The prevention and treatment of murine colitis using gene therapy with adenoviral vectors encoding interieukin-10

those in the mice treated with IL-IO alone. (2) Macroscopic and histological examinations revealed remarkable improvement of colitis in IL-IO- mice treated with GM-IL-IO. (3) Expressions of IL-12 mRNA and CD 40 in Mac-1 positive cells in the mice treated with GMIL-IO was significantly down-regulated compared with those in the mice treated with IL-IO alone. Conclusion: The therapeutic effects of GM-IL-IO was associated with decreasedexpression of IL-12 mRNA via down-regulation of CD40 expression on Mac-1 positive cells. GMIL-IO may be useful for the treatment of patients with IBD.

3706 The Prevention And Treatment Of Murine Colitis Using Gene Therapy Adenovirai Vectors Encoding Inferfeukfe-lO. ,James O. Lindsay, Cathleen J. Ciesielski, Tim Scheinin, Fionula M. Brennan, Imperial Coil Sch of Medicine, London United Kingdom; Humphrey J. Hodgson, Royal Free Hosp, London United Kingdom Introduction: tnterleukin*lO knockout (IL-IO - / - ) mice spontaneously develop a Thl T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-IO will prevent disease onset, but are unable to induce remission in IL-IO - / - mice with established disease.This lack of therapeutic efficacy may represent either the inadequatemucosal delivery of IL-IO or the fact that early exposure to IL-IO is required for immunoregulatory cell differentiation. Aims: To investigate the therapeutic efficacy and mechanism of action of gene therapy using replication deficient adenoviral vectors encoding 11-10(AdwnulL-lO) in the IL10 - / - mouse model of colitis. Results: A single systemic injection of lx108 plaque forming units (PFU) of AdvmulL-lO to 4 week old IL-IO - / - mice was able to prevent the onset of colitis for at least 10 weeks. Furthermore, a single injection of 5x10' PFU AdvmulL-lO was sufficient to induce clinical remission in ten week old IL-IO - / - mice with established disease. Histological scores were significantly lower in all AdvmulL-lO treated mice than age matched littermate controls treated with either saline or empty vector virus (p-
3708

Attenuation of Colon Inflammation Through the RXR/PPAR~,Heterodimer: A Basis for New Therapeutic Strategies Laurent Dubuquoy, Sophie Nutten, Lab de Research sur Ins MtCI, CHU, Lille France; Michel Peuchmaur, Service d'Anatomo-pethologie, Hosp Robert Debre, Paris France; Kristina Schuonjans, IGOMC, lllkirch France; Benoit Derijard, UMR 6548-CNRS, Nice France; Walter Wabli, Inst de Biology Animale, Univ de Lausanne, Lausanne Switzerland; Pierre Chambon, IGBMC, ltlkirch France; Mark D. Leibowitz, Ligand Pharmaceuticals, San Diego, CA; Jean-Frederic Colomhal, Lab de Research sur lea MICI, CHU, Lille France; Johan Auwerx, IGBMC, IIIkirch France; Pierre Desreumaux, Lab de Research sur les MICI, CHU, Lille France Background: The peroxisome proliferator-activated receptor -~ (PPAR-f) is highly expressed in colon mucosa and its activation has been reported to protect against colitis (1). To activate transcription, PPAR~ requires haterodimerizatlon with the retinoic X receptor (RXR). Proof of a role for the RXR/PPAR,y haterodimer in intestinal inflammation has been limited to pharmacological studies demonstrating the efficacy of PPAR~, agonists. Aim: To study the involvement of both RXR and PPAR-y in intestinal inflammatory response. Methods: Colitis was induced by intra-rectal administration of TNBS (150 mg/kg in 50% ethanol) in PPAR'y + / - and RXRa + / - mice and their respective wild type littermates (WT). A role for the RXR/PPAR.y heterodimer in the protection against colon inflammation was also explored by the use of selective RXR (rexinoid LG101305) and PPAR-y agonists (troglitazone and rosiglltazone). The intensity of colitis was evaluated by macroscopic and histologic scores and quantification in the colon of the myeloperoxidase (MPO)(Western blot), TNFa and IL1,8 mRNA concentrations (competitive RT-PCR), and the NF-KB, p38 and JNK activities. Results: PPAR-y + / - and RXR~ + / - mice both displayed a significantly enhancedsusceptibility to TNBS-induced colitis compared to their wr liffermates. TNBS-induced colitis was significantly reduced by the administration of both PPAR~,and RXR agonists. This beneficial effect was reflected by increasedsurvival rates, an improvement of macroscopic and histologic scores, a decrease in TNFa and IL-1/3 mRNA levels, a diminished MPO concentration and reduction of NF-KB DNA binding activity, JNK and p38 activities in the colon. When coadministred, a significant synergistic effect of the PPAR~ and RXR ligands was observed. Conclusion: These data demonstrate that actlvathon of the RXR/PPAR~,heterodimer protects against colon inflammation and suggest for the first time that the combination therapy with both RXR and PPAR-yligands might hold promise in the clinic due to their synergistic effects. 1: So eta/. J Cliff Invest 1999; 104:383

3709 OivergeM Effects of Chronic Nicotine Admlnlsiretion on JejuniUs and Colitis in Interlneldn lO-Ooflcient Mice. Rami Eliakim, Rambam Medical Ctr, Haita Israel; Qui-Xlang Fan, Kiran Nimagadda, Mark W. Babyatsky, Mount Sinai Medical Ctr, New York, NY Cigarette smoking alters the course of inflammatory bowel disease (IBD). Smoking protects against ulcerative colitis (UC), but aggravatesor has no effect on Crohn's disease (CD), While the etiology of this discrepancy remains unclear, differences between location of involvement in UC and CD have not been examined in these studies. We have previously shown that nicotine (12.5p.o/ml), a main active ingredient in cigarette smoke, protects against colonic injury but exacerbatesjejunal injury induced by iodoacetamide in rats. The aim of the current study is to examine the effects of nicotine administration on the course of jejunitis and colitis in interleuldn (IL) -10 deficient (KO) mice. Methods: Male C57/BI6 IL-IO KO and wildtype (WT) mice were given nicotine (12.5p.g/ml) in their drinking water at age 12-14 weeks when they had developed clinical signs of IBD. Sex and age-matched control mice received tap water alone. All mice were sacrificed after two weeks of treatment. Whole tissue sections of jejunum, proximal and distal colon were separatedand examinedby macroscopic and histologic score. RNA was prepared by Trizol reagent purification; Northern blots were examined for somatostatin (SST) and intestinal trefoil factor (ITF) gene expression, two peptides with protective properties in experimental IBD; blots were re-probed with/~-actin and MUC2 as RNNmucin expression controls. Results: At sacrifice at 14-16 weeks, IL-IO KO untreated control mice developed both jejunitis (macroscopic score (macro) = 1.4 + 0.5, microsocpic score (micro) = 2.0+0.2) and colitis (macro=2.0+O.2, micro=5.9+O.9). IL-IO KO mice treated with 2 weeks of nicotine had significantly reduced macro 1.4+0.6) and micro (2.2 + 0.15) colonic scores (p = 0.01). In contrast, the jejunum was more severely damaged with macro scores 2.6 + 0.4 and micro 4.0 + 0.3 (p = 0.01). Nicotine significantly increased both SST and ITF mRNA expression in colon but not in jejunum; no effect was noted on MUC2 or ~-actin mRNA expression. Conclusions: 1. Two weeks of nicotine administration leads to contrasting effects on jejunal and colonic inflammation in IL-lO KO mice. 2. Nicotine ameliorated inflammation in the colon, which was associated with enhanced expression of two protective peptides. 3. Regional effects of nicotine on gut inflammation may, at least partially, explain divergent effects of cigarette smoking seen in UC and CD.

3707 Adhesion Molecule Expression and Blockade in the TNFL~RE Model of Crohn's Disease Jesus Rivera-Nieves, Marco Marini, Digest Health Ctr Unlv of Virginia Health System, Charlottesville, VA; Christopher A. Moskaluk, Dept of Pathology, Univ of Virginia, Charlottesville, VA; Cynthia Nast, Dept of Pathology, Cedars-Sinai, Los Angeles, CA; Klaus Ley, Oept of Blomed Engineering, Univ of Virginia, Charlottesville, VA; George Kolllas, institute of immunology; Alexander Fleming, Biomedical Sciences Research Center, Athens Greece; Theresa T. Pizarro, Digestive Health Center, University of Virginia, Charlottesville, VA; Fabio Cominelli, Digest Health Ctr, University of Virginia, Charlottesville, VA BACKGROUND:Tumor necrosis factor (TNF)plays a central role in the pathogenesis of Crohn's disease (CD). This concept is supported by 2 main lines of evidence: 1) TNF blockade results in decreased inflammation in genetically and immunologically-manipulated murine models, as well as in two thirds of patients with CD; 2) targeted deletion of a 69 bp fragment in the 3'-AU-rich region of the murine TNF gene results in systemic TNF overexpression and in terminal ileitis closely resembling CD (TNFAARE model). TNF is also known to upregulate the cell surface expression of severalcell adhesion molecules (CAM) (e.g. P-selectin, E-selectin, ICAM-1, VCAM-1, MAdCAM), which regulate the migration of neutrophils, macrophages and tymphocytes into inflamed intestinal tissues. The TNFL~AREmodel provides a unique opportunity to study the role of CAM in a murine model of CD, which shows remarkable similarities to human disease. METHODS: Homozygous TNFAARE mice (8 week-old) and age-matched wild type controls were bled, sacrificed and flea harvested. Tissues were immunostained for ICAM-1 and VCAM-I. Circulating levels of slCAM-1 were determined by ELISA. A subset of 7 week old heterozygous mice received an anti-integrin alpha-4 antibody (150 p,g/mouse) for a total of 3 doses. Mice were sacrificed 7 days later, flea harvested and ileitis severity assessed by a semi-quantitative histological scoring system. RESULTS: Increased expression of ICAM1 and VCAM-1 was detected by immunohistochemistry in homozygous TNF,~ARE mice, compared to controls. ElevatedslCAM-1 levels were detected by ELISA in homozygous (82 /~g/ml) and heterozygous (40 pg/ml) TNFL~AREmice, compared to wild type controls (12 /Lo/ml). Increased expression of integrin alpha-e was detected by FACS in heterozygous TNFAARE (13.6 %) mesenteric lymph node cells compared to wild type controls (7.5%). Integrin alpha-4 blockade with a specific monoclonal antibody (PS-2) resulted in decreased active (4.5 _+ 2 vs. 0.5 _+ 0.5), chronic (2 _+ 0.5 vs. 1 _+ 0.5), and villus distortion (4.5 _+ 1.5 vs 2 _+ 0.7) inflammatory scores in haterozygous mice compared to littermate wild type controls. CONCLUSION:Overexpressionof TNF resulted in increased expression of 1CAM-l, VCAM-1, and alpha-e integrin. Antibody blockade of alpha-4 integrin significantly decreased intestinal inflammation, possibly by simultaneous blockade of the 04 ~I-VCAM and 04/37MAdCAM pathways. Our results further suggest that specific adhesion molecules play a key role in the pathogenesis of TNF-mediated intestinal inflammation.

3710 Prevention of murine colitis by feeding a soluble fraction of E. co/i Astrid Konred, Inselspital, Bern Switzerland; Michael Maehler, Medicine Hochschule Hannover, Hannover Germany; Beatrice Flogerzi, Bernhard Egger, Markus Kalousek, Frank Seibold, Inselspital, Bern Switzerland Background: Immune reactivity towards the bacterial intestinal flora plays an important role in inflammatory bowel disease. Several studies showed the positive influence of disease by influencing the bacterial flora, e.g. by administration of problotic bacteria. Aim: We sought to investigate the efficiency of a soluble bacterial extract from Escherichia (E.) coil (strain Laves, 1951) on disease activity of murine colitis. Methods: Ten 4- and ten 8-week-old IL1 0 - / - mice C3H/HeJBir genetic background were treated either with a soluble bacterial extract form 2.7 x 108/ml E. coil or with placebo (6 mice) for one week, then 2% DSS for five days to induce colitis, an again E. coil extract versus placebo for a period of one week. Weight and clinical signs of colitis were monitored twice a week. Colonic and cecal inflammation

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