monolayers and rotavims infected piglet jejunal tissue, using immunohistochemical tech- niques. Methods Confluent intestinal epithelial cells (IEC-6) were infected with Ad5FAK- CD (dominant negative version of FAK lacking the C-terminal kinase domain) or Ad5Luc (contains the luciferase reporter gene 5' of the adenovirus promoter). Cells were subsequently razor-injured, and surface area where cells had migrated was quantified at 24h. Western blots, using an antibody directed to the carboxy-terminus of FAK, revealed a dense band at MW 42-44 Kda, indicating FAK-CD expression. Immunostaining of p70s6k was performed on razor-injured IECG-cells and normal and virus- infected piglet jejunal epithelium. Results. FAK-CD transfection did not entirely block basal migration, but reduced migration by 33%. FAK-CD transfection inhibited by 100% the cell migration stimulated by ARC,. In contrast, control transfection with adeno-luciferase had no significant inhibitory effect, with cells migrating at >3.5x the control rate after treatment with ARG. Rapamycin (10 nM) blocked the cell migration response to ARG. immunoreactivity of p70sGk was noted in the cytoplasm and nucleus and of Ser427-phosphorylated-p70sGk mainly in the nucleus of all cells, espe- cially those at the leading edge. In intestinal epithelia, immunoreactivity of p70s6k was much higher in rotavirus-infected piglet jejunal epithdium than that in normal tissue. The site of maximal p70s6k immunoreactivity was at the restituting villus tip in the epithelial cells. Conclusions: FAK and ribosomal p70s6k play critical roles in intestinal cell migration. The immunoreacti'aty of pT0sGk is strongly localized to rotavirus-infected villus epithdinm, which suggests that p70s6k plays an important rule in repair of the intestinal epithelium in viral enteritis.

861

Pioglitazone, a Specific PPAR~y Ligand, Accelerates Ulcer Healing. Importance of Proinflammatory Cytokines, Cyclooxygenase-2, Inducible NO-Synthase and Heat Shock Protein 70 Expression Peter Konturek, Tomasz Brzozowski, Joanna Kania, Robert Pajdo, Wieslaw Pawlik, Eckhart G. Hahn, Stanislaw J. Konturek

The peroxisome proliferator-activa ted receptor gamma (PPAR'y) is a ligand-dependent nuclear receptor that has been implicated in the control of metabolism and numerous cellular processes including cell cycle control, carcinogenesis and inflammation. The present study was designed to determine the effect of increasing doses of pioglitazone, the specific PPAR'y ligand, on the healing of chronic gastric ulcers in rats and mucosal expression of mRNA and protein for proinflammatory cytokines (ILI~, TNFa), cyclooxygenases (COX-1 and COX-2), heat shock protein 70 (HSPT0), constitutive and inducible nitnc oxide synthases (cNOS and iNOS) and PPAR'y by RT-PCR and Western Blot Rats with ulcers produced by acetic acid method (ulcer area 28 ram2) were treated daily with vehicle (saline) or pioglitazone applied i,g in graded doses of 5, 20 and 40 mg/kg, Animals were sacrificed 7 days after ulcer induction, the ulcer area was measured by planimetry, and the gastric blood flow (GBF) was determined by H2-gas clearance method. Treatment with pioglitazone reduced the area of gastric ulcers in dose-dependent manner and this effect was accompanied by a significant increase in GBF at the ulcer margin. The mucosal expression of ILl]3, TNFa, iNOS and COX-2 either for mRNA and protein was significantly attenuated while expression of COX-1 and cNOS mRNA and protein were not influenced. HSP70 mRNA and protein expression was significantly upregulated in animals treated with pioglitazone. The PPARy mRNA and protein expression was significantly increased in the ulcerated mucosa as com- pared to those detected in non-ulcerated gastric mucosa. We conclude that pioglitazone accelerates the healing of preexisting ulcers due to the hyperemia at ulcer margin and the antiinflammato D' action including suppression of proinflammatory cytokines, downregula- tion of COX-2 and iNOS at the level of mRNA and protein and an overexpression of HSP70, which plays an essential role in the mechanism of gastric defense and ulcer healing.

862

Reciprocal Regulation of HO-1 and iNOS in Intestinal Epithelial Cells in Response to Oxidative Stress Gerard Dijkstra, Lisene Bok, Manon Homan, Hans Blokzijl, Peter Jansen, Han Moshage

Background: Inducible Nitric Oxide Sypthase (iNOS) is expressed in intestinal epithelial cells of patients with active inflammatory bowel disease and in epithelial cells of endotoxemic rats. The induction of iNOS in epithelial cells is a NF-kB mediated survival pathway of epithelial cells. Inflammation induced oxidative stress might compromise this NF-kB medi- ated survival response. The transcription factor AP-1 is induced by oxidative stress and one of its target genes is heine oxygenase 1 (HO- 1 ) The enzyme HO- 1 produces carbon monoxide (CO) which may attenuate the inflammatory response. Aim: To determine the effects of oxidative stress on iNOS and HO-1 induction in intestinal epithelial cells. Methods: In endotoxemic rats (in vivo model) the thiol-modifying agent diethylmaleate (DEM) and in human coloncarcinome cells DLD-1 (in vitro model) both DEM and the lipid peroxidation end product 4-hydroxynonenal (4-HNE) were used to induce oxidative stress. Rats were treated with DEM (4mmol/kg ip) 0.5 hr before and 3 hrs after LPS injection (5mg/kg ip). Rats were sacrificed 6 hr after LPS administration. DLD-1 human colon carcinoma cells were exposed to a cytokine mix (CM) composed of IL-118, TNF-,x and 1FN-y for 8 hr. DEM (1 mM) or 4-HNE (100 ~M) were added 0.5 hr prior to and 4 hr after addition of CM iNOS expression was evaluated by RT-PCR, Western blot and immunohiatology, the HO- 1 expression was evaluated by RT-PCR. Results: In vivo: LPS strongly induced iNOS and protein and mRNA expression in intestinal epithelial cells of ileum and colon but did not induce expression of HO-1 mRNA Combined treatement with LPS and DEM completely abolished iNOS expression but strongly induced HO-1 mRNA. In vitro: Cytokines induced iNOS mRNA and protein expression but did not induce HO-I mRNA in DLD-1 ceils. DEM and 4-HNE treatment prevented iNOS induction but increased the HO-1 mRNA in CM- exposed DLD-1 cells Conclusion: In the presence of oxidative stress NF-kB mediated iNOS expression is switched off and AP-1 mediated HO-1 expression is switched on in intestinal epithelial cells. These findings indicate that AP-1 and NF-kB regulate complementary defense mechanisms. AP-1 mediated defense mechanisms, like HO-1 are more important in inflam- matory conditions accompanied by oxidative stress, e.g. when antiomdant status is poor, whereas NF-kB-regulated defense mechanisms like iN OS are more important in inflammatory"

conditions accompanied by restricted oxidative stress exposure, e.g when antioxidant status is still intact.

863

Bone Marrow Derived Cells Contribute to a Population of Fibroblasts and Myofibroblasts which Engraft to Multiple Sites, Including the Gastrointestinal Tract Natalie C. Direkze, Stuart Forbes, Mairi Brittan, Toby Hunt, Rosemary Jeffery, Sean Preston, Richard Poulsom, Kairbaan Hodivala-Dilke, Malcolm Alison, Nicholas Wright

Our previous work has shown that the bone marrow can make an important contribution to myofibroblast populations in the lamina propria of the gut after bone marrow transplanta- tion in ammals and man (Gut 2002;50:752-7). Here we show that the bone marrow can also contribute to fibroblast populations m sites of injury, and that in fact, the engraftment of cells destined to be myoflbroblasts from the bone marrow appears to be a systemic phenomenon not confined to gastrointestinal tissues Methods C57/black female mice were irradiated with 12 Gray in divided doses to ablate the bone marrow followed immediately by tail vein injection of male wild type whole bone marrow. The mice received the lethal irradiation required prior to whole bone marrow transplantation and a further group received 2 doses of paracetamol at 5 and 8 weeks post transplantatmn at a dose of 400mg/kg I.P. Formalin-fixed paraffin-embedded sections were examined using in situ hybridisation to detect Y-chromosome and immunohistochemistry for intermediate filaments and cytoskeletal elements. Results: examination of the intestine and stomach showed numerous myofibroblasts of bone marrow origin distributed throughout the gut wall. In addition, areas of fibrosis contained numerous Y chromosome positive cells with a fibroblastic phenotype. Myofi- broblast engraftment was also demonstrated in the skin, adrenal capsule, lung and kidney. Conclusions: we conclude that bone marrow provides a circulating population of cells which can contribute to myofibroblasts in healing tissues. Our data also suggest that these cells can then adopt a fibroblastic phenotype and contribute to fibrosis. We therefore hypothesize that circulating bone marrow-derived precursors are available which are able to colonize injured tissues and give rise to myofibroblasts and then fibmblasts. These conclusions have important connotations for tissue repair in gastrointestinal and other tissues.

864

Increased Kinase Suppressor of RAS Activity in Inflamed Intestinal Mncosa Fang '/an, Sutha K John, Guinn Wilson, D.-Brent Polk

Background: Cytokines such as tumor necrosis factor (TNF) play a pathogenic rol~ in the development of inflammatory bowel diseases (IBD). Yet, the relationship between cytokine- induced signal transduction pathways and the development of IBD remains unclear. We reported kinase suppressor of Ras (KSR) is an essential upstream regulatory Thr/Ser kinase for TNF-stimulated cell survival signaling pathways, including ERKAVlAPK, NFkB and Akt. The purpose of this study is to evaluate the kinase activity of KSR in inflamed intestinal mucosa using the interleukin 10 deficient (IL-10 /) mouse, which spontaneously develops chronic IBD around 12 weeks of age. Methods: Wild type (w0 Balb/C or IL-10" (Bulb/C) mice were raised under conventional housing. 1L-1W mice developing rectal prolapse, characteristic of IBD in this model, their well litter controls and age-matchad wt Balb/C mice were sacrificed and 1 cm segments of the colon were fixed for histological analysis and injury score, immunohistochemistry, and the remainder prepared for biochemical analysis. Mucosal homogenates were prepared for immunoisolation of KSR and Raf-1 or Western blot to detect the activation state of signaling molecules or the Thr and Ser phosphorylation of KSR and Raf-1. Results: IL1W mice with prolapse show increased (P < 0001) mean inflammatory index of 11 (Max 15) compared to well littermates or age-matched wt Balb/ C (3, 1.1 respectively). KSR is predominantly expressed in the intestinal epithelial ceils of both normal and inflamed mouse colon without detection in the submucosa. Both RSR and RaM imm~noprecipitated from inflamed ILl0 / colon mucosa show increased Thr phosphorylation, and increased kinase activiues using in vitro kinase assays, compared to those from Balb/C and healthy ILl0" littermates. The KSR-regulated signals, ERK, NFKB, and Akt are all increased in the inflamed IL-10 -/- mucosa compared to both controls. The p38 MAPK activity is increased only in the inflamed mucosa, as expected. Conclusions: The kinase activities of KSR and RaM and their downstream targets are all increased in the inflamed mucosa of mouse colon. Based on our previous findings that KSR kinase activity ~s required for intestinal cell survival in a high-TNF environment, we postulate increased KSR kinase activity may have a protective role in inflamed colon mucosa in chronic IBD. These findings have significant implications for understanding the mechanisms of intestinal epithelial cell survival mechanisms during the inflammatory response.

865

Importance of the a1131 Integrin in Acute and Chronic Intestinal Inflammation Kevin P. Pavlick, Christian F. Krieglstein, F. Stephen Laroux, Laura Gray, Jason M. Hoffman, D. Nell Granger, Antonin De Fougerolles, Matthew B. Grisham

Background: Recent evidence suggests that interaction between the leukocyte-associated collagen-binding integrms (e.g. e ,~ ) and components of the extracefiular matrix (ECM) promote migration and/or activation of extravasated leukocytes (e.g. T-cells, monocytes, neutrophila). Therefore, the objective of this study was to assess the role of leukocyte- associated Cll~ 1 in initiating and/or perpetuating acute and chronic colitis in vivo. Methods: Acute colonic inflammation was induced via oral administration of dextran sulphate sodium (DSS) to BALB/c mice for seven days. Transfer of CD4+CD45RB h'~h T-cells, from either wdd type (wt) or a~ deficient (oq / ) BALB/c mice, into Recombinase Activating Gene-l-deficient (RAG-1 ~) BALB/c recipients was used to induce chronic colitis. Disease activity (DA), blinded histopathology and cytokine mRNA levels were used to quantify colonic inflammation induced in both models of colitis. Results: Using DSS to induce acute colitis, we found that DA for DSS-treated cq" mice was significantly reduced by 62% (3.5 vs. 1.33 for wt vs. cx( mice, respectively). This attenuation in DA correlated with reductions in colon weight/length

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