- Email: [email protected]
Protection of acute TNBS colitis by an IL-2-immunoglobulin fusion protein (FP) in mice
GASTROENTEROLOGY e e l . 114, No. 4
A1060 AGA ABSTRACTS
• G4339 A P55 TUMOR NECROSIS FACTOR-u (TN1D RECEPTOR IMMUNO-
ADHESIN PREVENTS T CELL-MEDIATED INTESTINAL INJURY BY INHIBITING MATRIX METALLOPROTEINASE PRODUCTION. S.L.F. Pender, J.M.C. Fell, A. Ashkenazi* and T.T. MacDonald. Department of Paediatric Gastroenterology, St. Bartholomew's and the Royal London School of Medicine and Dentistry; *Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA. Anti-tumor necrosis factor-ix (TNF-u) antibody therapy has been shown to be of benefit in the treatment of active Crohn's disease, but the tissue-injuring processes in the gut mediated by TNF-u which might be inhibited by neutralising antibody are unknown. In this work we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury (J. Immunology 1997; 158:1582-1590) which ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin pokeweed mitogen (PWM). Following T cell activation and associated with mucosal injury there is a marked elevation of soluble TNF-u in organ culture supematants and a large increase in TNF-u mRNA transcripts. The addition of TNFR-IgG at the onset of cultures almost completely prevented PWM-induced tissue injury. When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-u and IL-113 added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFRIgG. These results suggest that one of the ways in which TNF-u causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases, especially stromelysin-1. Neutralisation of this activity should help maintain tissue integrity. A. Ashkenazi, Employee; T.T. MacDonald, Research Consultant, Genentech, San Francisco, CA. This work was funded by the Wellcome Trust, Joint Research Board of St. Bartholomew's Hospital and the Crohn's in Childhood Research Association, UK. G4340 SERUM LEVELS OF INTERLEUKIN I RECEPTOR ANTAGONIST (IL-1RA) AND SOLUBLE TUMOR NECROSIS FACTOR a-RECEPTOR (sTNFct-R) IN SEVERE ATrACKS OF ULCERATIVE COLITIS. D, prrrVerge, J. Bienvenu, B. Flouri~, L. Descos, Services d'HepatoGastroentrrologie et d'Immunologie, Centre Hospitalier Lyon Sud, 69495 Pierre-Brnite, FRANCE. In ulcerative colitis (UC) severity of relapses remains often difficult to assess objectively. In this work we wanted to know whether semm levels of cytokines in conjunction with biological parameters of inflammation could help in the assessment of UC severity. Methods: C reactive protein (CRP), orosomucoid (OROSO), interleukin 6 (IL-6), soluble interleukin 6-receptor (slL-6R), interleukin 1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor u p75-receptor (sTNFu-R) were measured in serum from 42 patients with UC (26 M, 16 F, mean age 38 yrs). Severity was assessed according to Truelove and Witts' criteria. Dosages were also performed in 20 healthy humans. Results: mean _+SEM, a * b : p < 0.05 by ANOVA Severity severe moderate mild n 12 6 7 CRP g/l 60±15 a 19±6 8±3 b OROSO g/1 1.9 ± 0.2 a 1.3±0.2 1.0± 0.1 IL-6 pg/ml 117±40 a 22±4 17±4 slL-6R ng/ml 70 ± 11 45 ± 6 65 ± 7 IL-1Ra pg/ml 1458± 418 a 258 ± 55b 258 -+70b sTNFct-R ng/ml 6.2 ± 0.8a 3.8 ± 0.8b 2.4 ± 0.2b
quiescent 17 4±1 b 0.8 ± 0.1 b 22±3 b 79 ± 8 116± 28b 2.3 ± 0.2b
normals 20 3±1 0.8±0.1 3±1 58 _+6 130±12 3.5±0.1
Serum levels of IL-1Ra and sTNFu-R were significantly higher (p<0.05) in patients with severe attacks of UC compared to those with moderate and mild relapses. Conclusion: In severe attacks of UC, serum levels of IL-1Ra and sTNFu-R are increased providing an objective biological diagnosis of severity. The course of serum levels of IL-1Ra and sTNFct-R under treatment could be useful for decision making, ie. indication and time of colectomy. • G4341 MOLECULAR MECHANISM OF INTERLEUKIN-11 IN THE HLA-B27 RAT MODEL OF INFLAMMATORY BOWEL DISEASE. R. L. Peterson L. Wang, L. M. Albert, J. C. Keith, A. J. Domer. Preclinical Research and Development, Genetics Institute, Inc, Andover, MA. Recombinant human Interleukin-ll(rhlL-11)is a pleiotropic cytokine with effects on multiple cell types. In addition to thrombopoetic activity, rhlL-11 has anti-inflammatory activity. Treatment with rhlL-I 1 reduces the gross and histological lesions of inflammatory bowel disease (IBD) in rats expression human HLA-B27. We investigated the effects of rhlL-11 at the molecular and cellular level to elucidate the mechanism of action of rhlL-11 in this model of IBD. METHODS: 20 week old male HLA-B27 rats were treated with 37.5 tag/kg rhIL-11 two times per week, control animals were treated with vehicle. Stool character was assessed daily. Animals were sacrificed after 1, 2.5 and 4
weeks of dosing. Colonic and cecal tissue was harvested for mRNA analysis by reverse transcriptase polymerase chain reaction (RT-PCR) and myeloperoxidase assays. Spleen cells from rhIL-11-treated and vehicle-treated animals were cultured in the presence of anti-CD3 antibody for 24 hours and the levels of TNFct, INF7 and IL-2 protein in the conditioned media were determined by ELISA. RESULT: RTP-CR analysis of colonic RNA revealed that treatment with rhlL-11 caused a reduction in the levels of pro-inflammatory cytokine messages such a TNFu, INF'/ and IL-lfl (49%, 58% and 61% respectively). Treatment with rhlL-11 also reduced the level of myeloperoxidase activity in the cecum by 31 to 42 % (P<0.001). Following stimulation in vitro with anti-CD3 antibody, spleen cells derived from rhlL-11-treated animals produced significantly less IFN~/, TNFu and IL-2 protein (P<0.001) than cultures derived from vehicle-treated animals. These molecular and cellular effects of rhlL- 11 were seen at all times of treatment and illustrate that rhlL-11 effects multiple parameters of intestinal inflammation which correlate with the amelioration of clinical disease symptoms. CONCLUSIONS: These results further support the role of rhlL-11 as an anti-inflammatory molecule to treat IBD and have identified potential markers of rhlL-11 activity applicable for clinical monitoring. Authors employed by Genetics Institute, Inc. G4342
EI~'FECT OF T IMEBUTINE MALEATE ON EXPERIMENTAL COLITIS IN RATS. F. P6toux, E. Chevalier, M. Chovet, A. Lan~,lois Jouveinal, Parke-Davis, Fresnes, France. Trimebutine maleate (TMB) displays similar binding affinity to type-2 sodium channel as lidocaine. The use of local anesthetics had been proposed in the treatment of distal ulcerative colitis. The aim of the present study was to evaluate the effect of TMB on trinitrobenzene sulfonic acid (TNBS)induced colitis in rats. Methods: Colitis was induced by a single intracolonic administration of 50 mg/kg TNBS dissolved in 30 % ethanol in male Sprague Dawley rats (250-300g). TMB or lidocaine were locally administered before (- 30 min) and after (+ 24 and 48 hours) the induction of colitis. Three days after TNBS, the severity of colitis was evaluated by colonic wet weight (CW), colonic myeloperoxidase (MPO) content and colonic damage using a computerized morphometric analysis. Colonic damage was expressed by the percent of colonic byperemia (HYP) and necrosis (NEC). Results: TNBS dramatically increased colonic wet weight, colonic myeloperoxidase content and colonic damage. TMB significantly improved colitis in TNBS-treated rats (see Table below). Similar results were obtained with lidocaine. Table: Effect of TMB and lidocaine on TNBS-induced colitis. Results are expressed as the ;rcentage of inhibition Trimebutine maleate Lidocaine (mg/kg) (mg/kg) 3 I I0 30 1 3 10 CW 41_+9" 56_+10" 65_+6* 40_+13 59_+7* 63_+4* MPO 37+_20 45+_14" 85_+6* 16+33 71+10" 86+4* 73+11" 75+_11" HYP 69+_7* 62+_13" 72+_13" 42_.+16 NEC 70+_16" 84+_6* 85+12" 0 8 8 + _ 1 0 " 95+_3* *: P < 0.05, Mann and Whitney U-test Conclusion: Local treatment with TMB improved TNBS-induced colitis in rats and this effect could be explained by its inhibitory activity on type-2 sodium channel. • G4343 PROTECTION OF ACUTE TNBS COLITIS BY A N IL-2-IMMUNOGLOBULIN FUSION PROTEIN (FP) IN MICE. K. Pflster, C. Peters, L Hoffmann, R. Duchmann, #S. Bulfone-Pauss, *U. Kunzendorf, M. Zeitz, A. Stallmach. Dep. Med. II, Homburg Saar, #Institut of Immunology, Berlin, *Dep. Med. IV, Erlangen, Germany. A chimeric protein consisting of mouse interleukin-2 (IL-2) fused to the mouse IgG2b Fc domains suppresses both cellular and humeral immune • response after immunization with sheep erythrocytes (J Clin Invest 1996; 97: 1204). The aim of this study was to evaluate the effects of this IL-2-IgG2b FP in an established experimental granulomatous (TNBS) colitis, which is a Thl-mediated animal model of human Crohn's disease. Methods: Male BALB/c mice were divided into 3 experimental groups: control (n=5), TNBS (n=23), and TNBS+IL-2-IgG2b (8 pg/ip, every 12 hours for 3-5 days) (n=22). Colitis was induced by intracolonic administration of TNBS (2 mg in 50% ethanol). Animals were sacrifled after 3 or 5 days. Changes in body weight, histology, and cellular infiltrates (FACS analysis) were determined Results: IL-2-IgG2b treatment significantly increased survival rate, reduced TNBSinduced weight loss and mucosal alterations compared to TNBS alone. TNBS treatment resulted in an increased activation of colonic CD4 ÷ T cells. Interestingly, after treatment with IL-2-IgG2b an increase of both, splenic and colonic CD4/CD25 ÷ T cells was observed.
April 1998
Immunology, Microbiology, and Inflammatory Disorders A1061 Control
TNBS
survival rate 100% 43 % 1.5 -+0.5 -1.7 -+0.9 Wilt change (g) 73 (45-87) 88 (50-90) spleen Wgt (mg) 19 (15-42) 30 (16-49) CD4+/CD25+ (colon) CD4+/CD25+ (spleen) 14 (11-18) 10 (9-12) * p < 0.05 compared to TNBS treated animals
TNBS+IL-~ IgG2h 64% -0.3 -+0.7* 155 (120-207)* 37 (25-63)* 49 (47-51)*
Summary and conclusion: IL-2-IgG2b substantially attenuates colonic injury and inflammation induced by TNBS in mice. The increase of CD4/CD25 + T cells suggests that activation of immunosuppressive cells rather than cytolysis of T ceils prevents colonic inflammation. This is the first indication that IL-2-IgG2b FP may be beneficial in reducing the severity of Thl-mediated colitis. • G4344 TERTIARY REFERRAL PATIENTS WITH CROHN'S DISEASE HAVE EARLIER AGE OF DISEASE ONSET, SHORTER DISEASE DURATION BUT EQUIVALENT FAMILIALITY COMPARED TO COMMUNITYBASED PATIENTS. M F. Picc0*, J S Mann* J Reed IIl**, L Rosen**, C D'Angelo**, ML Harris*, TM Bayless*. *Johns Hopkins University, Baltimore, MD.**Lehigh Valley Hospital, Allentown, PA. Purpose: Studies of Crohn's disease have been largely based on university populations with a perceived referral bias in favor of greater disease severity. Genome wide screens seeking susceptibility genes are currently being done based on these populations. Differences between referral and community populations could influence genetic analyses. We have compared Johns Hopkins referral based population with a community based population from Lehigh Valley, PA with regard to disease severity, family history of inflammatory bowel disease and Jewish ethnicity. Methods: 345 consecutive patients seen from 1992-1997 at the Meyerhoff Center for Inflammatory Bowel Disease at Johns Hopkins Hospital were compared to 186 patients obtained from the Lehigh Valley Inflammatory Bowel Disease Registry with Crohn's disease. Case ascertainment for the registry is based on hospital admissions to 16 area hospitals in the Lehigh Valley between 1981-1987 and has been maintained through 1992 and surveyed in 1997. Comparison of severity between groups was based on age at diagnosis of disease, disease duration, family history and use of azathioprine. Results: Variable
Family History Positive Jewish Ethnicity Age at Diagnosis Disease Duration Use of Imuran
Group Lehigh Valley Johns Hopkins Registr~ 35% (n = 186) 30% (n = 333) 22% (n = 41) 27.8 yrs (n = 333) 12.2 yrs (n = 333) 61% (n = 28)
p-value
NS
6% (n = 35) 0.046 34 yrs (n --- 186) < 0.0001 17.3 ~'rs (n = 186) < 0.0001 35% (n = 49) 0.03
Patients seen at Johns Hopkins had an age of diagnosis that was 6.4 yrs earlier, duration of disease that was 5 years shorter than the community based population. The proportion with positive family history was similar in both groups with Jewish ethnicity being approximately four times more common in the Johns Hopkins population. The usage of azathioprine at Johns Hopkins was nearly twice that of the community. Conclusion: Our results are compatible with the conclusion that referral populations have more severe disease based on age of diagnosis, duration of disease and use of azathioprine than a community based populations. Although the community based and referral based populations have similar frequencies of positive family histories for inflammatory bowel disease there are potentially important differences in ethnicity and severity of disease that will require careful stratification of populations when analyzing for phenotypes and genotypes. Disclosure: This research was funded by the Meyerhoff Center for Digestive and Inflammatory Bowel Diseases at Johns Hopkins and the National Institutes of Health
G4345 PERIANAL (PA) MANIFESTATIONS IN AFRICAN-AMERICAN (AA) AND CAUCASIAN PEDIATRIC PATIENTS WITH CROHN'S DISEASE (CD). M. Pickles, G.B. Burgess, P. Sutabutra, J. Cho, B.S. Kirschner. Depts of Pediatrics and Medicine, The Pritzker School of Medicine, The University of Chicago, Chicago, IL. Introduction: Pediatric studies by others have suggested a high frequency of severe perianal complications in AA pts. with CD. [Markowitz J e t al. J Pediatr Gastroenterol Nutr 1995;21: 149-153] In our study, a large group of AA children and adolescents with CD was compared with non-AA patients. Methods: The medical histories of 60 AA children with CD were reviewed. All patients were followed by the pediatric GI service as outpatients and/or inpatients at the University of Chicago Children's Hospital. Results: The mean age at diagnosis of Crohn's was 12.4 years, range 5 to 18 years. Twenty-four (40%) patients had perianal disease with 5 presenting with PA manifestations at diagnosis and 1 patient with a perianal abscess drained 3 years prior to diagnosis. Anal fissures were the commonest finding: 15160 (25%), fistulae were present in 9 (15%), abscesses in 5 (8%), skin tags in 3 (5%), and intemal hemorrhoids in 1 (2%). Table 1 compares the prevalence of PA disease of this AA population with predominantly Caucasian populations of Chicago, Toronto and New York. Eight patients required surgical intervention for perianal disease, including one diverting colostomy. The incidence of intestinal resection was higher in those with perianal disease (38%) versus no perianal disease (22%). Highly destructive perianal disease (HDPD or cavitating ulcerations) occurred in 2/60 (3.3%) of AA and 4/117 (3.4%) of Caucasian pts vs. 5/11 (45%) AA pts. cited in the above ref. Table 1 - comparison of PA disease in AA and Caucasians Chicago Chicago Toronto a NYb AA Caucasian n=60 n=l17 n=325 n=149 Perianal Lesions 40% 69% 62% 49% Perianal Fistulae 15% 15% 15% 7% Abscesses 8% 9% 13% 8% Anal Fissures 25% 34% 51% 34%* Skin Tags 5% 46% 35% * * skin tags and anal fissures combined a) Palder SB et al, J Pediatr Surgery 1991; 26:513-515 b) Markowitz J et al, Gastroenterology 1984; 86:829-833
p value
< 0.001 NS NS NS < 0.001
Conclusion: The prevalence of PA fissures, fistulae, abscesses, and HDPD in this large series of AA children with CD did not differ from our age-matched Caucasian CD population. We were not able to substantiate the high frequency of HDPD previously described by others in pediatric AA pts with Crohn's disease. G4346 IS THERE IMPAIRED DETOXICATION OF HYDROGEN SULPHIDE IN ULCERATIVE COLITIS? R Picton, MJS Langman, S Sin~,h. Dept. of Medicine, University of Birmingham and Good Hope Hospital Sutton Coldfield, UK. Hydrogen sulphide (H2S) in vitro retards butyrate oxidation in colonocytes mimicking the defect seen in ulcerative colitis. The presence of bacteria (particularly anaerobic) is requisite in most models of colitis, and as H:S is synthesised by sulphate reducing bacteria in the large bowel, impaired detoxication of this highly toxic gas may play a role in colitis. H2S is detoxicated via methylation by the microsomal enzyme thiol methyltransferase (TMT), or by conversion to thiocyanate by the mitochondrial enzyme rhodanese (RHOD). We have sought activity for TMT and rhodanese in rectal biopsies from patients with colitis or controls (mostly patients with irritable bowel syndrome). In addition, as a control, we have determined activity of the cytosolic enzyme thiol purine methyltransferase (TPMT). This enzyme methylates sulphydryl groups on aromatic compounds e.g. 6-mercaptopurine, but is not able to detoxify H2S. TMT assay was performed using [3 mercaptoethanol as the methyl acceptor and co-factor, tritium labelled S-adenosyl methionine. TPMT assay was performed using 6-mercaptopurineas the methyl acceptor. Rhodanese activity was determined by measuring thiocyanate production from thiosulphate and cyanide.
Control (n = 21) Colitis (n = 11)
TMT fmol/min/ug 55.1-+5.0 80.1 -+ 16.5
RHOD pmol/min/ug 151.9-+17.6 143.9 -+ 16.3
TPMT fmol/min/ug 30.8-+2.4 42.2 -+ 4.4
The Table shows mean -+ SEM enzyme activity. TMT and TPMT were both on average higher in colitics than in controls, although RHOD levels were similar. Severity of mucosal inflammation did not correlate with enzyme activity, although numbers studied currently are small. There is no evidence of impaired H2S detoxication due to impaired enzyme activity in rectal mucosa of colitics.
A1060 AGA ABSTRACTS
• G4339 A P55 TUMOR NECROSIS FACTOR-u (TN1D RECEPTOR IMMUNO-
ADHESIN PREVENTS T CELL-MEDIATED INTESTINAL INJURY BY INHIBITING MATRIX METALLOPROTEINASE PRODUCTION. S.L.F. Pender, J.M.C. Fell, A. Ashkenazi* and T.T. MacDonald. Department of Paediatric Gastroenterology, St. Bartholomew's and the Royal London School of Medicine and Dentistry; *Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA. Anti-tumor necrosis factor-ix (TNF-u) antibody therapy has been shown to be of benefit in the treatment of active Crohn's disease, but the tissue-injuring processes in the gut mediated by TNF-u which might be inhibited by neutralising antibody are unknown. In this work we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury (J. Immunology 1997; 158:1582-1590) which ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin pokeweed mitogen (PWM). Following T cell activation and associated with mucosal injury there is a marked elevation of soluble TNF-u in organ culture supematants and a large increase in TNF-u mRNA transcripts. The addition of TNFR-IgG at the onset of cultures almost completely prevented PWM-induced tissue injury. When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-u and IL-113 added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFRIgG. These results suggest that one of the ways in which TNF-u causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases, especially stromelysin-1. Neutralisation of this activity should help maintain tissue integrity. A. Ashkenazi, Employee; T.T. MacDonald, Research Consultant, Genentech, San Francisco, CA. This work was funded by the Wellcome Trust, Joint Research Board of St. Bartholomew's Hospital and the Crohn's in Childhood Research Association, UK. G4340 SERUM LEVELS OF INTERLEUKIN I RECEPTOR ANTAGONIST (IL-1RA) AND SOLUBLE TUMOR NECROSIS FACTOR a-RECEPTOR (sTNFct-R) IN SEVERE ATrACKS OF ULCERATIVE COLITIS. D, prrrVerge, J. Bienvenu, B. Flouri~, L. Descos, Services d'HepatoGastroentrrologie et d'Immunologie, Centre Hospitalier Lyon Sud, 69495 Pierre-Brnite, FRANCE. In ulcerative colitis (UC) severity of relapses remains often difficult to assess objectively. In this work we wanted to know whether semm levels of cytokines in conjunction with biological parameters of inflammation could help in the assessment of UC severity. Methods: C reactive protein (CRP), orosomucoid (OROSO), interleukin 6 (IL-6), soluble interleukin 6-receptor (slL-6R), interleukin 1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor u p75-receptor (sTNFu-R) were measured in serum from 42 patients with UC (26 M, 16 F, mean age 38 yrs). Severity was assessed according to Truelove and Witts' criteria. Dosages were also performed in 20 healthy humans. Results: mean _+SEM, a * b : p < 0.05 by ANOVA Severity severe moderate mild n 12 6 7 CRP g/l 60±15 a 19±6 8±3 b OROSO g/1 1.9 ± 0.2 a 1.3±0.2 1.0± 0.1 IL-6 pg/ml 117±40 a 22±4 17±4 slL-6R ng/ml 70 ± 11 45 ± 6 65 ± 7 IL-1Ra pg/ml 1458± 418 a 258 ± 55b 258 -+70b sTNFct-R ng/ml 6.2 ± 0.8a 3.8 ± 0.8b 2.4 ± 0.2b
quiescent 17 4±1 b 0.8 ± 0.1 b 22±3 b 79 ± 8 116± 28b 2.3 ± 0.2b
normals 20 3±1 0.8±0.1 3±1 58 _+6 130±12 3.5±0.1
Serum levels of IL-1Ra and sTNFu-R were significantly higher (p<0.05) in patients with severe attacks of UC compared to those with moderate and mild relapses. Conclusion: In severe attacks of UC, serum levels of IL-1Ra and sTNFu-R are increased providing an objective biological diagnosis of severity. The course of serum levels of IL-1Ra and sTNFct-R under treatment could be useful for decision making, ie. indication and time of colectomy. • G4341 MOLECULAR MECHANISM OF INTERLEUKIN-11 IN THE HLA-B27 RAT MODEL OF INFLAMMATORY BOWEL DISEASE. R. L. Peterson L. Wang, L. M. Albert, J. C. Keith, A. J. Domer. Preclinical Research and Development, Genetics Institute, Inc, Andover, MA. Recombinant human Interleukin-ll(rhlL-11)is a pleiotropic cytokine with effects on multiple cell types. In addition to thrombopoetic activity, rhlL-11 has anti-inflammatory activity. Treatment with rhlL-I 1 reduces the gross and histological lesions of inflammatory bowel disease (IBD) in rats expression human HLA-B27. We investigated the effects of rhlL-11 at the molecular and cellular level to elucidate the mechanism of action of rhlL-11 in this model of IBD. METHODS: 20 week old male HLA-B27 rats were treated with 37.5 tag/kg rhIL-11 two times per week, control animals were treated with vehicle. Stool character was assessed daily. Animals were sacrificed after 1, 2.5 and 4
weeks of dosing. Colonic and cecal tissue was harvested for mRNA analysis by reverse transcriptase polymerase chain reaction (RT-PCR) and myeloperoxidase assays. Spleen cells from rhIL-11-treated and vehicle-treated animals were cultured in the presence of anti-CD3 antibody for 24 hours and the levels of TNFct, INF7 and IL-2 protein in the conditioned media were determined by ELISA. RESULT: RTP-CR analysis of colonic RNA revealed that treatment with rhlL-11 caused a reduction in the levels of pro-inflammatory cytokine messages such a TNFu, INF'/ and IL-lfl (49%, 58% and 61% respectively). Treatment with rhlL-11 also reduced the level of myeloperoxidase activity in the cecum by 31 to 42 % (P<0.001). Following stimulation in vitro with anti-CD3 antibody, spleen cells derived from rhlL-11-treated animals produced significantly less IFN~/, TNFu and IL-2 protein (P<0.001) than cultures derived from vehicle-treated animals. These molecular and cellular effects of rhlL- 11 were seen at all times of treatment and illustrate that rhlL-11 effects multiple parameters of intestinal inflammation which correlate with the amelioration of clinical disease symptoms. CONCLUSIONS: These results further support the role of rhlL-11 as an anti-inflammatory molecule to treat IBD and have identified potential markers of rhlL-11 activity applicable for clinical monitoring. Authors employed by Genetics Institute, Inc. G4342
EI~'FECT OF T IMEBUTINE MALEATE ON EXPERIMENTAL COLITIS IN RATS. F. P6toux, E. Chevalier, M. Chovet, A. Lan~,lois Jouveinal, Parke-Davis, Fresnes, France. Trimebutine maleate (TMB) displays similar binding affinity to type-2 sodium channel as lidocaine. The use of local anesthetics had been proposed in the treatment of distal ulcerative colitis. The aim of the present study was to evaluate the effect of TMB on trinitrobenzene sulfonic acid (TNBS)induced colitis in rats. Methods: Colitis was induced by a single intracolonic administration of 50 mg/kg TNBS dissolved in 30 % ethanol in male Sprague Dawley rats (250-300g). TMB or lidocaine were locally administered before (- 30 min) and after (+ 24 and 48 hours) the induction of colitis. Three days after TNBS, the severity of colitis was evaluated by colonic wet weight (CW), colonic myeloperoxidase (MPO) content and colonic damage using a computerized morphometric analysis. Colonic damage was expressed by the percent of colonic byperemia (HYP) and necrosis (NEC). Results: TNBS dramatically increased colonic wet weight, colonic myeloperoxidase content and colonic damage. TMB significantly improved colitis in TNBS-treated rats (see Table below). Similar results were obtained with lidocaine. Table: Effect of TMB and lidocaine on TNBS-induced colitis. Results are expressed as the ;rcentage of inhibition Trimebutine maleate Lidocaine (mg/kg) (mg/kg) 3 I I0 30 1 3 10 CW 41_+9" 56_+10" 65_+6* 40_+13 59_+7* 63_+4* MPO 37+_20 45+_14" 85_+6* 16+33 71+10" 86+4* 73+11" 75+_11" HYP 69+_7* 62+_13" 72+_13" 42_.+16 NEC 70+_16" 84+_6* 85+12" 0 8 8 + _ 1 0 " 95+_3* *: P < 0.05, Mann and Whitney U-test Conclusion: Local treatment with TMB improved TNBS-induced colitis in rats and this effect could be explained by its inhibitory activity on type-2 sodium channel. • G4343 PROTECTION OF ACUTE TNBS COLITIS BY A N IL-2-IMMUNOGLOBULIN FUSION PROTEIN (FP) IN MICE. K. Pflster, C. Peters, L Hoffmann, R. Duchmann, #S. Bulfone-Pauss, *U. Kunzendorf, M. Zeitz, A. Stallmach. Dep. Med. II, Homburg Saar, #Institut of Immunology, Berlin, *Dep. Med. IV, Erlangen, Germany. A chimeric protein consisting of mouse interleukin-2 (IL-2) fused to the mouse IgG2b Fc domains suppresses both cellular and humeral immune • response after immunization with sheep erythrocytes (J Clin Invest 1996; 97: 1204). The aim of this study was to evaluate the effects of this IL-2-IgG2b FP in an established experimental granulomatous (TNBS) colitis, which is a Thl-mediated animal model of human Crohn's disease. Methods: Male BALB/c mice were divided into 3 experimental groups: control (n=5), TNBS (n=23), and TNBS+IL-2-IgG2b (8 pg/ip, every 12 hours for 3-5 days) (n=22). Colitis was induced by intracolonic administration of TNBS (2 mg in 50% ethanol). Animals were sacrifled after 3 or 5 days. Changes in body weight, histology, and cellular infiltrates (FACS analysis) were determined Results: IL-2-IgG2b treatment significantly increased survival rate, reduced TNBSinduced weight loss and mucosal alterations compared to TNBS alone. TNBS treatment resulted in an increased activation of colonic CD4 ÷ T cells. Interestingly, after treatment with IL-2-IgG2b an increase of both, splenic and colonic CD4/CD25 ÷ T cells was observed.
April 1998
Immunology, Microbiology, and Inflammatory Disorders A1061 Control
TNBS
survival rate 100% 43 % 1.5 -+0.5 -1.7 -+0.9 Wilt change (g) 73 (45-87) 88 (50-90) spleen Wgt (mg) 19 (15-42) 30 (16-49) CD4+/CD25+ (colon) CD4+/CD25+ (spleen) 14 (11-18) 10 (9-12) * p < 0.05 compared to TNBS treated animals
TNBS+IL-~ IgG2h 64% -0.3 -+0.7* 155 (120-207)* 37 (25-63)* 49 (47-51)*
Summary and conclusion: IL-2-IgG2b substantially attenuates colonic injury and inflammation induced by TNBS in mice. The increase of CD4/CD25 + T cells suggests that activation of immunosuppressive cells rather than cytolysis of T ceils prevents colonic inflammation. This is the first indication that IL-2-IgG2b FP may be beneficial in reducing the severity of Thl-mediated colitis. • G4344 TERTIARY REFERRAL PATIENTS WITH CROHN'S DISEASE HAVE EARLIER AGE OF DISEASE ONSET, SHORTER DISEASE DURATION BUT EQUIVALENT FAMILIALITY COMPARED TO COMMUNITYBASED PATIENTS. M F. Picc0*, J S Mann* J Reed IIl**, L Rosen**, C D'Angelo**, ML Harris*, TM Bayless*. *Johns Hopkins University, Baltimore, MD.**Lehigh Valley Hospital, Allentown, PA. Purpose: Studies of Crohn's disease have been largely based on university populations with a perceived referral bias in favor of greater disease severity. Genome wide screens seeking susceptibility genes are currently being done based on these populations. Differences between referral and community populations could influence genetic analyses. We have compared Johns Hopkins referral based population with a community based population from Lehigh Valley, PA with regard to disease severity, family history of inflammatory bowel disease and Jewish ethnicity. Methods: 345 consecutive patients seen from 1992-1997 at the Meyerhoff Center for Inflammatory Bowel Disease at Johns Hopkins Hospital were compared to 186 patients obtained from the Lehigh Valley Inflammatory Bowel Disease Registry with Crohn's disease. Case ascertainment for the registry is based on hospital admissions to 16 area hospitals in the Lehigh Valley between 1981-1987 and has been maintained through 1992 and surveyed in 1997. Comparison of severity between groups was based on age at diagnosis of disease, disease duration, family history and use of azathioprine. Results: Variable
Family History Positive Jewish Ethnicity Age at Diagnosis Disease Duration Use of Imuran
Group Lehigh Valley Johns Hopkins Registr~ 35% (n = 186) 30% (n = 333) 22% (n = 41) 27.8 yrs (n = 333) 12.2 yrs (n = 333) 61% (n = 28)
p-value
NS
6% (n = 35) 0.046 34 yrs (n --- 186) < 0.0001 17.3 ~'rs (n = 186) < 0.0001 35% (n = 49) 0.03
Patients seen at Johns Hopkins had an age of diagnosis that was 6.4 yrs earlier, duration of disease that was 5 years shorter than the community based population. The proportion with positive family history was similar in both groups with Jewish ethnicity being approximately four times more common in the Johns Hopkins population. The usage of azathioprine at Johns Hopkins was nearly twice that of the community. Conclusion: Our results are compatible with the conclusion that referral populations have more severe disease based on age of diagnosis, duration of disease and use of azathioprine than a community based populations. Although the community based and referral based populations have similar frequencies of positive family histories for inflammatory bowel disease there are potentially important differences in ethnicity and severity of disease that will require careful stratification of populations when analyzing for phenotypes and genotypes. Disclosure: This research was funded by the Meyerhoff Center for Digestive and Inflammatory Bowel Diseases at Johns Hopkins and the National Institutes of Health
G4345 PERIANAL (PA) MANIFESTATIONS IN AFRICAN-AMERICAN (AA) AND CAUCASIAN PEDIATRIC PATIENTS WITH CROHN'S DISEASE (CD). M. Pickles, G.B. Burgess, P. Sutabutra, J. Cho, B.S. Kirschner. Depts of Pediatrics and Medicine, The Pritzker School of Medicine, The University of Chicago, Chicago, IL. Introduction: Pediatric studies by others have suggested a high frequency of severe perianal complications in AA pts. with CD. [Markowitz J e t al. J Pediatr Gastroenterol Nutr 1995;21: 149-153] In our study, a large group of AA children and adolescents with CD was compared with non-AA patients. Methods: The medical histories of 60 AA children with CD were reviewed. All patients were followed by the pediatric GI service as outpatients and/or inpatients at the University of Chicago Children's Hospital. Results: The mean age at diagnosis of Crohn's was 12.4 years, range 5 to 18 years. Twenty-four (40%) patients had perianal disease with 5 presenting with PA manifestations at diagnosis and 1 patient with a perianal abscess drained 3 years prior to diagnosis. Anal fissures were the commonest finding: 15160 (25%), fistulae were present in 9 (15%), abscesses in 5 (8%), skin tags in 3 (5%), and intemal hemorrhoids in 1 (2%). Table 1 compares the prevalence of PA disease of this AA population with predominantly Caucasian populations of Chicago, Toronto and New York. Eight patients required surgical intervention for perianal disease, including one diverting colostomy. The incidence of intestinal resection was higher in those with perianal disease (38%) versus no perianal disease (22%). Highly destructive perianal disease (HDPD or cavitating ulcerations) occurred in 2/60 (3.3%) of AA and 4/117 (3.4%) of Caucasian pts vs. 5/11 (45%) AA pts. cited in the above ref. Table 1 - comparison of PA disease in AA and Caucasians Chicago Chicago Toronto a NYb AA Caucasian n=60 n=l17 n=325 n=149 Perianal Lesions 40% 69% 62% 49% Perianal Fistulae 15% 15% 15% 7% Abscesses 8% 9% 13% 8% Anal Fissures 25% 34% 51% 34%* Skin Tags 5% 46% 35% * * skin tags and anal fissures combined a) Palder SB et al, J Pediatr Surgery 1991; 26:513-515 b) Markowitz J et al, Gastroenterology 1984; 86:829-833
p value
< 0.001 NS NS NS < 0.001
Conclusion: The prevalence of PA fissures, fistulae, abscesses, and HDPD in this large series of AA children with CD did not differ from our age-matched Caucasian CD population. We were not able to substantiate the high frequency of HDPD previously described by others in pediatric AA pts with Crohn's disease. G4346 IS THERE IMPAIRED DETOXICATION OF HYDROGEN SULPHIDE IN ULCERATIVE COLITIS? R Picton, MJS Langman, S Sin~,h. Dept. of Medicine, University of Birmingham and Good Hope Hospital Sutton Coldfield, UK. Hydrogen sulphide (H2S) in vitro retards butyrate oxidation in colonocytes mimicking the defect seen in ulcerative colitis. The presence of bacteria (particularly anaerobic) is requisite in most models of colitis, and as H:S is synthesised by sulphate reducing bacteria in the large bowel, impaired detoxication of this highly toxic gas may play a role in colitis. H2S is detoxicated via methylation by the microsomal enzyme thiol methyltransferase (TMT), or by conversion to thiocyanate by the mitochondrial enzyme rhodanese (RHOD). We have sought activity for TMT and rhodanese in rectal biopsies from patients with colitis or controls (mostly patients with irritable bowel syndrome). In addition, as a control, we have determined activity of the cytosolic enzyme thiol purine methyltransferase (TPMT). This enzyme methylates sulphydryl groups on aromatic compounds e.g. 6-mercaptopurine, but is not able to detoxify H2S. TMT assay was performed using [3 mercaptoethanol as the methyl acceptor and co-factor, tritium labelled S-adenosyl methionine. TPMT assay was performed using 6-mercaptopurineas the methyl acceptor. Rhodanese activity was determined by measuring thiocyanate production from thiosulphate and cyanide.
Control (n = 21) Colitis (n = 11)
TMT fmol/min/ug 55.1-+5.0 80.1 -+ 16.5
RHOD pmol/min/ug 151.9-+17.6 143.9 -+ 16.3
TPMT fmol/min/ug 30.8-+2.4 42.2 -+ 4.4
The Table shows mean -+ SEM enzyme activity. TMT and TPMT were both on average higher in colitics than in controls, although RHOD levels were similar. Severity of mucosal inflammation did not correlate with enzyme activity, although numbers studied currently are small. There is no evidence of impaired H2S detoxication due to impaired enzyme activity in rectal mucosa of colitics.