcg and ir - p3 in combination with rhcg and then th1 polarization was performed . fig6 shows that ir - p inhibited the production of ifn - gamma in the th1 polarization assay and thereby inhibited the outgrowth of th1 cells under th1 polarizing conditions . there was moderate inhibition of the th1 polarization found with ir - p3 and rhcg alone , while the outgrowth of th1 cells was completely blocked with the combination of rhcg and ir - p3 ( fig6 ). we also stimulated spleen cells from these ir treated mice with anti - cd3 and then at different time points , ifn - gamma and il - 10 production was measured . fig6 shows that in vivo treatment with ir - p , and its fractions ir - p1 , ir - p2 , inhibited the in vitro anti - cd3 stimulated ifn - gamma production , while a moderate increase in ifn - gamma production was found with rhcg and ir - p3 . in addition , fraction ir - p3 in combination with rhcg was able to inhibit the production of ifn - gamma ( fig6 ). we also measured anti - cd3 stimulated il - 10 production ( t = 48 ) in splenocyte cultures of these in vivo treated mice . fig6 shows that all fractions ( ir - p , ir - p1 , ir - p2 , ir - p3 ) were able to increase the production of il - 10 . since ir and its fraction promote anti - cd3 proliferation of splenocytes in vitro , and in order to know the effect of in vivo treatment with ir on anti - cd3 stimulated proliferation in vitro , we also measured the anti - cd3 stimulated proliferation of splenocytes obtained from these ir treated mice at different time points ( t = 12 , 24 , 48 h ). fig6 shows that anti - cd3 stimulated splenocytes from nod mice treated with ir - p , and ir - p1 have a smaller capacity to proliferate in vitro . furthermore , splenocytes from ir - p3 and rhcg treated mice showed a higher capacity to proliferate as compared to the pbs treated control mice ( ctl ), while ir - p3 , in combination with rhcg , caused the same decrease in proliferation as ir - p . moderate effect was found in the anti - cd3 stimulated proliferation of splenocytes from ir - p2 treated nod mice . as mentioned above , dominant th1 polarization causes a b - cell switch from igm to igg2a production under the influence of massive production of ifn - gamma . therefore , we also measured igg2a production in lps stimulated splenocytes obtained from ir treated nod mice . fig6 shows that lps stimulated splenocytes from ir - p , ir - p1 and ir - p3 treated produced in vitro less igg2a , while moderate inhibition of igg2a was found with ir - p2 . furthermore , rhcg treatment was not able to decrease the production of igg2a while , in combination with ir - p3 , it did ( fig6 ). in order to determine the effect of ir on the maturation of dendritic cells ( dc ) from the bone marrow , we cultured bone marrow cells from 8 - wk - old nod mice for 7 days in the presence of gm - csf . under these conditions , the outgrowth of dc from bone marrow is more then 90 %. when we co - cultured dc in the presence of gm - csf and ir - p for 7 days , we observed that all dc treated with ir were less mature than control dc treated with gm - csf only . this was concluded from the decrease in cell surface markers cd1d , er - mp58 , f4 / 80 , cd14 , and the increase in cd43 , cd95 , cd31 and e - cad ( fig2 ). moreover , no change was observed in cell surface markers er - mp20 / ly6c , mhc i and ii ( fig2 ). in contrast , when dc were cultured with gm - csf for 6 days and at day 7 co - cultured with 300 iu / ml ir - p or 100 mg / ml ( fig3 ) of ir - u / lmdf ( fig3 ) for an additional 24 hrs , the dc became more mature and could function better as apc . this was concluded from the increase in cd1d , cd40 , cd80 , cd86 , cd95 , f4 / 80 , cd11c and mhc ii cell surface markers ( fig3 and 31 ). in order to test whether ir also has an effect on th2 phenotype mice , we tested ir - p and ir - u / lmdf in balb / c mice . after the ir treatment , we isolated cd4 + t - cells in the polarization assay . polarization assays revealed that cd4 + t - cells from ir - p and ir - u / lmdf treated mice have less ability to produce ifn - gamma ( fig3 and 33 ), while these cells produced more il - 4 as compared to cells from pbs - treated mice ( fig3 and 35 ). this suggests that due to the in vivo treatment with ir , t - cells are shifted more towards th2 phenotype . cd4 + t - cells from pbs treated and ir - p mice treated with different doses of ir - p showed an increase in ifn - gamma ( fig3 ) and a decrease in il - 4 ( fig3 ) production , which suggests a shift towards the th1 phenotype . in order to determine whether a shift of cd4 + t - cells towards the th2 phenotype is il - 10 or tgf - beta dependent , we also added anti - il - 10 and anti - tgf - beta in the polarization assays of cd4 + t - cells from ir - p treated mice . this caused an increase of ifn - gamma production under th1 polarization conditions of ir - p treated mice cells and of il - 4 production under th2 polarization conditions supported by anti - il - 10 addition ( fig3 and 39 ) which suggests an involvement of il - 10 in th1 / th2 polarization with ir - p . furthermore , no big differences were seen of il - 4 and ifn - gamma production in th2 and th1 polarization conditions with anti - tgf - beta in vitro treatment ( fig4 and 41 ) between the control and ir - p treated group . this proves that due to the ir treatment , il - 10 and tgf - beta are involved . moreover , purified cd4 + cell from ir - u / lmdf produce more tfg - beta then the cells from control mice ( fig4 ). when anti - il - 10 or anti - il - 6 was added in both cultures , cd4 + cells from control group mice produce more tgf - beta than ir - u / lmdf treated group . this suggests an involvement of il - 6 and il - 10 in tgf - beta production . this is consistent with our data which shows that lps stimulated spleen cells from ir treated mice produce a high level of il - 6 ( fig4 ) as compared to control mice . spleen cells from mice irradiated with uvb also produced more il - 10 and induced suppression of th1 cytokines . lps and anti - cd3 stimulation of spleen cells from these mice revealed they are less capable to proliferate . we also compared the lps and anti - cd3 stimulated proliferation of spleen cells from uvb and ir treated balb / c mice . reduction in lps and anti - cd3 induced proliferation was observed after culture of splenocytes from uvb treated balb / c mice ( fig4 and 47 ), while ir or combined treatment by ir and uvb - irradiation treatment increased the lps and anti - cd3 stimulated proliferation ( fig4 and 47 ). in order to determine whether this change in lps and anti - cd3 stimulated proliferation is il - 10 dependent , we treated il - 10 knockout mice with ir - p or uvb . no change in proliferation pattern was seen in anti - cd3 stimulated spleen cells when uvb - irradiated and ir - p treated balb / c mice were compared ( fig4 ), while the inverse pattern in proliferation was observed in anti - cd3 stimulated lymph node cells as compare to uvb - irradiated balb / c of both groups ( fig4 ). this shows that the decrease in anti - cd3 stimulated proliferation after uvb treatment or increase in proliferation after ir - p treatment of spleen cells is not completely il - 10 dependent , while this is true for anti - cd3 stimulated lymph node cells . when the lps stimulated proliferation of spleen cells was evaluated at 48 hours , we observed an increase of proliferation in the uvb and ir - p treated groups as compared to the control group ( fig5 ), while a decrease in proliferation was observed in both groups at 72 hours of proliferation ( fig5 ). in order to determine the influence of in vivo uvb or ir - p - treatment on the percentage of positive cells for cd4 , cd8 , b220 , m5 / 114 cell surface markers , we performed flow cytometry analysis on lymph node cells and spleen cells . reduction in b220 and m5 / 114 positive cells , and an increase in cd4 and cd8 positive cells was observed in the lymph nodes of ir - p - treated il - 10 knockout mice ( fig5 ), while an increase in cd4 , cd8 , b220 and m5 / 114 positive cells was observed in the spleen ( fig5 ). in the uvb treated group , an increase in cd8 positive cells and a decrease in cd4 , b220 , and m5 / 114 positive cells was seen in lymph nodes ( fig5 ), while no change in cell markers was observed among spleen cells , except for a moderate increase in cd8 positive cells ( fig5 ). in order to determine the effect of ir on the maturity of dendritic cells ( dc ) of the bone marrow , we cultured bone marrow cells from balb / c mice for 7 days in the presence of gm - csf . in this way , the outgrowth of dc from bone marrow is more than 90 %. when we co - cultured these dc in the presence of gm - csf and ir ( ir - p , ir - u , ir - u3 - 5 , ir - u / lmdf ) for 7 days , we observed that all dc treated with ir were less mature than control dc treated with gm - csf only . this was concluded from the decrease in cell surface markers cd1d , cd40 , cd80 , cd86 , er - mp58 , f4 / 80 , e - cad and mhc ii ( fig5 ). moreover , a moderate increase in cd95 was observed ( fig5 ). in contrast , when dc were cultured with gm - csf for 6 days and on day 7 the culture was supplemented with 300 iu / ml ir - p or 100 mg / ml ir - u ( ir - u , ir - u3 - 5 , or ir - u / lmdf ) for an additional 24 hrs , they became more mature and could function better as apc . this was concluded from the increase in cd1d , cd14 , cd40 , cd80 , cd86 , cd95 , er - mp58 , f4 / 80 , rb6 8c5 , e - cad and mhc ii cell surface markers ( fig5 ). in order to test the immunosuppressive activity of ir for instance , for transplantation purposes , we also performed allo - mlr with bm cells from 9 - wk - old female balb / c as mentioned above and cultured with gm - csf ( 20 ng / ml ) and ir ( ir - p , 300 iu / ml ; ir - u , 300 mg / ml ; ir - u3 - 5 , 300 mg / ml ; ir - u / lmdf , 300 mg / ml ) for 7 days . after 7 days these dc were irradiated ( 2 , 000 rad ) and co - cultured in various ratios with splenic cd3 + cells isolated from 9 - wk - old female c57bl6 / ly . t - cell proliferation was measured via [ 3 h ] tdr incorporation during the last 16 hrs in culture . proliferation data shows that ir treated dc in all dc versus t - cell ratios tested are able to suppress proliferation ( fig5 ). lower molecular weight fraction of ir obtained by purification method 2 ( ir - u / lmdf ) had also anti - shock activity ( fig5 ) and mice treated with this fraction remained alive . we also tested all three fractions obtained from superdex ® peptide , ir - p3 , and ir - a3 for anti - shock activity . the method for this activity screening is mentioned elsewhere in this document . our results showed that all three fractions from the superdex ® peptide column and ir - p3 had anti - shock activity , while ir - a3 had low to moderate activity ( data not shown ). fig1 . shows macrosphere gpc 60 å chromatogram of an ir - p sample . three selected areas were fractionated , ir - p1 which elutes apparently with molecular weight of & gt ; 10 kda , ir - p2 which elutes apparently with molecular weight between the 10 kda - 1 kda , and ir - p3 which elutes apparently with molecular weight & lt ; 1 kda . all these activities were tested for at least anti - shock activity and they all had anti - shock activity ( shown elsewhere in this document ). fig1 . shows macrosphere gpc 60 å chromatogram of ir - p and ir - a sample ( 500 iu of each sample was injected with a same injection volume ). the results revealed that ir - a contains large amount of ir - a3 fraction as compared to ir - p3 fraction in the ir - p sample . we have tested the same amount of ir - a and ir - p for their anti - shock activity . the results revealed that ir - a had low to moderate anti - shock activity compared to ir - p ( results not showen ). pooled urine was obtained from pregnant women during the first trimester of their pregnancy . after desalting on a fdc column in an fplc system and employing 50 mm ammonium bicarbonate as the running buffer , the pooled low molecular weight fractions ( lmdf ; & lt ; 5 kda ) were lyophilized . the lmdf sample ( 13 - 17 mg ) was suspended and applied on a bio - gel p - 2 column using water for the elution . the elution profile was segregated into 8 different peaks and the poled fractions were tested for bioactivity in the lps - induced septic shock ( method mentioned elsewhere in document ). based on the inhibition of lps shock , the activity was located in fractions ic (“?”), ii , iii , vi , and vii . these peaks comprised elution volumes between 40 - 45 ml ( peak ic “?”), 45 - 50 ml ( peak iii ), 60 - 65 ml ( peak vi ) and 65 - 70 ml ( peak vii ) ( fig9 ). a sample of ir - p ( pregnyl ) was applied on the macrosphere gpc 60 å column and eluted with ammonium bicarbonate . the third peak fraction ( fig1 ) ( ir - p3 ) was pooled and applied on the bio - gel p - 2 column and eluted with water into various peaks . testing for activity in the lps shock model revealed that the activity was located in the fractions located between the elution time of 7 and 9 hours ( fig9 ). a sample of ir - a ( apl ) was applied on the macrosphere gpc 60 å column and eluted with ammonium bicarbonate . the third peak fraction ( ir - a3 ) was pooled and applied on the bio - gel p - 2 column and eluted with water . testing for activity in the lps shock model revealed that the activity was located in the peaks 2 , 3 and 7 . these peaks comprised elution volumes between 105 - 115 ml ( peak 2 ), 115 - 120 ml ( peak 3 ) and 160 - 180 ml ( peak 7 ) ( fig9 ). survival curve : the most striking results from this experiment are the black and white differences between those animals treated with ir - p prior to tsst - 1 and d - gal treatment versus those that were not ( fig2 ). this is evident in the survival curve obtained from this experiment . while a 4 μg dose of tsst - 1 coupled with d - galactosamine sensitization was 100 % lethal by 32 hours ; animals pre - treated with ir prior to tsst - 1 exposure did not succumb to the effects of lethal toxic shock . lps - treated balb / c mice and sjl mice revealed different sensitivity to lps . 600 μg lps was 100 % lethal by 48 hours and 36 hours in balb / c and sjl , respectively , while ir pre - treated balb / c and sjl mice remained alive . we also pre - treated balb / c mice with ir - u fractions , namely , ir - u1 , ir - u2 and ir - u3 - 5 [ pooled ] and then treated with lps . these experiments showed that ir - u1 and ir - u2 pre - treated mice were very sick by 48 hours and were killed along with lps group . however , mice treated with ir - u3 - 5 remained alive . a group of balb / c mice were treated twice with 700 iu ir - p after the injection of lps . the control group mice ( only lps ) were killed after 48 hours because of their severe sickness . mice treated with ir - p remained alive , except two ( 2 / 6 ) mice were killed at 60 hours time point . illness kinetics : visible signs of sickness were apparent in all of the experimental animals , but the kinetics , and obviously the severity of this sickness were significantly different . ir - p pre - treated balb / c mice group did not exceed the sickness level 2 in tsst - 1 exotoxin model ( fig2 ) and also in lps endotoxin model in addition to ir - u3 - 5 pre - treated mice . ir - p pre - treated sjl mice and ir - p post - treated balb / c mice in lps model did not exceed the sickness level 3 . all mice in both models were killed when they exceeded the sickness level 5 . shock induced weight loss in tsst - 1 : ir pretreatment also resulted in significantly reduced weight loss of survivors of toxic shock . weight loss data from this experiment was combined with that from another experiment which followed identical illness kinetics ( data not shown ), but resulted in two survivors of the 4 ug tsst - 1 & amp ; d - gal without ir pre - treatment group . ( fig1 .) when this weight loss data was statistically analyzed using a 2 - sample t - test ( using minitab statistical software , version 11 . 21 ), significant differences ( p ( ho : μ1 = μ2 )& lt ; 0 . 05 ) in weight loss were observable at 32 and 48 hours despite low n numbers , indicating an even higher possible significance if n were increased : two sample t - test and confidence interval two sample t for weight loss at 32 hours ( group 1 = tsst1 & amp ; d - gal ; group 2 = t & amp ; d with ir pre - treatment ) wbc and platelets counts : white blood cell levels in blood ( fig2 ) were significantly higher in tsst - and d - gal treatment alone ( bar # 2 ) versus wbc counts in regular mice ( bar # 1 ) and ir - p pre - treated mice ( bar # 3 ). this indicates , as expected , a higher level of immune activation in the mice suffering from lethal toxic shock . there is still a normal level of wbc in the ir - p group . such a finding also fits our other results , as this group did not show severe visible signs of illness . blood platelet counts ( fig2 ) were also reduced in tsst - 1 d - gal treated mice . elevated platelet counts were seen in ir - p treated mice . a major goal of transplantation research is the development of strategies to inhibit allograft rejection and , even better , to induce allo - specific tolerance . for this purpose , animal models have been widely used and it has become clear that skin allograft rejection may be one of the most difficult to prevent . mhc - disparate graft loss is inevitable if alloreactivity is not suppressed by immunosuppressive agents . currently , immunosuppressive protocols are based upon the combined use of multiple immunosuppressive agents which may potentially interfere with distinct steps of the rejection process , including antigen recognition , t - cell cytokine production , cytokine activity and t - cell proliferation , macrophages , nk cells and cytotoxic t - cell . in experimental settings , many drugs and monoclonal antibodies ( mab ) have been and are being evaluated for their immunosuppressive capacity . among these are mizorbine , rs - 61443 , 15 - deoxyspergualin , brequinar sodium and mab against lfa - 1 , icam - 1 , cd3 , cd4 and il - 2r . cytokines produced by many cell types , such as t - cells , macrophages and nk cells , may influence the rejection process . because of their central role in graft rejection , cd4 + t - cells and the cytokines they produce have been widely studied in rejection and acceptance of allografts . cd4 + t - lymphocytes can be subdivided into at least two subsets , th1 and th2 cells , based on their cytokine production pattern . th1 cells , which produce il2 , tfn - gamma and tnf - beta , play a role in delayed type hypersensitivity ( dth ) reactions and cellular cytotoxicity , whereas th2 cells , which produce il - 4 , il5 , il - 6 and il - 10 , are effective stimulators of b - cell differentiation and antibody production . these two th subsets can regulate each other &# 39 ; s proliferation and function . while ifn - gamma inhibits th2 cell proliferation and antagonizes il - 4 effects , il - 10 inhibits th1 cytokine production . there are indications for the existence of regulatory t - cells which can also regulate these two subsets . graft rejection is thought to be mediated by th1 cells that may stimulate dth and ctl activity . on the other hand , suppression of alloreactive th1 cells may lead to graft acceptance . immunosuppression may be achieved by neutralizing pro - inflammatory cytokines by administration of anti - cytokine mab or soluble cytokine receptors . alternatively , “ skewing ” of t - cell differentiation towards one of the th subsets can be achieved by varying the cytokine environment . for example , ifn - gamma ( th1 , nk cells ) and il - 12 ( macrophages , b - cells ) promote th1 cell differentiation , whereas il - 4 ( th2 ) enhances th2 cell development . changing the in vivo cytokine environment by anti - cytokine mab or cytokines , may have a similar effect . moreover , induction of regulatory cells like th3 and tr1 and like dc1 and dc2 , also reduce transplant rejection and induce tolerance for graft . results : treatment of balb / c recipients with ir - p prolonged c57bl / 6 skin graft survival as compared to the untreated control group . the control recipients rejected skin graft within 12 days ( fig9 ) while ir - p treated recipients were able to prolonged the graft until 22 days after transplantation ( fig9 ). fig9 and 96 show one such prolonged graft ( picture taken on day 19 ) due to the ir - p treatment and a rejected graft from the control mice . mice treated with pbs only lost weight during the first three weeks ( fig7 ). these mice all had clinical signs of eae of at least 2 and longer duration of the disease , except for one mouse which remained resistant to disease during the whole experiment ( fig7 ). in the ir treated mice group there was less weight loss observed during the experiment ( fig7 ) and two mice were free of disease during the experiment . sick mice in this group had maximum clinical scores of 2 , had short duration of the disease , and recovered faster from eae symptoms then the pbs treated group ( fig8 ). ir treated mice are resistant to lbs - induced shock : to determine the effect of high - dose lps treatment in ir treated mice , balb / c mice ( n = 30 ) were injected intraperitoneally with lps ( 150 mg / kg ) and survival was assessed daily for 5 days . pbs - treated balb / c mice succumbed to shock between days 1 and 2 after high - dose lps injection , with only 10 % of mice alive on day 5 ( fig5 ). in contrast , 100 % of ir - p , or its fractions ir - p1 or ir - p3 , treated mice were alive on day 5 ( p & lt ; 0 . 001 ) ( fig5 ), while groups of ir - p2 , ir - a and dexamethasone treated mice demonstrated around 70 % of survivors ( fig5 ). blood test : major manifestations of systemic response on lps in shock is severe inflammation in organs , leading to organ failure or organ system dysfunction , initially in the liver . therefore , we measured enzymes like alat , asat , ldh1 as well as wbc and platelets . fig5 shows that ir - a , ir - p and its fraction ir - p1 , ir - p3 have all platelets counts within normal range ( 100 - 300 × 10 9 / ml ., while control , ir - p2 and dexamethasone treated mice have platelets counts below normal range . fig6 - 62 show that mice treated with ir - a , ir - p and its fraction ir - p1 , ir - p2 or ir - p3 had relatively low levels of alat , ldh1 and asat enzymes in the plasma as compared to control and dexamethasone treated mice . these enzymes were present in higher concentrations in blood during shock due to organ damage . these results are consistent with our surviving results ( fig5 ). in addition , during shock , low numbers of wbc were found in blood because of their migration to the sites of inflammation . our results in fig6 show that mice treated with ir - a , ir - p and its fractions have moderate to normal levels of wbc at t = 48 hours than control and dexamethasone treated mice , suggesting weaker inflammatory responses in ir treated mice . fig6 shows inhibition of ifn - gamma production in th1 polarization assay with cd4 + cells isolated from nod mice treated with ir - p or ir - p3 in combination with rhcg , while moderate inhibition was found in th1 polarization by rhcg and ir - p3 alone . this shows that treatment with ip - p3 in combination with rhcg gives massive inhibition of th1 outgrowth in nod mice . this suggests that ir - p3 fraction needs rhcg for its maximum inhibition of the th1 subset . fig6 shows inhibition of ifn - gamma production in anti - cd3 stimulated spleen cells obtained from nod mice treated with ir - p , ir - p1 , ir - p2 or with ip - p3 in combination with rhcg as compared to pbs treated mice . rhcg and ir - p3 separately did not have the same effect as in combination . this suggests again that ir - p3 fraction needs rhcg for its ifn - gamma inhibition . fig6 shows anti - cd3 stimulated proliferation at different time points ( t = 12 , 24 , 48 h ) of spleen cells obtained from nod mice treated with ir - p , its fractions , rhcg , or ir - p3 in combination with rhcg . again , the results are consistent with the previous ifn - gamma inhibition ( fig6 ). here , the ir - 23 fraction is also needed rhcg for its inhibitory effect on anti - cd3 induced proliferation of spleen cells from in vivo treated nod mice . fig6 shows that ir - p and its fractions promote il - 10 production of anti - cd3 stimulated spleen cells from treated nod mice as compared to pbs treated mice . fig6 shows that igg2a production is not inhibited by in vivo treatment of nod mice with ir - p2 or rhcg , while ir - p , ir - p1 , ir - p3 and ir - p3 in combination with rhcg did inhibit the igg2a production . since , ir - p3 in combination with rhcg has the same characteristics as ir - p , it is thinkable that this combination can also be used for the induction of pregnancy , ivf , prevention of abortions or related problems . the determining event in the pathogenesis of diabetes i is the destruction of insulin - producing pancreatic beta cells . there is strong evidence that the progressive reduction of the beta - cell mass is the result of a chronic auto - immune reaction . during this process , islet - infiltrating immune cells , islet capillary endothelial cells and the beta cell itself are able to release cytotoxic mediators . cytokines , and in particular nitric oxide ( no ), are potent beta - cell toxic effector molecules . the reactive radical no mediates its deleterious effect mainly through the induction of widespread dna strand breaks . this initial damage presumably triggers a chain of events terminating in the death of the beta cell . diabetes induced in rodents by the beta - cell toxin streptozotocin ( sz ) has been used extensively as an animal model to study the mechanisms involved in the destruction of pancreatic beta cells . sz is taken up by the pancreatic beta cell through the glucose transporter glut - 2 . this substance decomposes intracellularly , and causes damage to dna either by alkylation or by the generation of no . the appearance of dna strand breaks leads to the activation of the abundant nuclear enzyme poly ( adp - ribose ) polymerase ( parp ), which synthesizes large amounts of the ( adp - ribose ) polymer , using nad + as a substrate . as a consequence of parp activation , the cellular concentration of nad + may then decrease to very low levels , which is thought to abrogate the ability of the cell to generate sufficient energy and , finally , to lead to cell death . reactive radicals also play an important role in the pathogenesis of many diseases like nephropathy , obstructive nephropathy , acute and chronic renal allograft rejection , auto - immune diseases ( like sle , rheumatoid arthritis , diabetes , ms ), aids , diseases related to angiogenesis , atherosclerosis , thrombosis and type ii diabetes mellitus . for instance , recently increased oxidative damage to dna bases has been shown in patients with type ii diabetes mellitus which contribute to the pathogenesis and complications of diabetes . we tested whether ir also has the capacity to delay the induction of stz - induced diabetes and thus also has an effect on cellular reactive radical forming and protection . in hd - stz models , the induction of diabetes is due to direct effect on beta cells of pancreatic tissue by inducing activation of parp . consequently , decrease of nad + and abrogation of the ability of the cell to generate sufficient energy finally leads to the cell death . this suggests that there is not any immunological component involved in this process . in contrast , in the md - stz model , strong immunological components are present . fig6 and 70 show that ir - p treatment is able to delay the induction of diabetes in both models . the mechanism behind this delay is probably of a different nature . the immune system has a remarkable capacity to maintain a state of equilibrium even as it responds to a diverse array of microbes and despite its constant exposure to self - antigens . after a productive response to a foreign antigen , the immune system is returned to a state of rest , so that the numbers and functional status of lymphocytes are reset at roughly the pre - immunization level . this process is called homeostasis , and it allows the immune system to respond effectively to a new antigenic challenge . the size and the repertoire of the pre - immune lymphocyte subpopulations are also closely regulated , as new emigrants from the generative lymphoid organs compete for “ space ” with resident t - cells . lymphocytes with receptors capable of recognizing self - antigens are generated constantly , yet normal individuals maintain a state of unresponsiveness to their own antigens , called self - tolerance . in auto - immune diseases , the immune system inappropriately recognizes “ self ,” which leads to a pathologic humoral and / or cell - mediated immune reaction . in a normal , nonauto - immune state , self - reactive lymphocytes are deleted or made unresponsive to peripheral self ligands . populations of potentially autoreactive cells can be demonstrated , yet appear not to give rise to apathogenic auto - immune reactions to their ligands . a picture of auto - immune disease is emerging wherein these autoreactive cells are activated through molecular mimicry , given that t - cell receptor ( tcr ) interactions can be degenerated and t - cells can be activated by a diversity of ligands ( 1 , 2 ). there is evidence that under appropriate conditions , activation of autoreactive t - cells is facilitated by the induction of cytokines and the up - regulation of particular co - stimulatory molecules ( e . g ., cd80 / cd86 and cd40 ), leading to autoimmunity . when the immune system mistakes self tissues for nonself and mounts an inappropriate attack , the result is an auto - immune disease . there are many different auto - immune diseases . some examples are wegener &# 39 ; s granulomatosis , multiple sclerosis , type 1 diabetes mellitus , and rheumatoid arthritis . moreover , infection can also induce immune responses that lead to the induction of immune diseases , while the infection itself is not dangerous to the host . for example , the role of tubercle bacilli in tuberculosis , in which the immune system reacts top aggressively on tubercle bacilli , resulting in inflammatory illness and tissue destruction due to its own immune response . the same is also true , for example , for lepta tuberculoid . auto - immune diseases can each affect the body in different ways . for instance , the auto - immune reaction is directed against the brain in multiple sclerosis and the gut in crohn &# 39 ; s disease . in other auto - immune diseases , such as sjögren disease and systemic lupus erythematosis ( lupus ; sle ), affected tissues and organs may vary among individual with the same disease . many auto - immune diseases are rare . as a group , however , they afflict many people in western societies . many auto - immune diseases are more prevalent in women than in men . the sexual dimorphism covers a broad range of auto - immune disorders , ranging from organ - specific ( such as graves &# 39 ; s disease ) to generalized such as sle . in ms , there is a female - to - male preponderance approaching 2 : 1 to 3 : 1 . the reasons for the sex bias in ms and other auto - immune diseases are unclear but many include factors as sex - related differences in immune responsiveness to infection , sex steroid effects , and sex - linked genetic factors . it is recognized that ms , sjögrens , sle , and ra are different diseases and probably differ in etiology . however , the common link is the overwhelming prevalence of these diseases in women . considering that each of these diseases is auto - immune , the effects of sex hormones and gender may be similar , making a comparison of these diseases useful . auto - immune diseases strike women , particularly during their working age and their childbearing years . however , the clinical course of these diseases are surprisingly less severe , or even remission is seen , during pregnancy . during pregnancy , women undergo immunologic changes consistent with weakening of cell - mediated immunity ( th1 responses ) and strengthening certain components of humoral immunity ( th2 responses ). this th2 - biased like - response by the maternal system during pregnancy introduces a status of temporary immunosuppression or immuno - modulation , which results in suppression of maternal rejection responses against the fetus but maintain , or even increase , her resistance to infection . in addition , decreased susceptibility to some auto - immune diseases , especially th1 - cell - mediated immune disorders have also been observed . for instance , approximately 77 % of women with rheumatoid arthritis ( predominantly a th1 - cell - mediated auto - immune disorder ) experience a temporary remission of their symptoms during gestation , which are apparent from the first trimester in the majority of cases . hence , clinical improvement during gestation in th1 - cell mediated auto - immune diseases should probably be related to physiologic immune changes during the early pregnancy . since our ir is able to inhibit the development of auto - immune disease in animal models such as nod and eae , we treated few patients with immune diseases . all patients were treated because of refractory disease and after informed consent . wegener &# 39 ; s granulomatosis is an auto - immune vascular disease that can affect both men and women and although it is more common in persons in their middle age , it can affect persons of any age . the initial manifestations generally involve the upper and lower respiratory tract , with a chronic , progressive inflammation . the inflammation may form lumps or granulomas in the tissues or in the skin . it may progress into generalized inflammation of the blood vessels ( vasculitis ) and kidneys ( glomerulonephritis ). a restricted form of the disease that does not involve the kidneys may occur . the vasculitis is the result of an auto - immune reaction in the wall of small and medium - sized blood vessels . chronic vasculitis causes a narrowing of the inside of the blood vessel and can result in obstruction of the flow of blood to the tissues . this situation may cause damage to the tissues ( necrosis ). auto - immune diseases occur when these reactions inexplicably take place against the body &# 39 ; s own cells and tissues by producing self - reactive antibodies . in wegener &# 39 ; s granulomatosis , an autoantibody is directed toward components in the cytoplasm of certain white cells . the cause of wegener &# 39 ; s granulomatosis remains unknown . though the disease resembles an infectious process , no causative agent has been isolated . anti - neutrophilic cytoplasmic antibody ( anca ) is found in the majority of patients , and its level appears to correlate with the disease activity . wegener &# 39 ; s granulomatosis is a quite rare disease , especially in europe and in dark people ( africans , south americans , asian people ). the exact number of patients is not known , but a rough estimate is two new cases per million americans per year , or about 500 new cases diagnosed every year in the united states . the disease can occur at any age ; however , it has its peak in the 4th or 5th decade of life it affects males and females equally 85 % of the patients are above age 19 the mean age of patients is 41 ( current age range is 5 - 91 ) 97 % of all patients are caucasian , 2 % black and 1 % are of another race the symptoms of wegener &# 39 ; s granulomatosis and the severity of these symptoms vary from one patient to another , although most patients first notice symptoms in the upper respiratory tract . a common manifestation of the disease is a persistent rhinorrhea (“ runny nose ”) or other cold - like symptoms that do not respond to standard treatment , and that become progressively worse . rhinorrhea can result from sinus drainage and can cause upper respiratory obstruction and pain . complaints include discharge from the nose , sinusitis , nasal membrane ulcerations and crusting , inflammation of the ear with hearing problems , cough , coughing of blood and pleuritis ( inflammation of the lining of the lung ). other initial symptoms include fever , fatigue , malaise ( feeling ill ), loss of appetite , weight loss , joint pain , night sweats , changes in the color of urine , weakness . most wegener &# 39 ; s patients do not experience all of the above symptoms , and the severity of the disease is different with each patient . fever is often present , sometimes resulting from bacterial infection in the sinuses . one third of the patients may be without , symptoms at the onset of the disease . laboratory tests are not specific for wegener &# 39 ; s granulomatosis and only suggest that the patient has an inflammatory disease . blood tests often show anemia ( low red blood cell count ) and other changes in the blood . chest x - rays and kidney biopsy are important tools used in diagnosing wegener &# 39 ; s granulomatosis . for effective treatment , early diagnosis is critical . asymptomatic patients can be diagnosed by anca blood tests and ct scans of sinuses and lungs . it takes 5 - 15 months , on average , to make a diagnosis of wegener &# 39 ; s granulomatosis . 40 % of all diagnoses are made within less than 3 months , 10 % within 5 - 15 years . erythrocyte sedimentation rate is generally elevated complete blood count will often shows anemia , elevated white counts , elevated platelet counts urinalysis is often considered as a screening test for kidney involvement 24 - hour urine collection is used in certain patients to assess kidney function c - anca is characteristic , measuring proteinase - 3 antibodies our initial results of treatment of patient with ir - p . the patient was treated because of refractory disease and after informed consent . case : a 34 year old male patient known with relapsing wegener &# 39 ; s granulomatosis for 5 years . this patient was treated with high dosage steroids , cyclosporine ( 5 mg / kg ) and cyclophosphamide ( 1 - 2 mg / kg ). because of progressive disease in july 1998 he was treated with ir ( pregnyl ), 5000 i . u , s . c . daily . fig8 shows that before ir treatment the patient was immuno - compromised due to the high doses of steroids . after ir treatment , the levels of t - lymphocytes ( cd4 , cd8 ) were increased and within normal range , except for b - cells . we also measured cytokines in lps and pma / ca stimulated pbmc obtained from patient during the ir treatment . we observed that lps stimulated pbmc produced more tnf - alpha , il - 1o and il - 12 during treatment ( fig8 a ), while pma / ca stimulated pbmc produced less ifn - gamma ( fig8 b ). accordingly , we show that ir treatment increases the production of anti - inflammatory cytokines ( il - 10 , tnf ) while it decreases the production of inflammatory cytokine ( ifn - gamma ). this is consistent with our clinical observation that during 3 months of treatment , no further progression was observed as measured by sinal inflammation activity . these results suggest a beneficial effect of ir - p . definition : a systemic connective tissue disease , which occurs through t - cell mediated inflammation causing destruction of muscle fibers . other possible causes of these syndromes include complement activation , infection , drugs , stress , vaccines . it can affect people at any age , but most commonly occurs in those between 50 to 70 years old , or in children between 5 to 15 years old . it affects women twice as often as men . muscle weakness may appear suddenly or occur slowly over weeks or months . there may be difficulty with raising the arms over the head , rising from a sitting position , or climbing stairs . the voice may be affected by weakness of the larynx . joint pain , inflammation of the heart , and pulmonary ( lung ) disease may occur . a similar condition , called dermatomyositis , is evident when a dusky , red rash appears over the face , neck , shoulders , upper chest , and back . a malignancy may be associated with this disorder . the incidence of polymyositis is 5 out of 10 , 000 people . case : a 50 year old woman who suffered for two years from systemic sclerosis with an active polymyositis component . she was treated with dapsone , steroids , methotrexate and cylosporine . because of refractory myositis as measured by the creatin phosphate level , she was treated for three months with a combination of prednisone , zyrtec and pregnyl 5000 l . u ., s . c . during treatment , the cpk level dropped from 1100 to 750 . this reflects a decrease in disease activity . fig8 shows that due to the ir - p treatment the number of lymphocytes , t - cells ( cd4 , cd8 ) and b - cells were decreased which indicates the down - regulation of the hyperactive immune system due to the treatment . this is also consistent with our cytokine data ( fig8 ) which shows inhibition of lps stimulated il - 12 and tnf - alpha by pbmc . moreover , there was an increase in il - 10 production , during the treatment , which is an anti - inflammatory cytokine ( fig8 ). in addition , the elevated cpk and liver enzymes ( asat , alat ) were also decreased ( fig8 and 85 ). this all reflects a decrease in the disease activity . diabetes mellitus is a chronic disorder characterized by impaired metabolism of glucose and other energy - yielding fuels , as well as the late development of vascular and neuropathic complications . diabetes mellitus consists of a group of disorders involving distinct pathogenic mechanisms with hyperglycemia as the common denominator . regardless of cause , the disease is associated with insulin deficiency , which may be total , partial , or relative when viewed in the context of co - existing insulin resistance . lack of insulin plays a primary role in the metabolic derangements linked to diabetes and hyperglycemia , in turn , plays a key role in the complications of the disease . in the united states , diabetes mellitus is the fourth most common reason for patient contact with a physician and is a major cause of premature disability and mortality . it is the leading cause of blindness among working - age people , of end - stage renal disease , and of nontraumatic limb amputations . it increases the risk of cardiac , cerebral , and peripheral morbidity and mortality . on the bright side , recent data indicate that most of the debilitating complications of the disease can be prevented or delayed by prospective treatment of hyperglycemia and cardiovascular risk factors . insulin - dependent diabetes mellitus ( iddm ) is one of the clinically defined types of diabetes and develops predominantly in children and young adults , but may appear in all age groups . the major genetic susceptibility to iddm is linked to the hla complex on chromosome 6 . these genetic backgrounds interact with environmental factors ( possibly certain viruses , foods and climate ) to initiate the immune - mediated process that leads to beta cell destruction . while non - insulin dependent diabetes ( niddm ), which is another clinically defined type of diabetes , is the most common form of diabetes , the prevalence of niddm varies enormously from population to population . the greatest rates have been found in the pima indians . the major environmental factors identified as contributing to this form of diabetes are obesity and reduced physical activity . niddm shows strong familial aggregation in all populations and is clearly the result of an interaction between genetic susceptibility and environmental factors . before niddm develops , insulin concentrations are high for the degree of glycemia and of obesity , reflecting the presence of insulin resistance . as insulin resistance worsens , glucose levels increase , with the appearance of glucose intolerance and , finally , of niddm , when insulin response cannot compensate for insulin resistance . since our preliminary mice data shows that ir has the ability to shift th1 phenotype cytokines towards th2 phenotype and ir is also able to inhibit diabetes in nod mice , we postulated that it should also have positive clinical effects in human immune diseases like diabetes . case : patient is a 21 year old male suffering from diabetes mellitus since 3 months . he was treated with insulin ( actrapid and insultard ). high level of anti - islet cell antibodies was in his blood . he was treated with pregnyl 5000 i . u . s . c . for three months . during his treatment , the insulin needed to maintain euglycemia decreased as shown in fig8 . after withdrawal of pregnyl , his insulin need raised again ( fig8 ). in this patient with the new onset of diabetes mellitus , the insulin need dropped significantly during treatment with ir - p and also improvement of glucose control was found , supported by a decrease in glycosylated hbalc level during ir - p treatment ( fig8 ) and decrease in inflammatory cytokines ( il12 , tnf - alpha , ifn - gamma ) produced by lps stimulated pbmc ( fig8 ). furthermore , increase in il - 10 ( anti - inflammatory cytokine ) was also observed during the treatment ( fig8 ). accordingly , this suggests an improvement of the islet cell function and , eventually , also better glucose regulation . multiple sclerosis ( ms ) is a disorder of unknown cause , defined clinically by characteristic symptoms , signs and progression , and patholologically by scattered areas of inflammation and demyelination affecting the brain , optic nerves , and spinal cord . the first symptoms of ms most commonly occur between the ages of 15 and 50 . the cause of ms is unknown , but it is now widely believed that the pathogenesis involves immune - mediated inflammatory demyelination . pathologic examination of the ms brain shows the hallmarks of an immunopathologic process — perivascular infiltration by lymphocytes and monocytes , class ii mhc antigen expression by cells in the lesions , lymphokines and monokines secreted by activated immune cells , and the absence of overt evidence for infection . additional evidence for an auto - immune pathogenesis includes ( 1 ) immunologic abnormalities in blood and cerebrospinal fluid ( csf ) of ms patients , notably selective intrathecal humoral immune activation , lymphocyte subset abnormalities , and a high frequency of activated lymphocytes in blood and csf ; ( 2 ) an association between ms and certain mhc class ii allotypes , ( 3 ) the clinical response of ms patients to immunomodulation tends to improve with immunosuppressive drugs and worsens with interferon - gamma treatment , which stimulates the immune response ; and ( 4 ) striking similarities between ms and experimental auto - immune encephalomyelitis ( eae )— an animal model in which recurrent episodes of inflammatory demyelination can be induced by inoculating susceptible animals with myelin basic protein or proteolipid protein . epidemiologic studies suggest environmental and genetic factors in the etiopathogenesis of ms . the uneven geographic distribution of the disease and the occurrence of several point - source epidemics have suggested environmental factors ; however , intense study over the past 30 years has failed to establish an infectious cause . migration studies have shown that exposure to undefined environmental factors prior to adolescence as required for subsequent development of ms . a genetic influence is well - established by excess concordance in monozygotic compared with dizygotic twins , clustering of ms in families , racial variability in risk , and association with class ii mhc allotypes . in caucasians , the hla class ii haplotype dr15 , dq6 , dw2 appears strongly and consistently associated with an increased risk of ms . the evidence — immunologic , epidemiologic , and genetic — supports the concept that exposure of genetically susceptible individuals to an environmental factor ( s ) during childhood ( perhaps any one of many common viruses ) may lead eventually to immune - mediated inflammatory demyelination . the precise interplay between genetic , environmental and immunologic factors and the nature of the environmental trigger ( s ) remains to be elucidated . we isolated pbmc from ms patients and stimulated these with lps or pma / ca . after 24 hours of culture , supernatants were collected for cytokine analysis ( tgf - beta , il - 10 , ifn - gamma ). ms patient 1 ( in vitro ): there was an increase in production of tgf - beta and il - 10 in lps stimulated pbmc treated with ir - p ( figs . h and i ). no differences were observed in tgf - beta and il - 10 production in cultures stimulated with pma / ca and treated with ir - p ( fig8 and 90 ), while ir - p inhibited the production of ifn gamma in pma / ca stimulated pbmc ( fig9 ). ms patient 2 ( in vitro ): pbmc obtained from patient 2 showed a decreased production of tgf - beta and ifn - gamma in cultures treated with ir - p as compared to tpa / ca stimulation alone , while ir - p treatment increased lps stimulated tgf - beta production ( fig9 and 93 ). il - 10 production was inhibited with ir - p in both lps and tpa / ca stimulated cultures ( fig9 ) the stimulating effect of ir - p on the production of anti - inflammatory cytokines by pbmc from ms patients in vitro and the inhibitory effects on the production of inflammatory cytokines correlated with the beneficial clinical effects of ir - p treatment of sjl mice in which eae was induced ( see elsewhere in this document ). human bronchial epithelial cell line beas 2b ( asthma in vitro data ): diseases characterized by airway inflammation affect a substantial proportion of the population . these diseases include asthma and chronic obstructive pulmonary disease ( copd ). in the european union , copd and asthma , together with pneumonia , are the third most common cause of death . the production of cytokines and growth factors in response to irritants , infectious agents and inflammatory mediators play an important role in the initiation , perpetuation and inhibition of acute and chronic airway inflammation . airway inflammation is associated with excessive production and activity of several mediators and cytokines released by inflammatory and resident t - cells in the airways . now it is clear that the epithelium is not only an important target for the action of mediators of inflammation , but also an active participant in the inflammatory process itself . bronchial epithelial cells are able to recruit inflammatory cells to the airways through the release of chemoattractants , to direct inflammatory cell migration across the epithelium through the expression of cell adhesion molecules , and to regulate the inflammatory activity of other cells through the release of mediators , like cytokines , chemokines , arachidonic acid metabolites and relaxant and contractile factors . bronchial epithelial cells not only form a passive barrier but also play an active role in the immune response . they are able to produce a variety of mediators that may act either pro - or anti - inflammatory . in addition , bronchial epithelial cells may express adhesion molecules for many different t - cell types , thereby contributing to their recruitment . tnf - alpha produced by inflammatory cells present in the air ways can trigger other inflammatory cytokines and chemokines like rantes and il - 6 . it can also down regulate the production of anti - inflammatory cytokines and thereby damage the barrier function of epithelial cells . glucocorticoids inhibit the transcription of most cytokines and chemokines that are relevant in asthma , including il - 6 , rantes , il - 4 . this inhibition is at least partially responsible for the therapeutic effects of glucocorticoids . our results ( fig7 - 73 ) are consistent with these findings , and show that dexamethasone is able to inhibit tnf - alpha induced il - 6 and rantes production in the beas 2b - cell line . ir - p is also able to inhibit the production of tnf - alpha induced inflammatory cytokines . moreover , dexamethasone was able to restore tnf - alpha induced down - regulation of anti - inflammatory tgf - beta cytokine , while ir - p not only restores tgf - beta production but also promotes this anti - inflammatory cytokine further ( fig7 ). in addition , dexamethasone and ir - p were both able to inhibit ifn - gamma induced production of rantes ( fig7 ). tnf - alpha can also induce cell adhesion markers , such as hla - dr and icam - 1 on the surface of epithelial cells which then recruit inflammatory cells . in this way epithelial cells can also function as antigen presenting cells ( apc ). our results show that dexamethasone and ir - p both were able to down - regulate the tnf - alpha induced expression of hla - dr and icam - 1 ( fig7 and 76 ). these results show that ir - p also has the ability to affect the clinical course of diseases characterized by th2 - type cytokine phenotype - like allergy , asthma and particular parasitic diseases . non - obese diabetic ( nod ) mice naturally develop an insulin - dependent diabetes ( iddm ) with remarkable similarity in immunopathology and clinical symptoms to human iddm patients . as a result , nod mice have become a valuable tool for studying the underlying immunobiology of iddm and the complex genetics that control it . through their study , we now know that diabetes is caused by a disbalance in the ratio of the th1 / th2 subsets and consequently , the destruction of insulin producing β - cells . this destruction is coordinated by β - cell antigen - specific cd4 + t - cells that produce proinflammatory cytokines like ifn - γ , tnf - alpha / β , and il - 1 . a growing number of studies has now correlated diabetes ( in mice and in humans ) with a preferential development of th1 - like cells . in contrast , pregnancy is thought to be a selective th2 phenomenon , and surprisingly during pregnancy the severity of many immune - mediated diseases has been seen to reduce . in contrast , gallo et al . have shown that hcg mediated factor ( s ) ( haf ) present in the urine of first trimester pregnancy have an anti - tumor ( and anti - viral ) effect , which is possibly achieved by a direct cytotoxic effect on tumor cells and , according to these authors , not by an immune - mediated response . here , we show an immunoregulator obtainable , for example , from urine of first trimester pregnancy not only affects the above mentioned immune deviation during pregnancy , but also affects the development of diabetes in nod mice . our results show that , for example , pregnyl , a partially purified hcg preparation from urine of first trimester pregnancy , can delay the onset of diabetes , for example in 15 - wk - old nod when treated for only 3 times a week during four weeks . in addition , spleen cells isolated from these treated mice upon transfer have also the potential to delay the onset of diabetes in immuno - compromised nod . scid mice . we fractionated a pregnyl preparation to assess whether this anti - diabetic activity resides in hcg itself , its subunits , β - core ( natural break - down product of β - hcg ) or in unidentified factors ( haf ). it is worth knowing that pregnyl is one of the most purified hcg preparations available and it contains only low amounts of β - core fragments . we found that most of the anti - diabetic activity resided in a fraction without hcg . furthermore , we showed that human recombinant alpha - hcg and β - hcg also had no effect . however , we do not exclude the possibility that hcg can synergize with other factors in diabetes and other immune - mediated diseases . imnmunohistological analysis of the presence of insulin and infiltration in the pancreas of nod mice showed that nod mice treated with 600 iu pregnyl did not reveal a significant infiltrate . moreover , new insulin islets were seen in the pancreas , which shows a possible regeneration process induced by this treatment . as mentioned before , normally at the age of 9 weeks , infiltrating cells penetrate into the islets and the islets become swollen with lymphocytes . in our experiments , the nod mice were 15 - wk - old and the pbs treated control mice had many infiltrating cells and almost no insulin - producing cells at that time in their pancreas . in addition , pbs treated mice had also an elevated ratio of cd8 / cd4 in their spleen and many t - cells in their pancreas . since our treated mice had a normal cd8 / cd4 ratio in their spleen and no infiltration was found in their pancreas , the elevated cd8 / cd4 ratio was due to selective recruitment of cd4 + cells into the pancreas . ifn - γ and tnf - alpha are involved in the recruitment of t - lymphocytes ( rosenberg et al . 1998 ). our results show that treatment of nod mice with 600 iu pregnyl for four weeks had dramatic effects on the morphology and function of their otherwise inflamed pancreas . furthermore , our 300 iu pregnyl nod mice were kept alive until the age of 28 weeks without treatment and remained non - diabetic . the 600 iu pregnyl nod mice were also examined for symptoms of generalized auto - immune diseases , like sjögren &# 39 ; s disease , which were not found . our in vitro experiments with total spleen cells and purified cd4 + cells of nod are consistent with the in vivo data . there was marked inhibition of ifn - γ , il - 1 and tnf - alpha release by spleen cells ( data not shown ) from nod mice treated in vitro with pregnyl , f3 - 5 , and to a lesser extent with human recombinant β - hcg . increase in il - 4 production was also observed implying a shift of th1 to th2 type response with the treatment . however , doses above 800 iu pregnyl caused opposite results and may be due to the presence of a high amount of hcg itself . the immune system is clearly involved in the onset of diabetes . treatment with pregnyl affects the immune system and thereby can reduce the disease activity in nod mice . in order to separate the immune - modulating activity of pregnyl from its beneficial clinical effect , we treated healthy balb / c mice . this strain is generally considered to react upon stimulation with a th2 driven immune response . our results suggest that purified 0d4 + t - cells obtained from pregnyl - treated balb / c mice display a further th2 skewing . the same cells , when restimulated with pregnyl in vitro , showed an enhancement of ifn - γ production and a decrease in il - 4 production . this implies that pregnyl affects different regulatory t - cell subsets upon treatment in vivo versus in vitro . we suggest that treatment in vivo stimulates the outgrowth of a population of presumably cd4 + tr1 cells , characterized by selective production of tgf - 13 and a lower or no production of il - 10 . these cd4 + tr1 cells have been shown ( o &# 39 ; garra et al . 1997 ) in different models of th1 driven diseases including diabetes and ms , to selectively inhibit the activity of th1 cells , thereby also decreasing the disease severity . similarly cd4 + t - cells from pregnyl treated balb / c mice restimulated in vitro with pregnyl showed an increase of th1 cells concomitant with a decrease of th2 cells . this is consistent with a preferential stimulation of the cd4 + th3 cells characterized by a high production of il - 10 and a low production of tgf - β . these regulatory cells are inhibitors of ifn - γ production by th1 cells as well as the outgrowth of th2 type cells . it has also been shown that in nod . scid mice a steady increase of th2 cells is responsible for the less severe hyperglycemia and the different nature of the infiltrates in the pancreatic islets . our results of the 300 iu pregnyl treated nod and our reconstituted nod . scid mice showed a similar slow increase in blood glucose , particularly in nod . scid , and a different nature of the infiltrates as compared to pbs - treated nod . in nod mice , the activity of pregnyl might well be mediated with the induction of th3 cells inhibiting both th1 and th2 cells . these th3 cells may suppress the disease activity for prolonged periods of time at the very least . in nod . scid mice , having no functional t - cells , reconstitution with pregnyl - treated spleen cells is mediated with selective induction of th1 cells , thereby inhibiting the th1 subset only . after prolonged periods , the steady outgrowth of diabetogenic th2 cells is responsible for the late onset of a less severe form of diabetes . similarly , our f3 - 5 , but not f1 - 2 , displays the above - discussed phenomenon , arguing that hcg cannot be responsible for the observed affects . this f3 - 5 is principally pointing towards a decisive effect on the immune response in the onset of auto - immune diabetes and is an active component for immunotherapy of this disease and other immune - mediated disorders . in addition , pregnyl and immunoregulators functionally equivalent thereto , is effective in non - insulin - diabetes mellitus ( niddm ). the essential problem in niddm patients is the insulin resistance and obesity . it has been shown that tnf -( alpha ) is the cause of the insulin resistance and obesity of niddm ( miles et al . 1997 , solomon et al . 1997 , pfeiffer et al . 1997 , hotamisligil et al . 1994 ), argiles et al . 1994 ). this insulin resistance induced by tnf - alpha can be reversed by recently developed medicines like pioglitazone and metformin , and with engineered human anti - tnf - alpha antibody ( cdp571 ) ( solomon et al . 1997 , ofei et al . 1996 ), which possibly achieved their beneficial action by lowering the tnf - alpha - induced free fatty acid ( ffa ) concentration of the blood and / or by stimulating glucose uptake at an intracellular point distal to the insulin receptor autophosphorylation in the muscle . furthermore , the presence of retinopathy ( pfeiffer et al . 1997 ) ( one of the late complications of diabetes ) has been mediated with significantly elevated plasma tnf - alpha and is sex - dependent ( pfeiffer et al . 1997 ). the increased tnf - alpha occurs in male but not in female niddm and may participate in the development of retinopathy and other complications like neuropathy , nephropathy or macroangiopathy ( pfeiffer et al . 1997 ). since pregnyl and fraction 3 - 5 have immune modulating potential and , in particular , inhibit tnf - alpha directly or indirectly , pregnyl and its fraction 3 - 5 also have beneficial effects in niddm patients . lower incidence of diabetes complications among females could implicate the involvement of female hormones . a key pathogenic cytokine indicated in sepsis or septic shock is the immunological mediator tnfα which occupies a key role in the pathophysiology associated with diverse inflammatory states and other serious illnesses including sepsis or septic shock and cachexia . when tnf is produced by t - cells ( for example , by t - cell activation through superantigen [ exotoxin ] or by macrophages through endotoxin ), it mediates an inflammatory response that may alienate and repel the attacking organisms . when the infection spreads , the subsequent release of large quantities of tnf into the circulation is catastrophic , damaging the organ system and triggering a state of lethal shock . these toxic effects occur by direct action of tnf on host cells and by the interaction with cascades of other endogenous immunological mediators including il - 1 , ifn - gamma . this has been shown by induction of shock - like symptoms in mice sensitized with d - galactosamine and treated with tnfα as well as inhibition of both lethality and visible signs of disease after concurrent infusion of anti - tnfα mabs following tsst - 1 and d - galactosamine treatment . in the low dose endotoxin model and in the exotoxin model , d - galactosamine treatment is necessary to inhibit the transcription of acute phase proteins that allow the liver to detoxify the high levels of tnfα present following shock induction . the lack of these acute phase proteins leads to increased susceptibility of murine hepatocytes to tnfα mediated apoptosis induction . this apoptosis , and inability to neutralize the inflammatory effects of tnfα eventually lead to death . we have shown that factors ( ir ) with or without hcg present in , for example , the urine of the first trimester of pregnancy ( ir - u ) and in commercial hcg preparations ( ir - p ) have immune regulatory effects . in particular , they have the potential to inhibit auto - immune and inflammatory diseases . since tnf and ifn - gamma are pathologically involved in sepsis or septic shock and also in auto - immune and inflammatory diseases , ir also has the ability to inhibit tnf and ifn - gamma in acute inflammatory states like shock . our results show that ir inhibits sepsis or septic shock in balb / c or sjl , treated with lps ( endotoxin model ) or with tsst - 1 ( exotoxin model ). ir not only has the potency to inhibit chronic inflammatory diseases , but it can also suppress acute inflammatory diseases like shock . moreover , we also show that even post - treatment with ir inhibits the shock . furthermore , our ir fraction data show that most of the anti - shock activity resides in fractions ir -( u / p ) 3 - 5 [ pooled ] which contain mostly individual chains of hcg , homodimers of these chains or beta - core residual chains , breakdown products of these chains and other molecules (& gt ; 30 kda ). we have also shown that the same fractions ir - u / p3 - 5 have anti - diabetic effect in nod mice models . thus , the endotoxin and exotoxin model serves as a fast readout model for the determination of anti - diabetic activity in nod mice and nod . scid mice . with the help of endotoxin and exotoxin models , we can check for anti - diabetic activity in ir fractions within 48 hours . thus , ir such as pregnyl and its fraction 3 - 5have high potency to suppress auto - immune diabetes by modulating the immune system by effecting regulatory t - cell subsets . our nod and balb / c data show that they have the potential to restore the t - cell subset balance ( th1 -& gt ; th2 / th2 -& gt ; th1 ). therefore , pregnyl and its fraction 3 - 5 are effective in modulating the severity of other immune - mediated diseases too , like diseases where th1 cytokines are dominant such as rheumatoid arthritis ( ra ), multiple sclerosis ( ms ), niddm , systemic lupus erythematosis ( sle ), transplantation models and diseases like allergies and asthma where th2 cytokines responses are dominant . animal models of these diseases ( like eae model for ms , bb - 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