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Symposium: Gut flora in IBD—practical importance 105–107

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105 DIAGNOSIS OF CROHN'S DISEASE IS ASSOCIATED WITH INCREASED LEVELS OF ANTIBIOTIC USE OVER THE PRECEEDING FIVE YEARS

T.R. Card1, R.F.A. Logan1, L.C. Rodrigues2, J.G. Wheeler2.1Division of Public Health Sciences, University of Nottingham, Queen's Medical Centre; 2London School of Hygiene and Tropical Medicine, UK

Background: Although it is generally accepted that Crohn's disease has both genetic and environmental determinants, few environmental determinants are well established. In view of the widely supported notion that dysfunction of the gut is inter-linked with its flora, we explored the potential importance of the use of antibiotics as a risk factor.

Methods: We selected records of incident cases of Crohn's disease from the General Practice Research Database. All cases were diagnosed after 1992, had no history of Ulcerative Colitis and at least five years of GPRD data prior to diagnosis. Controls with five years complete data were randomly selected. Data were extracted on smoking, presentation to GP with a diagnosis of infection, oral contraceptive use (OCP) and antibiotic use 3–5 years prior to the date of diagnosis of Crohn's disease (and for a comparable period for controls). Data on antibiotic use was restricted in time to exclude those that could have been prescribed as treatment of premonitory symptoms. Logistic regression was used to investigate the relationship between antibiotic use and Crohn's disease.

Results: 601 Crohn's disease and 1460 controls were available for analysis. We found statistically significant associations between Crohn's disease and use of OCP in the year before diagnosis (Odds ratio 1.7, P=0.003) and smoking (Odds ratio 1.5, P=0.001) despite incomplete data for the latter (these are consistent with the literature). Antibiotic use 3–5 years pre-diagnosis was significantly greater in cases than controls. Only 29% of cases compared to 42% of controls received no antibiotics (P<0.001), and the mean number of courses was 2.6 in cases and 1.9 in controls (P<0.0001). Adjusting for age, sex, smoking and use of OCP, antibiotic use had an odds ratio of 1.6 (1.3–2.0) (P<0.001). There was a highly significant trend with increasing numbers of courses (P=0.001). The population attributable fraction for antibiotic use was 28% (18%-36%).

Conclusions: There is a highly significant association between Crohn's disease and prior antibiotic use in this data. This is unlikely to be explained by reporting bias in view of the prospective recording of all prescriptions in GPRD. If this association is causal then it may explain about a quarter of all Crohn's disease.

106 MICROBIAL CELL WALL OLIGOMANNAN INHIBITS THE NEUTROPHIL RESPIRATORY BURST: A POSSIBLE MECHANISM FOR GRANULOMA FORMATION IN CROHN'S DISEASE?

C.M. Mpofu, J.M. Rhodes, S.W. Edwards.Department of Medicine and School of Biological Sciences, University of Liverpool, Liverpool L69 3GA, UK

Background: Crohn's like intestinal lesions occur in Chronic Granulomatous Disease, a condition caused by a defect in phagocyte function. Crohns disease patients commonly have serum antibodies to baker's yeast (Saccharomyces cerevisiae). The epitope for this antibody is oligomannan which is present in bacterial and yeast cell walls. Our hypothesis is that oligomannan, shed by intramucosal bacteria, may inhibit neutrophil and macrophage function within the mucosa, leading to the granulomatous lesions seen in CD.

Methods: The effect of S. cerevesiae oligomannan on phorbol ester (PMA) induced-respiratory burst of neutrophils from healthy volunteers was measured by luminol-amplified and isoluminol-amplified chemiluminescence and by the cytochrome c reduction method. Neutrophils were isolated using a one-step centrifugation method and suspended in HBSS buffer.

Results: See fig 1. At the highest concentration used (1 mg/mL), oligomannan inhibited luminol dependent chemiluminescence by 68 % (± 5 %, n = 6). Luminol chemiluminescence measures the combined activities of the neutrophil NADPH oxidase and myeloperoxidase, and so the oligomannan could be acting on either or both of these activities. However, oligomannan (at 1 mg/mL) also inhibited PMA-stimulated isoluminol-amplified chemiluminescence and cytochrome c reduction. As isoluminol and cytochrome c reduction largely measure extracellular superoxide secretion, the oligomannan may have an effect on NADPH oxidase activity.

Conclusion:S cerevesiae oligomannan causes dose-related inhibition of the PMA-induced respiratory burst. It has extracellular and probably intracellular effects. This supports the hypothesis that microbial oligomannans impair mucosal phagocyte function, thus generating the granulomatous phenotype of Crohn's disease.

107 REDUCED BIFIDOBACTERIA AND INCREASED E. COLI IN RECTAL MUCOSA-ASSOCIATED FLORA IN ACTIVE INFLAMMATORY BOWEL DISEASE

N. Rayment, M. Mylonaki, B. Hudspith, J. Brostoff, D.S. Rampton.Infection and Immunity Group, Division of Life Science, Kings College London; Academic Dept of Adult & Paediatric Gastroenterology, Barts & The London School of Medicine, London, UK

Background: Colorectal bacteria probably contribute to the pathogenesis of IBD. To test the hypothesis that potentially protective bacteria are reduced and pathogenic flora increased, we have compared the mucosa-associated flora in IBD and controls.

Methods: Snap-frozen rectal biopsies were taken at routine colonoscopy from patients with ulcerative colitis (UC), Crohn's and controls with normal colorectal mucosa. Fluorescent in situ hybridisation was used separately to count numbers of mucosa-associated bifidobacteria, lactobacillus, E.coli, clostridia and sulphate-reducing bacteria.

Results: Bacteria were sited on, and superficially within rectal mucosa. Mucosa-associated bifidobacteria counts (median 14/hpf (range 4–65), n=13) in active UC were lower than in controls (79 (0–146), n=24, P=0.008); the difference from quiescent UC (38 (0–118), n=19) did not reach statistical significance (P=0.07). Conversely, E.coli counts were higher in active UC (80/hpf (30–186)) than in controls (0 (0–16), P=0.0002) and inactive disease (2 (0–144), P=0.003). Similar results were found in patients with active Crohn's colitis (n=4). In 6 patients with UC who had biopsies from 2 sites, inflamed mucosa always showed fewer bifidobacteria (P=0.008) and more E.coli (P=0.008) than normal-looking more proximal colon. There were no differences, between IBD and controls, of quantitative counts of other bacteria.

Conclusions: The reduction in mucosa-associated bifidobacteria and increase in E.coli in active IBD supports the hypothesis that a deficiency of potentially beneficial bifidobacteria and excess of E.coli could play a role in the causation of IBD.

107A LOCAL IL-10 GENE THERAPY INDUCES COLONIC IL-10 RELEASE AND IS THERAPEUTIC FOR MURINE COLITIS

J.O. Lindsay, C.J. Ciesielski, T. Scheinin, F. Brennan, H.J.F.Hodgson.The Division of Medicine and The Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, London, UK

Introduction: Interleukin-10 knockout (IL-10 -/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, the intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long-term disease suppression with profound systemic immunoregulatory changes.

Aims: To determine whether the rectal delivery of AdvmuIL-10 will induce localised colonic IL-10 expression without systemic immune-suppression, and assess its therapeutic efficacy in IL-10 -/- mice with established colitis.

Results: A single rectal infusion of 5x108 PFU AdvmuIL-10 to 10 week IL-10 -/- mice induced a median of 27.3 pg/mg IL-10 in colonic homogenates harvested after one week. In contrast, the IL-10 concentration of liver and spleen homogenates did not differ significantly from the background seen in PBS treated controls. IL-10 -/- mice with established colitis were treated with 5x108 PFU AdvmuIL-10, empty cassette adenovirus (Adv0), or PBS vehicle by rectal infusion (n=10/group). The mean clinical score in the AdvmuIL-10 group fell from 1.8 ± 0.13 to 0.4 ± 0.16, whereas the clinical scores increased from 1.4 ± 0.27 to 2.5 ± 0.27 and from 1.8 ± 0.22 to 2.6 ± 0.13 in the PBS and Adv0 treated groups respectively (p<0.001, 2-way ANOVA). In addition, the stool concentration of IL-1β over the four-week experiment was significantly higher in mice treated with saline or Adv0 than those treated with AdvmuIL-10 (p<0.01). Finally, local AdvmuIL-10 therapy had no effect on TNF-α release from stimulated splenocytes.

Conclusion: Local AdvmuIL-10 therapy reverses colitis in IL-10 -/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.

107B INFLAMMATORY BOWEL DISEASE IS ASSOCIATED WITH FUNCTIONAL TNF POLYMORPHISM AFFECTING OCT1/NF-Kb INTERACTION

D.A. van Heel, I.A. Udalova, A.P. De Silva, D.P. McGovern, Y. Kinouchi, J. Hull, N.J. Lench , L.R. Cardon, A.H. Carey, D. Kwiatkowski, D.P. Jewell.University of Oxford, UK

Introduction: The tumour necrosis factor-alpha (TNF) gene lies within a replicated inflammatory bowel disease (IBD) genetic susceptibility locus (6p21, IBD3); and TNF is clearly implicated in IBD pathogenesis.

Aims: To assess genetic associations of TNF promoter variants in IBD and study the functional biology of associated variants.

Methods: Association studies of the common TNF polymorphisms (-1031,-863,-857,-308). Two independent cohorts were used (set A, 457 IBD families: 294 Crohn's disease (CD) trios, 252 ulcerative colitis (UC) trios; set B 130 IBD families and 278 healthy controls (HC)). Functional studies of –857C/T used: LPS stimulated whole blood TNF ELISA in 46 healthy controls; monocyte nuclear extract/ promoter construct electro-mobility shift assays; in vitro GST pull-down assays of transcription factors OCT1, NF-kB, and deletion mutants; in vivo COS cell immuno-precipitation and COS cell luciferase reporter gene analysis.

Results: TNF –857C was associated with IBD in both set A and replication set B, using case control and family based (TDT) association analyses. Numbers homozygous for TNF-857C were IBD=90.9% (n=587 unrelated cases, P=0.001 vs HC 83.1%), NOD2 negative CD=92.1% (n=241, P=0.002), UC=92.1% (n=304, P=0.001). Higher stimulated TNF production was seen in whole blood from TNF-857C homozygotes (P=0.03) at 2 hours. The OCT1 transcription factor bound the TNF-857T but not the TNF-857C allele, adjacent to a NF-kB site. The DNA binding domains of OCT-1 and NF-kB p65 interacted in vitro and in vivo. OCT1 diminished NF-kB induced reporter gene expression.

Conclusion: The TNF-857C allele is associated with IBD, and TNF-857C homozygotes show higher TNF production. We have identified a molecular mode of action, through allele specific binding of OCT1 to the TNF promoter and interaction between OCT1 and NF-kB.

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