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sCXCL11, CCL5, CCL23 and CCL21 identified epithelial and/or stromal cell localisation.Conclusion: Dysregulation of several inflammatory genes is a key feature of pre-cancerouscolonic polyps. These chemokines are of biological interest as most have a central role ininflammatory response and are involved in key cell processes that are linked to the develop-ment of malignancy. The study of these markers will provide further insight into how chronicinflammation may mediate neoplastic progression in sporadic colorectal carcinogenesis.

S1997

Paxillin Produces a Molecular Switch to Converge Helicobacter pylori-InducedIntracellular SignalingFazal H. Tabassam, David Y. Graham, Yoshio Yamaoka

Background: Helicobacter pylori infection modulates motility, cytoskeletal reorganization,and adhesion in gastric epithelial cells. Initial signaling events causing these phenotypicchanges remain unknown. Paxillin, a multidomain adaptor protein plays an important roleto regulate actin cytoskeletal organization, focal adhesion formation, and cell motility. Aim:To investigate role of paxillin in H. pylori-mediated phenotypic changes in gastric epithelialcells. Methods: Gastric epithelial cells AGS, MKN28 or MKN45 were co-cultured with livewild type H. pylori or their isogenic cag pathogenicity island (PAI) or oipA mutants to assessthe effects on paxillin activation and changes in cytoskeleton by immunoblot, immunoprecip-itation and fluorescence microscopy. Results: H. pylori induced tyrosine phosphorylationof paxillin sites Y31 and Y118 in a dose-and time-dependent manner. Interestingly, phos-phorylated paxillin, colocalized with H. pylori-induced actin stress fibers, formed signalingcomplexes with focal adhesion kinase(FAK)and H. pylori injected CagA to influence down-stream signaling. FAK-specific siRNA or selective inhibitors of EGFR, Src or PI3K significantlyreduced H. pylori-induced paxillin activation and actin cytoskeletal changes suggesting theyhave roles in paxillin activation and development of the hummingbird phenotype. Infectionof gastric cells with oipA mutants significantly reduced phosphorylation of paxillin Y31 andY118, resulted in inhibition of stress fiber formation and altered cell morphology. In contrast,cag PAI mutants only reduced phosphorylation of paxillin Y118 with less effect on stressfiber formation compared to oipA mutants. Conclusion: OipA plays an important role inH. pylori-induced earlier cellular events through involvement of EGFR/PI3K and Src signalingresulting in phosphorylation of paxillin Y31 and Y118 and phenotypic changes related tocellular phenotype and/or changes in cell motility. We propose that activation of paxillinproduces a molecular switch to converge H. pylori-induced intracellular signaling leadingto the morphologic changes describe as the hummingbird phenotype.

S1998

Sustained Procoagulant and Inflammatory Response Following Esophagectomy,and the Impact of Neoadjuvant ChemoradiationMiriam Byrne, John V. Reynolds, Donal Hollywood, Graham P. Pidgeon, Barry White,Narayanasamy Ravi, Patrick J. Byrne

Objective: To assess the procoagulant and inflammatory response in esophageal cancer to(a) neoadjuvant chemoradiation (nCRT) and (b) esophagectomy Background Data: Theprocoagulant and inflammatory response are intimately related, and may be associated withclinical sequelae in sepsis, trauma or following surgery. The impact of esophagectomy andthe potential added influence of nCRT on the procoagulant pathway is unknown, andvenous thromboembolism (VTE) prophylaxis remains empirically-based and in most centresrestricted to the in-hospital postoperative period. Methods: In a prospective study patients(n = 80) with localized esophageal cancer had the following studies performed pre andduring nCRT and on postoperative days (POD) 1, 3, 7, 14, 21, 28, and at 3, 6 and 9months: D-dimers; PT; APTT; fibrinogen; factor-VIII; protein C; protein S; antithrombin;thrombin anti-thrombin complex (TATC); prothrombin fragment (PF) 1+2 ; a panel of 10cytokines and growth factors; and NFκB activation in peripheral blood mononuclear cells(PBMCs) . All patients had standard in-hospital peri-operative prophylaxis with low molecularweight heparin (LMWH). Results: Ten thromboembolic events were observed. Neoadjuvanttherapy significantly (p<0.05) increased PT, APTT, Fibrinogen, D-dimers, TATC, PF1+2 , andFactor-VIII levels, and these markers were further (p < 0.05) augmented by esophagectomy, aneffect sustained for 3 to 6 months. Surgery decreased (p<0.05) Protein C,S and antithrombinfor one week. NFκB activation followed nCRT and was evident postoperatively for twoweeks along with raised IL-6, IL- 10, EGF, VEGF and MCP-1. Conclusions: Esophagectomyis associated with early systemic immunoinflammation, inhibition of natural anticoagulationmechanisms, and a sustained prothrombotic pattern that persisted for up to 6 months.Neoadjuvant therapy activates the procoagulant pathway. These data suggest that VTEthromboprophylaxis during neoadjuvant therapy and beyond the in-hospital postoperativeperiod may merit evaluation.

S1999

Tumor Suppressor FOXO3A Is Involved in TNFα-Induced Regulation of IL-8in Intestinal Epithelial CellsLobke Snoeks, Christopher R. Weber, Kaarin Wasland, Suzana D. Savkovic

Introduction: Tumor suppressor Foxo3a belongs to the family of Forkhead transcriptionalfactors and regulates the expression of genes that modulate the metabolic state, proliferation,and apoptosis. In the absence of a stimulus, Foxo3a is retained in the nucleus. Stimulation withgrowth factors induces Foxo3a translocation to the cytosol and transcriptional inactivation.Proximally, proinflammatory IKK may regulate Foxo3a, moreover Foxo3a deficient micedevelop systemic inflammation. However, the regulation of Foxo3a and its role in inflamma-tion in intestinal epithelia is unknown. Aim: To examine the effect of TNFα on Foxo3a inintestinal epithelia and its role in inflammation. Methods: In Vitro studies were performedusing human HT-29 intestinal epithelial cells. We studied the effect of TNFα on Foxo3alocalization, phosphorylation and degradation by using immunofluorescent staining andimmunoblot. IKK inhibitor PS1145 was employed to examine proximal Foxo3a regulation.Silencing Foxo3a with siRNA was used to examine its role in IL-8 regulation. For In Vivo

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study we utilized C57BL/6J mice treated with 2.5% DSS for 5 days; Foxo3a distribution incolonic epithelia was assessed by immunohistostaining. Results: First, we assessed the effectof TNFα on Foxo3a in HT-29 cells. After TNFα treatment, nuclear Foxo3a translocates tothe cytosol and consequently degrades (51±6% at 3 h) in HT-29 cells. In Vivo, in untreatedcolonic epithelia, there is prominent nuclear Foxo3a localization, which is lost in DSS-treated colonic epithelia. These data suggest that in proinflammatory conditions Foxo3a isinactivated both In Vitro and In Vivo in colonic epithelia. Next, we examined the contributionof the TNFα-dependent IKK pathway in Foxo3a regulation. During TNFα treatment, IKKand Foxo3a interaction, as well as Foxo3a phosphorylation on IKK specific Ser644 site,increased. Also, IKK inhibition attenuated Foxo3α degradation, implicating IKK-dependentTNFα-induced Foxo3a inactivation. Subsequently, we examined the role of Foxo3a in TNFα-induced expression of proinflammatory cytokines such as IL-8 by siRNA experiment. InHT-29 cells with silenced Foxo3a, TNFα-induced IL-8 production increased ~70% (withoutsiRNA: control 27±2 pg/ml, TNFα 187±8 pg/ml; with siRNA: control 40±4 pg/ml, TNFα318±19 pg/ml, n=4, p=0.003 for TNFα treated without siRNA vs. with siRNA). Conclusions:1. TNFα induces Foxo3a inactivation in intestinal epithelia; 2. TNFα-induced Foxo3ainactivation is IKK dependent; 3. Foxo3a is additional regulator of IL-8 expression.

S2000

Expression of Apurinic/Apyrimidinic Endonuclease-1 (APE-1) in H. pyloriAssociated Gastritis, Gastric Adenoma and Gastric CancerSeiji Futagami, Tetsuro Hiratsuka, Tomotaka Shindo, Tatsuhiko Hamamoto, MasafumiKusunoki, Kazumasa Miyake, Taku Tsukui, Katya Gudis, Sheila E. Crowe, ChoitsuSakamoto

Background & Aims:Apurinic/apyrimidinic endonuclease-1 (APE-1) is a key enzyme in DNAbase excision repair (BER). However, little is known about the localization of APE-1 in H.pylori-infected gastric mucosa or its role in the development of gastric cancer. To investigatethe role of APE-1 in the development of gastric cancer, we examined APE-1 expression andlocalization in cultured cells and gastric biopsies from patients with H. pylori-infected gastritisor gastric adenoma and gastric cancer. Methods: APE-1 mRNA and protein expression weredetermined in H. pylori (CagA+) water-extract protein (HPWEP)-stimulated MKN-28 cells,AGS cells and human peripheral macrophages by real-time PCR and western blot analysis.APE-1 expression and 8-OHdG as a measure of oxidative DNA damage were evaluated byimmunostaining. Localization of APE-1 and IκBα phosphorylation in gastric adenoma andgastric cancer tissues were evaluated by single and double label immunohistochemistry.Results: In Vitro, HPWEP-stimulation significantly increased APE-1 mRNA expression levelsin both MKN-28 cells and human peripheral macrophages. Hypo/reoxygenation treatmentsignificantly increased APE-1 protein expression in HPWEP-stimulated MKN-28 cells.HPWEP stimulation significantly increased both APE-1 expression and IκBα phosphorylationlevels in MKN-28 and AGS cells. MG132 significantly reduced IκBα phosphorylation levelsbut not APE-1 expression in HPWEP-stimulated MKN-28 and AGS cells. In human tissue,APE-1 expression in H. pylori infected-gastritis without goblet cell metaplasia was significantlyincreased as compared to that in tissues from uninfected subjects. Eradication therapysignificantly reduced both APE-1 and 8-OHdG expression levels in the gastric mucosa. APE-1 expression localized mainly in epithelial cells within gastric adenoma and in mesenchymalcells of gastric cancer tissues. APE-1 expression in gastric cancer tissues was significantlyreduced compared to that in H. pylori-infected gastric adenoma, while 8-OHdG indexand IκBα phosphorylation levels did not differ between these two neoplastic tissue types.Colocalization of APE-1 and IκBα phosphorylation was observed in gastric adenoma cellsbut not in gastric cancer cells. Conclusion: H. pylori infection is associated with increasedAPE-1 expression in human gastric cancer cell-lines and in gastric tissues from subjects withgastritis and gastric adenomas. The distinct expression patterns of APE-1 and 8-OHdG anddifferent colocalization of APE-1 and IκBα phosphorylation in gastric adenoma and gastriccancer tissues may provide insight into the progression of these conditions and warrantsfurther investigation.

S2001

Not Only Reflux Esophagitis But H. pylori Infection May Be a Causal Factorfor the Gastroesophageal Junction Cancer in Japanese PeopleTomoyuki Koike, Shuichi Ohara, Yasuhiko Abe, Toru Horii, Katsunori Iijima, AkiraImatani, Tooru Shimosegawa

Background and Aims: We have reported that H. pylori (HP) infection prevents refluxesophagitis (RE) and Barrett's esophagus (BE) by decreasing gastric acid secretion in Japanesepeople (Gut 2001;49:330-4, Am J Gastroenterol 2004;99:1213-21). Gastroesophageal junc-tion adenocarcinoma (GEJ Ca), including BE cancer, has been thought to be a complication ofRE. Recent studies have confirmed the RE-BE (inflammation)-metaplasia-dysplasia-carcinomasequence for the development of BE Ca in RE patients. On the other hand, the inflammationof the gastric cardia is closely associated with HP infection. However, the inflammatory leveland pattern of BE is not well investigated in Japanese people. The aim of this study was toperform a histological assessment of the inflammation in the cardia and BE and to evaluatethe effect of HP infection and RE on it in patients with BE and GEJ Ca. Materials andMethods: A total of 104 patients with BE and 16 patients with GEJ Ca were enrolled in thisstudy. BE was endoscopically diagnosed by the presence of a red and tongue-like columnarepithelium in the distal esophagus. Of 104 patients with BE, the length of BE was 3cm ormore in 16 patients and less than 3cm in 88 patients. Biopsies were taken from the proximalmargin of the gastric folds (cardia), BE, the gastric body and the gastric antrum. Theinflammation was scored from 0 to 6 by the histological assessment. The status of gastricHP infection was determined by histology, rapid urease test, and detection of serum IgGantibodies. RE was diagnosed according to the Los Angeles classification. Patients who weretaking PPI were excluded. Results: The inflammation score of the cardia was significantlyhigher in the patients with HP than in those without HP regardless of the presence orabsence of RE (P<0.05). The inflammation score of BE was significantly higher in the patientswith RE than in those without RE when examined in the patients without HP (P<0.05),whereas it was not different between the patients with and without RE infection whenexamined in the patients with HP. The inflammation score of BE was significantly higher