April 1998 G3854 IL-8 IN EARLY AND CHRONIC ILEAL LESIONS OF CROHN'S DISEASE (CD). Eric Brandt l, Pierre Desreumanx ~,2, Dominique Emilie 3, Jean-Frtd&ic Colombel 2, Monique CapronL l: Unit6 INSERM U167, Institut Pasteur de Lille, France; 2: Laboratoire de Recherche sur les MICI (CRI4U004B), CHU de Lille, France; 3:Unit6 INSERM U131, Institut Paris Sud sur les Cytokines, Clamart, France. Background/aim: Distinct mucosal immunoregulatory cytokine patterns are observed during the evolutive stages of CD (1). 1L-8 upregulation is associated with an increased neutrophilic infiltrate in chronic colonic lesions of patients with CD. The aim of this study was to compare IL-8 mRNA levels in the colonic and ileal mucosa of patients with CD during the different clinical stages of the disease. Methods: 18 patients with CD who had ileocolonic resection with anastomosis were studied. No patient received immunosuppressive or steroid therapy during the study. Biopsies were obtained during surgery from the healthy ileal and colonic mucosa in 18/18 patients and from chronic ileal lesions in 15/18 patients. Endoscopic ileal biopsies were also taken from early recurrent ileal lesions (scored i3 or i4 according to Rutgeerts criteria) occurring three months after surgery in 9 patients. IL-8 mRNA levels were quantified by competitive PCR and expressed as the mean number of eDNA molecules of IL-8 per molecule of [3-actin. Results: IL-8 mRNA Healthy Healthy Early ileal Chronic ileal ileon colon lesions lesions n=18 n=18 n=9 n=15 mean (SEM) 225 (57) 105 (22) < 1 1204 (388) p p < 0.05 p < 0.01 Conclusions: Mucosal IL-8 expression in CD is downregulated in early post surgical ileal lesions and upregulated in chronic ileal lesions. These results further suggest a time dependent cytokine expression in CD with potential implication regarding anti-inflammatory treatments. [1] Desreumaux et al. Gastroenterology 1997; 113; 118-26. • G3855 LPS INDUCES UPREGULATION OF CD14 EXPRESSION AND TYROSINE PHOSPHORYLATION OF A 40 KD PROTEIN IN AN INTESTINAL EPITHELIAL CELL LINE. S.L. Brandwein. H.C. Reinecker, and D.K. Podolsky. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, MA. BACKGROUND; Recent reports support the importance of the normal enteric bacteria in the development of intestinal inflammation in a number of animal models. However, the mechanisms of bacterial modulation of the epithelium are not well defined. Previous studies of interleukin-2 deficient mice have demonstrated that intestinal epithelial cells express CD14, the receptor for lipopolysaccharide (LPS); furthermore, CD14 expression was upregulated in intestinal epithelial cells prior to the onset of colitis. The purpose of the current study was to characterize intestinal epithelial cell responses to LPS using a model cell line. METHODS: CMT-93 cells, a murine intestiaal epithelial cell line, were stimulated with increasing concentrations of LPS derived from E. coil (0 to 2 lag/ml). Cells were harvested after LPS exposure to assess CD14 expression and tyrosine phosphorylation of cellular proteins. CD14 mRNA expression was determined using RT-PCR on stimulated and unstimulated CMT-93 cells. CD14 protein expression was measured by Western blotting of cell lysates. Tyrosine phosphorylation was assayed by immunoblotting with anti- phosphotyrosine antibodies. RESULTS: RT-PCR results demonstrated constitutive expression of CD14 mRNA by CMT-93 cells. CD14 protein at baseline was not detectable by Western blot analysis. However, increasing concentrations of LPS induced dose-related increases in CD14 protein expression by CMT-93 cells. Production of CD14 was also time-dependent; CD14 expression increased in CMT-93 cells with time of incubation with LPS. LPS stimulation of CMT-93 cells was associated with tyrosine phosphorylation of a 40 kDa protein. This phosphorylation peaked at three minutes after stimulation and disappeared by 30 minutes. CONCLUSION: These studies demonstrate that a murine intestinal epithelial cell line, CMT-93, responds to LPS stimulation by increasing CD14 expression. Thus, LPS may regulate the expression of its own receptor on intestinal epithelial cells. In addition, the LPS stimulation of CMT-93 cells caused intracellular signaling as demonstrated by the tyrosine phosphorylation of a 40 kDa protein. Interaction of intestinal epithelial cells and enteric bacteria may be regulated by receptor mediated mechanisms resulting in specific intracellular signaling events that lead to functional changes in the intestinal epithelium. Immunology, Microbiology, and Inflammatory Disorders A941 • G3856 A GENOME WIDE SCREEN OF CROHN'S DISEASE IN A LARGE PEDIGREE SHOWS EVIDENCE FOR LINKAGES TO CHROMO- SOMES 11, 16, 8 AND 15. Steven R. Brant 1, Dan Nicolae 2, Michele C. LaBuda 1, Romulo Baltazar 1, Carter Fields, 2 Geoffrey Ravenhill, 1 Mike Pickles 2, Patrick M. Rohal 1, Ethylin Wang Jabs l, Stephen B. Hananer 2, Theodore M. Bayless 1, Judy H. Cho. 2 lJohns Hopkins Univ. Baltimore, MD. 2University of Chicago Hospitals Chicago, IL. Mapping studies in Crohn's disease (CD) have been largely limited to pedigrees containing two or three affected relatives without power from any individual pedigrees to identify susceptibility loci or suggest a mode of inheritance. We identified a pedigree containing seven relatives with confirmed diagnoses of CD: the proband (generation IV) has one affected sibling, three affected first cousins - two of whom are siblings, an affected uncle (the father of the sibling cousins), and an affected first cousin once removed (generation III). The three sets of first cousins in generation IV were raised in geographically separate areas. All affected relatives have relatively severe CD; five required surgical resections, the other two required azathioprine. Six have ileal disease, four have colonic disease with only one having disease limited to the colon. Methods: A genome wide screen was performed with 365 autosomal markers on DNA samples from the 7 affected and 12 unaffected relatives. Multipoint non-parametric linkage analysis was performed with the program Genehunter. Haplotypes were generated using genehunter which utilizes the maximum- likelihood set of inheritance vectors that explain the data. Results: Regions on chromosomes 11, 16, 8, and 15 had p-values < 0.03 at the following markers: Dlls1986, Z = 3.23, p = 0.004; D16s403, Z = 2.76, p = 0.014; Dgs264; Z = 2.50, p = 0.025; and D15s652, Z = 2.41, p = 0.025. Genotyping additional markers on chromosome 16 demonstrated that the locus is inherited in a dominant pattern and that six of the seven affecteds share a four marker haplotype (D16s403, D16s401, D16s769, and D16s753) that spans 13 cM. This region overlaps the previously defined IBD] CD susceptibility locus (Hugot et al., 1996). The boundaries of the IBD1 locus are defined by flanking markers D16s764 and D16s411. Six of the seven affecteds (not the same six) share a three marker common haplotype on chromosome 11 that spans 18cM, Conclusion: IBD1 and at least one other susceptibility gene are likely responsible for CD in this family, as one affected (child of the uncle with CD) does not appear to inherit the common IBD1 haplotype. Studies in additional pedigrees will be needed to determine if the regions on chromosomes 11, 8 and 15 define new CD susceptibility loci. • G3857 CLINICAL EFFICACY OF ADDITIONAL FXIII TREATMENT IN STEROID REFRACTORY PATIENTS WITH ULCERATIVE COLITIS. N. Bregenzer. I. Caesar, T. Andus, H. Malchow, S. Schreiber., J. Schtlmerich, and the Factor XIII study group, Department of Internal Medicine University of Regensburg, Germany. Introduction: Patients with acute ulcerative colitis have decreased levels of FXIII activity. Recent uncontrolled studies showed a beneficial effect of Factor XIII which is important for cell formation and wound healing on clinical symtoms of ulcerative colitis, in particular intestinal bleeding. The objective of this study was to evaluate the benefits of additional FXIII treatment in steroid refractory patients with ulcerative colitis in a prospective, double blind, placebo-controlled study. Methods: Primary objective was the time until cessation of visible intestinal bleeding within 14 days after the start of treatment, secondary efficacy criteria were the CAI Score (Rachmilewitz), a colo-/sigmoidoscopy score (Baron) and a histological score (Truelove). Patients were treated over 10 days either by i.v. application of FXIII concentrate (3750 U day 0. 1250 U days 1-9) or by placebo. The study was prematurely stopped in December 1996 after a total of 28 patients had been enrolled, because a planned blinded interim analysis of the primary and secondary efficacy criteria showed no differences between group A (placebo) and B (FXIII). Results: The study was designed to demonstrate a reduction of the bleeding probability on day 14 from 70% to 30% at the 5% significance level with a power of 90%. Interim analysis of the primary efficacy criteria showed, that 71% of the F XIII and 73% of placebo treated patients were still bleeding (p=0,8). Additional exploratory analysis did not reveal noteworthy differences between groups. No serious adverse events were reported. Since this analysis makes it obvious that no significant effect could be expected with the planned patient sample size the study was terminated. Conclusion: Overall the study showed no beneficial effect of FXIII substitution on active steroid refractory ulcerative colitis, these results do not confirm to previous open label studies which reported an improvement of clinical symptoms. This research was funded by Centeon Pharma GmbH.