A1062 AGA ABSTRACTS • G4347 IL-18, A NOVEL IMMUNOREGULATORY CYTOKINE, IS UPREGULATED IN CROHN'S DISEASE: EXPRESSION AND LOCALIZATION IN INTESTINAL MUCOSAL CELLS. T.T. Pizarro, M. Huybrechts, M. Bentz, E. Foley, C.A. Moskaluk, S.J. Bickston and F. Cominelli. University of Virginia Health Sciences Center, Charlottesville, VA. IL-18, also known as interfernn-T-inducing factor (IGIF), is a novel cytokine originally isolated from mice sequentially injected with P. acnes and LPS. IL-18 is a potent inducer of IFNT production and plays an important role in Thl responses. Therefore, IL-18 may play a central role in the pathogenesis of chronic inflammation, particularly in Crohn's disease. The aim of the present study was to characterize the expression and localization of IL-18 in intestinal specimens as well as isolated mucosal ceils from patients with inflammatory bowel disease (IBD). Intestinal mucosal biopsies were obtained from non- involved and involved areas from Crohn's disease (CD) and ulcerative colitis (UC) patients, as well as from non-inflamed controls, to measure IL-18 protein levels by Western blot analysis (samples were standardized to 20 lag total protein/lane). To localize human IL-18 in the gut, surgically resected whole thickness intestinal tissues were obtained from patients categorized in the same aforementioned experimental groups, and processed for histological analysis and immunohistochemical staining for IL-18. Total cellular RNA was isolated from intestinal epithelial cells (IEC) and lamina propria mononuclear cells (LPMC) obtained from CD, UC and non-inflamed control patients and analyzed by semi-quantitative (multiplex) RT-PCR using IL-18 and GAPDH as the target and housekeeping genes, respectively. An 18 kD band, consistent with both recombinant and mature human IL-18, was found predominantly in CD vs. UC intestinal mucosal biopsies; a second band of 24 kD, consistent with inactive, pro-IL-18, was detected in unaffected areas from both CD and UC biopsies, and was the sole form found in normal uninflamed controls. Immunohistochemical analysis localized IL-18 to both IEC and LPMC, which appear to be consistent with a macrophage-like phenotype. Staining was more intense in CD specimens compared to UC and controls, and in the affected areas compared to the non-affected areas. Increased IL-18 mRNA transcripts were detected in freshly isolated IEC and LPMC from CD and UC patients compared to controls. Interestingly, IL-18 mRNA transcripts were more abundant in IEC compared to LPMC. In summary, our studies demonstrate that: 1) mature IL-18 expression is increased in CD intestinal biopsies compared to UC, but not detectable in controls, 2) conversely, the inactive "pro" from of IL-18 is increased in UC and control compared to CD, 3) IL°18 protein is localized to both IEC and LPMC, 4) IL-18 mRNA transcripts are more abundant freshly isolated IEC compared to LPMC. These results indicate that IL-18 may be an important cytokine in the pathogenesis of CD and that IEC may be a primary source of IL- 18 in the gut mucosa. Supported by NIDDK 42191 & 45740 (FC) and NIAID 40303 (TTP). • G4348 SLPI SUPPRESSES NF-KB ACTIVATION AND IL-8 EXPRESSION IN HUMAN COLONIC EPITHELIAL CELLS. T.T. Pizarro*, M.S. Le*, and F. Cominelli, University of Virginia, Charlottesville, VA 22908. *Share first name authorship. Secretory leukoprnteinase inhibitor (SLPI) is a potent serine proteinase inhibitor found primarily in the mucous secretions of surface-lining epithelium. Recent evidence suggests that SLPI may play an important role in reducing inflammation within the mueosal epithelial microanvironment. In fact, our group has previously reported the anti-inflammatory effects of SLPI in an animal model of acute colonic inflammation through a mechanism which involves cytokine immunomodulation. The precise mechanism(s), however, by which SLPI decreases gut inflammation has not yet been fully elucidated. In the present study, we examined the effect(s) of SLPI on the expression of inflammatory genes in colonic epithelial cells. Caco-2 cells, a human colonic cell line which expresses characteristics of enterncytic differentiation when grown to confluency, were preincubated for lh in the presence or absence of human recombinant (hr) SLPI (10 lag/ml) and subsequently stimulated with hrlL-1[3 (10 ng/ml). Cells were collected at 0, 0.5, 1, 1.5, and 2h following IL-113 stimulation, and nuclear extract and total cellular RNA were isolated for electrophoretic mobility shift assay (EMSA) and RT-PCR analyses, respectively. EMSA was performed for detection of the transcriptional factor NF-KB and demonstrated an almost 3-fold decrease in IL-1 induced NF-KB activation with SLPI pre-treatment of Caco-2 cells, with maximum inhibition seen at lh after IL-1 stimulation. IL-1 induced expression of the IL-8 gene, a known cytokine under NF-KB regulation, was determined by RT-PCR and showed that IL-8 mRNA transcripts were present as early as 0.5h and persisted up to 2h following IL-1 stimulation of Caco-2 cells. Pretreatment with SLPI, however, decreased IL-1 induced IL-8 gene expression more than 3-fold at 1.5h and up to 6-fold at 2h after IL-1 stimulation. In summary, our results demonstrate that SLPI functions as an anti-inflammatory factor by inhibiting NF-~:B activation as well as decreasing steady-state levels of IL-1 induced IL-8 gene expression in human colonic epithelial ceils. These data provide further evidence that SLPI plays an important role in early barrier immune responses and may possess significant therapeutic value against inflammatory assaults to the gut epithelium. Supported by NIAID40303 (TFP) and NIDDK42191 & 45740 (FC). GASTROENTEROLOGY Vol. 114, No. 4 G4349 COMPLICATIONS AND ACCEPTANCE OF DIAGNOSTIC COLON- OSCOPY IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE. C, P0hl M. Behnke, T. TepaB, P. Bernards and W. Kruis. Innere Abteilung, Evangelisches Krankenhaus Kalk, Cologne, Germany. Background: Endoscopic colonoscopy is a diagnostic procedure with low complication rates. As inflammatory bowel disease represents generally with a structurally damaged and inflamed intestinum higher complication rates and higher patient discomfort are to be expected. No data are available on complications and acceptance of diagnostic colonoscopy in patients with inflammatory bowel disease. Methods: In an open prospective study 105 consecutive patients with known inflammatory bowel disease underwent ileocolonoscopy. Pain and discomfort was assessed by examiner and patient independently using a 0-5 point scale. Clinical complications as perforation, bleeding, hypotension or discomfort/pain were determined at 3 and 24h after the end of the procedure. Further the degree and extension of inflammation, examination time and technical difficulty, number of biopsies and dosage of analgosedation was documented. Results; Among the 105 procedures in patients with Morbus Crnhn (n61, activity in 20 severe, 21 moderate, 12 mild, 7 in remission) and ulcerative colitis (n44, average CAI 5,93, 95% range 4,7-7,1, min 0, max 12) no severe complications were observed at 3 and 24h after the examination. Four minor complications (Ix mild hypoxia, lx mild hypotension, lx mild bleeding after biopsy, lx colicky pain) were observed at 3 hours which all had resolved on symptomatic treatment at 24h. The terminal ileum or the neoterminal ileum after surgery (8/105) respectively was reached in 89/105 cases (85%). Causes for incomplete examination (16/105) were stenosis (4), pain (2), insufficient cleansing (2) and technical problems (8). Of the latter 4 were encountered by the least experienced examiner. The average examination time was 19,5 min (95% range 17,9-21,8, min 4, max 95), an average of 12 biopsies (95% range 11-14, min 0, max 37) was taken during this time. The average discomfort experienced by the examination was judged as 0,7 (95% range 0,51-0,86, min 0, max 5) by the examiners and 0,7 (95% range 0,47-0,93, min 0, max 4) by the patients on a 0 (no pain) to 5 (unbearable pain) point scale. No significant discrepancy between patient and examiner assessment was found. The experience of the examiner (< 5.000 examinations, 500-5.000 examinations, < 500 examinations) had no influence on complication rate nor on patient acceptance, examination time and dosage of analgosedation. Conclusion: Our results demonstrate that diagnostic colonocscopy has a remarkably low rate of complication and good acceptance even in patients with active inflammatory bowel disease that compares well to the complication rates reported for unselected populations. G4350 WHAT CAN URINE TEACH US ABOUT INTESTINAL DEFENSE? MA Poles. E Martin-Porter, JS Lee, J Naitoh, P Anton, T Ganz UCLA School of Medicine, Division of Digestive Diseases, Microbiology and Immunology, and Urology. The gastrointestinal (GI) mucosa, the largest surface of interaction between humans and their environment, constitutes an effective barrier to microbial penetration. Some components of this barrier, e.g. IgA and lysozyme, have been extensively investigated. More recently, small antibiotic peptides, defensins, have been identified as an integral component of innate mucosal defense. They are cationic peptides with a broad spectrum of antimicrobial activity. Human intestinal defensin (HD-5) has been immunolocalized to the Paneth ceils of the small intestine by means of polyclonal antibodies elicited against recombinant HD-5. Purification of naturally-occurring intestinal defensins and the investigation of their role in GI immunity have been hampered by difficulty obtaining sufficient material for study. To circumvent this problem we studied urine collected from ileal neobladders. These neobladder constructs, formed after bladder resection for carcinoma, are known to retain their Paneth cell population. We analyzed urine from 5 patients before and after neobladder formation and from 5 normal subjects. The urine was mixed with a weak cation exchange matrix to extract the defensins which were subsequently purified by reverse phase HPLC. Isolation of natural HD-5 was facilitated by means of polyclonal antiserum against its recombinant form. Since HD-6 has previously been described only at the nucleic acid level, the corresponding peptide was identified by its similarity to other defensins with respect to mobility in acid-urea PAGE and retention time in reverse phase HPLC. The patterns of defensin production varied between subjects with some overlap. No defensins were detected in the urine before neobladder formation or in urine from the normal subjects. Ileal neobladders are an excellent source of human intestinal defensins. This study describes, for the first time, isolation and sequencing of naturally- occurring HD-5 and HD-6, and suggests methods for further characterization of their role in GI defense. G4351 COMPARISON OF ENZYME-LINKED IMMUNOSORBENT ASSAY AND WESTERN BLOTTING IN THE DETECTION OF ANTI- CRYPTOSPORIDIAL ANTIBODIES IN THE INVESTIGATION OF A WATERBORNE OUTBREAK OF CRYPTOSPORIDIOSIS. RCG Pollok. MM Aljenaibi, J McLauchlin 1, MP Kelly, MJG Farthing. Digestive Disease Research Centre, St Bartholomew's & The Royal London School of Medicine & Dentistry, 1PHLS, London, UK. Introduction. Waterborne outbreaks of cryptosporidiosis have become a major problem in both the USA and Europe. Data on population exposure and