AGA Abstracts of 6-TGDP and 6-TGTP levels after 2 gram 5-ASA daily during steady-state thiopurine therapy and causes a reduction in 6-MMP levels during use of 4 grams 5-ASA daily. 504 A Double-Blind, Double-Dummy, Randomized, Controlled, Multicenter Trial On the Efficacy and Safety of Azathioprine Vs mesalamine for Prevention of Clinical Relapses in Crohn's Disease Patients with Postoperative Moderate or Severe Endoscopic Recurrence Walter Reinisch, Sieglinde Angelberger, Wolfgang Petritsch, Klaus Herrlinger, Olga Shonova, Milan Lukas, Simon Bar-Meir, Matthias Schwab, Roland Greinwald, Ralph Mueller, Eduard Stange Background: After curative intestinal surgery, Crohn's disease (CD) patients experience endoscopic recurrence at the site of the neoterminal ileum and the ileocolonic anastomosis. A moderate or severe endoscopic recurrence of i 2-4 according to Rutgeerts et al. has been deviced as a predictor for clinical relapse. Aim: To evaluate the efficacy and safety of mesalamine or azathioprine for the prevention of clinical relapse in CD patients with postoper- ative moderate or severe endoscopic recurrence. Design: In this double-blind, double- dummy, randomized, controlled trial CD patients with a CDAI<200, but with endoscopic recurrence of i 2-4 within 6 to 24 months after “curative” surgery with ileocolonic anastomosis, were randomized to a 52-weeks treatment with 4g mesalamine (Salofalk ® tablets) or 2.0- 2.5mg/kg/BW azathioprine (Azafalk ® tablets) per day. The primary endpoint was therapeutic failure - defined as a CDAI ≥ 200 and an increase of ≥ 60 points from baseline - or discontinuation due to lack of efficacy at the discretion of the investigator, or intolerable adverse drug reaction. The major secondary endpoint was endoscopic improvement defined as ≥ 1 point drop in the Rutgeerts score. Results: 78 patients were randomized. Both treatment groups showed comparable demographic, clinical, and endoscopical baseline characteristics. The primary efficacy results (ITT) are presented in Table 1 below. Endoscopic improvement according to ITT was observed in 19 of 41 patients (46.3%) with azathioprine vs 11 of 37 patients (29.7%) with mesalamine (p=0.1321), whereas the corresponding figures from the completer analysis were 19 of 30 patients (63.3%) with azathioprine vs 11 of 32 patients (34.4%) with mesalamine (p=0.0226). Conclusion: No statistically significant difference in the rates of therapeutic failures between patients treated with azathioprine or mesalamine could be shown. However, a higher proportion of azathioprine treated patients achieved endoscopic improvement. Supported by Dr. Falk Pharma GmbH, Freiburg, Ger- many Table 1: Primary efficacy endpoint (ITT): A-70 AGA Abstracts 505 Reorganization of Cytoskeleton As Basis for Apoptosis Yaohong Wang, Sudeep P. George, Kamalakkannan Srinivasan, Mohammad R. Siddiqui, Sijo Mathew, Langzhu Tan, Lester Vanmiddlesworth, Qi Xi, Jonathan H. Jaggar, Seema Khurana The homeostatic balance between proliferation and apoptosis is essential for the intestinal epithelium to function as a physiological and structural barrier. While normal apoptosis is essential for the hierarchical organization of the intestinal epithelium, defects in apoptosis are associated with several gastrointestinal disorders including colorectal cancer and inflam- matory bowel disease. Using drugs that affect actin turnover, several studies have established a link between actin and apoptosis but not a causal link between microfilament organization and cell survival. Using gene knock out mice, biochemical and cell biological assays we identify for the first time a role for the intestinal and renal actin-binding protein villin and its homologous partner gelsolin, in intestinal epithelial cell survival. Our data demonstrate that deletion of both villin and gelsolin in mice results in increased susceptibility of mice to both γ-radiation and dextran sodium sulfate-colitis induced injury and apoptosis. Trans- mission electron micrographs of the small intestine show a dramatic change in the mitochon- drial morphology in the villin, gelsolin double knock out mice which include damaged mitochondria undergoing autophagy and a significant inclusion of lipofuscin both of which are indicative of perturbed mitochondrial function. Detailed analysis using functional mutants of villin allows us to demonstrate that both villin and gelsolin function as anti-apoptotic proteins by regulating F-actin severing thus maintaining normal F- to G-actin ratio in cells during apoptotic injury. High calcium (100-200 μM) has been reported to regulate the actin severing activity of both these proteins however, using fluorescence calcium imaging, we demonstrate that while intracellular calcium levels are elevated (<200 nM) during apoptosis they are not sufficient to activate the severing activities of either of these proteins. Further, we demonstrate that tyrosine phosphorylation which releases the autoinhibited conformation of villin and allows it to sever actin at physiological calcium concentration, regulates villin's anti-apoptotic function. Like villin, gelsolin is also tyrosine phosphorylated by c-src kinase. Consistent with this, villin mutants that lack the phosphorylation sites failed to protect epithelial cells from apoptotic injury. The results of our studies shed new light on the previously unrecognized function of villin and gelsolin in the regulation of apoptosis in the gastrointestinal epithelium. 506 L-Arginine Availability Is Essential for Colonic Epithelial Restitution Kshipra Singh, Rupesh Chaturvedi, Roxanne J. Wadia, Mohammad Asim, Nuruddeen D. Lewis, Daniel P. Barry, Keith T. Wilson Background: Disorders of epithelial integrity have been implicated in inflammatory bowel disease (IBD). Wound repair has been shown to require polyamines. L-arginine (L-Arg) is the substrate for arginase, which generates L-ornithine that is converted into polyamines by ornithine decarboxylase (ODC). We have reported that in mouse models of colitis, L-Arg supplementation improves disease, while inhibition of arginase or ODC worsens colitis. Our aim was to assess the role of L-Arg in epithelial restitution. Methods: C57BL/6 mice were treated with 4% dextran sulfate sodium (DSS) in the drinking water for 7 days to induce colitis. Young adult mouse colon (YAMC) cells were activated with the colitis-inducing pathogen Citrobacter rodentium as an In Vitro model. Expression of the L-Arg transporter cationic amino acid transporter (CAT)2 was assessed by real-time PCR. Mouse colonic epithelial cells (MCECs) were isolated by disassociation and filtration and were >90% positive for the marker E-cadherin. L-Arg transport was measured by uptake of [ 14 C]-L-Arg in MCECs and YAMC cells. YAMC monolayers were grown on fibronectin-coated plates and wounded with a silicone-coated drill tip, and % restitution determined after 24 h by image analysis. Results: There was a 2.5 ± 0.2-fold increase in CAT2 mRNA in DSS colitis tissues (p < 0.01). In MCECs from colitis tissues, there was an increase in ex vivo uptake of L-Arg from 1.6 ± 0.3 to 2.6 ± 0.1 nmol/min/mg protein (p < 0.05). In YAMC cells, C. rodentium increased CAT2 expression by 2.2 ± 0.2-fold (p < 0.01), and increased L-Arg uptake from 1.8 ± 0.3 to 4.3 ± 0.9 (p < 0.05); uptake was abolished by lysine (L-Lys, 20 mM), a competitive inhibitor of L-Arg uptake. In YAMC cells, restitution was dependent on L-Arg concentration in the medium; levels were increased from 20.3 ± 1.1% with 0 mM L-Arg to 41.9 ± 1.6% (p < 0.01) at 0.05 mM, and 54.6 ± 1.7% at 0.1 mM (p < 0.01), the serum concentration of L-Arg in mammals; at 1.6 mM L-Arg, there was only a small additional increase to 59.4 ± 1.4%. C. rodentium decreased restitution at all concentrations of L-Arg, by 15-25% (p < 0.05-0.01). L-Lys inhibited restitution by 46.6 ± 15.3% and 57.8 ± 11.1% in control and C. rodentium-stimulated cells (p < 0.01 for both). The arginase inhibitor BEC (60 μM) blocked restitution by 55.9 ± 14.5% (p < 0.01) in control and 38.2 ± 10.6% (p < 0.05) in C. rodentium-treated cells. Conclusions: Wound repair in CECs requires uptake of L-Arg from the extracellular environment and its metabolism by the arginase-ODC pathway. These findings indicate that L-Arg supplementation has a beneficial effect in colitis by ameliorating defective epithelial integrity. 507 Parallel Roles for Human Beta-Defensin 2 (Hbd2) and the Inflammatory Chemokine CCL20 in Regulating Restitution of Model Intestinal Epithelium Rebecca A. Vongsa, Michael B. Dwinell Background & Aims: Pathologic intestinal inflammation is exacerbated by break downs in the epithelial barrier and subsequent penetration of luminal microbes into the underlying mucosa. An intact barrier, chemokine signaling, and antimicrobial peptides provide the first line of protection against invading organisms. Due to its antimicrobial activities, human beta-defensin-2 (HBD2) is a critical host defense mechanism that has also been shown to stimulate migration of dendritic cells through binding to the chemokine receptor CCR6. As human colonic epithelium expresses CCR6 we sought to investigate the potential for HBD2 to stimulate intestinal epithelial migration. Methods: Epithelial migration of wounded mono- layers was assessed in non-transformed rat IEC6 cells and human intestinal CaCo2 cells