activation significantly decreased basal pH levels and inhibited NHE1/NHE2 activity in aconcentration dependent manner (0.01-100 μg/ml; from 93±3 to 78±3% of control values).The level of TLR2 protein expression was not affected by short-term treatment with 10 μg/ml LTA. Long-term (18 h) TLR2 activation significantly decreased basal pH levels andinhibited NHE1/NHE2 activity (0.01-100 μg/ml; from 92±2 to 79±3% of control values);a significant increase in TLR2 protein expression (113% of control) was observed after long-term treatment with 10 μg/ml LTA. Inhibition of protein kinase A (PKA; with 10 μM H89),inhibition of phospholipase C (PLC; with 3 μM U73,122), and down regulation of proteinkinase C (PKC; with 100 nM PDBu for 18h), prevented the short- and long-term TLR2-mediated inhibition of NHE1/NHE2 activity. Short- and long-term treatment with LTA (10μg/ml) produced a significant increase in cAMP levels (142±4% and 117±2% of controlvalues, respectively). An increase in adenylyl cyclase 3 (AC-3) expression was observed afterlong-term, but not after short-term, exposure to 10 μg/ml LTA (122±7% of control values).Inhibition of NHE1/NHE2 activity was observed after treatment of T84 cells with forskolin(3 μM), db-cAMP (200 μM) or the PLC agonist m-3M3FBS (50 μM). Conclusions: Activationof TLR2 exerts marked inhibition of NHE1/NHE2 activity in intestinal epithelial cells.Transduction mechanisms set into motion during short- and long-term TLR2-mediatedinhibition of NHE1/NHE2 activity involves PKA, PLC and PKC. However, short- and long-term TLR2 activation might use different signaling pathways, since involvement of AC-3was limited to long-term TLR2-mediated inhibition of NHE1/NHE2 activity.

Tu1819

Stem Cell Related Phenotype of Hepatocyte-Derived Growth Factor ReceptorPositive Leukocytes in Blood and Biopsy Samples of Ulcerative ColitisFerenc Sipos, Györgyi Muzes, Gabor Valcz, Orsolya Galamb, Sándor Spisák, AlexandraKalmar, Tibor Krenács, Béla Molnár, Zsolt Tulassay

Background: Hepatocyte-derived growth factor receptor (HGFR) is a strong epithelial mitog-enic factor. In ulcerative colitis, the number of HGFR positive leukocytes in the inflamedcolon is elevated. These cells are primarily located to the isolated lymphoid aggregates whichare known to be involved in mucosal repair and thought to be the target place of bone-marrow derived stem cell homing in case of severe colonic inflammation. The role andlineage of HGFR positive subepithelial leukocytes in colonic repair are not clearly known.Aims: We examined the number and stem cell related phenotype of HGFR positive leukocytesin peripheral blood and colonic tissue of ulcerative colitis (UC). Materials and methods:Peripheral blood smears and histological sections were done using blood/biopsy samplesfrom 18 severely active UC and 20 healthy control patients. HGFR, CD133 mesenchymalstem cell marker, Thy-1 lymphoid stem cell marker and CDX2 epithelial stem cell markerdouble fluorescent immunohistochemistries (HGFR/CD133, HGFR/CDX2, HGFR/Thy-1,CD133/CDX2) were performed on all samples, and digitalized for virtual microscopic evalu-ation. In selected cases, real-time RT-PCR validation was also carried out. Results: Thenumber of HGFR positive leukocytes in active ulcerative colitis was significantly elevatedin both peripheral blood smears/lamina propria compared to normal (17.6±3.8/38±7.2 vs.2.4±1.1/9.4±1.5 per high power field /hpf/, p<0.05). CD133/Thy-1 expressions were alsofound to be significantly correlated to the inflammation in colonic tissue (26.5±5.2/17.4±3.1vs. 6.3±1.7/2.7±0.4 per hpf, p<0.05), while in blood only Thy-1 expression showed correla-tion to the inflammation (12.6±1.7 vs. 0.9±0.5, p<0.05). CDX2 expression was found inbiopsy samples in UC (2.1±0.3) and healthy (0.8±0.3) samples (p<0.05), and was rare inUC blood smears. In blood, HGFR and CD133/Thy-1 double immunoreactive leukocyteswere not found, while HGFR/CDX2 double immunopositive leukocytes were rarely presentin UC (1.0±0.1 vs. 0; p<0.05). In biopsies, HGFR/CD133 and CD133/CDX2 double positiveleukocytes were rarely found in UC, in lymphoid aggregates. The protein expression altera-tions of HGFR, CD133 and Thy-1 were also validated by real-time RT-PCR. Conclusions:The presence of HGFR/CDX2 double immunopositive leukocytes in blood and subepitheliallayer of UC suggests that these cells may be involved in epithelial regeneration in case ofinflammation. Subepithelial HGFR positive leukocytes seems to show mesenchymal charac-teristics. The presence of CD133/CDX2 double immunoreactive cells may indicate thatepithelial-to-mesenchymal transition may take place in lymphoid aggregates in case ofsevere inflammation.

Tu1820

Lymphotoxin-Beta Expression Predicts Response to Anti-TNF Therapy inActive Crohn's DiseaseGarrett Lawlor, Awais Ahmed, Aiping Bai, Nielsen Q. Fernandez-Becker, Glen A. Doherty,Dusan Hanidziar, Adam S. Cheifetz, Simon C. Robson, Alan C. Moss

Background & Aims Clinical response to anti-TNF therapy is variable in Crohn's disease,for reasons that are unclear. Our aim was to identify immunological predictors of subsequentresponse to anti-TNF therapy in patients with active Crohns disease before therapy. MethodsCD4+ T-cells were isolated from whole blood of patients commencing anti-TNF therapy foractive Crohn's disease. RNA was extracted and hybridized to a PCR array that evaluateddifferential gene expression of 84 genes within the TNF pathway. Response to therapy wasdefined as a drop in Harvey Bradshaw Index (HBI) score of 3 or greater after 6 weeks oftherapy. To evaluate gene function In Vitro, CD4+ cells were cultured in increasing concentra-tions of TNF, and assessed for gene expression. Results Blood from 6 responders and 6non-responders was analyzed. There were no significant differences in the pre-treatmentHBI score or CRP level between the two groups. Patients who had a clinical response toanti-TNF therapy demonstrated a 44% lower gene expression of Lymphotoxin-beta (LT-b)in their CD4+ T-cells compared to non-responders (P=0.02). Concomitantly, there was 82%lower expression of TNF in responders (P=0.03). In order to determine the effect of TNFon LT-b expression, we incubated CD4+ T-cells in varying concentrations of TNF. TNF ledto an increase in LTB expression in CD4+ T-cells by 77% (P=0.02) at concentrations above10nM. Conclusions Patients with pre-treatment low relative expression of LT-b and TNFin CD4+ T-cells are more likely to experience a clinical response than patients with highlevels. LT-b levels may be a marker for a mechanism of action independent of TNF. In Vitrowork showed that TNF potentiates LTB expression, suggesting a positive feedback loopbetween both pathways.

S-841 AGA Abstracts

Tu1821

Human Neutrophil Peptides Induce IL-8 and ICAM-1 in Intestinal EpithelialCellsKazunari Ibusuki, Toshio Sakiyama, Yuji Iwashita, Shinichi Hashimoto, Shuji Kanmura,Hirohito Tsubouchi

Background and aims: Human neutrophil peptides (HNPs) are antimicrobial peptides thatare stored in the azurophilic granules of neutrophils and released upon activation. Wepreviously reported that HNPs were increased in the sera of patients with active ulcerativecolitis (UC) and are potential biomarkers that predict the disease outcome (Inflamm BowelDis 2009;15:909-917). One of the key histological features of UC is the accumulation ofneutrophils in the lumen of crypts. Previous reports have shown that IL-8 and intercellularadhesion molecule-1 (ICAM-1) expression is increased in the colonic tissues of UC patients.Therefore, these factors may play an important role in neutrophil transepithelial migrationin the UC intestine. However, it is still unclear how IL-8 and ICAM-1 expression is regulatedin the UC mucosa. In this study, we investigated the effects of HNPs on IL-8 and ICAM-1expression in human intestinal epithelial cells. Methods: Total RNA and protein wereextracted from untreated and HNP-1 (100 μg/ml)-treated HT-29 human intestinal epithelialcells. IL-8 expression was measured by real-time PCR. IL-8 production in the culturesupernatants was measured by ELISA. ICAM-1 expression was measured by real-time PCRand Western blot analysis. Activation of mitogen-activated protein kinases (MAPK) wasmonitored by Western blot analysis. Results: HNP-1 significantly increased IL-8 expressionand production from HT-29 cells and the phosphorylation of ERK1/2 and p38 MAPK. IL-8 expression was significantly suppressed by the ERK1/2 inhibitor, U0126, and the P2-purinergic receptor antagonist, suramin. HNP-1 also significantly increased the expressionof ICAM-1 in HT-29 cells. Conclusions: HNPs increased IL-8 expression in intestinal epithelialcells, probably through the P2-purinergic receptor and ERK1/2-dependent pathway. HNPsreleased by infiltrating neutrophils in the UC intestine may stimulate additional neutrophilaccumulation by inducing IL-8 and ICAM-1 expression.

Tu1822

A New Assay for Monitoring Functional Serum Anti-Tumour Necrosis FactorAntibody Level in Crohn's Disease Patients Who Maintained and Those WhoLost Response to Anti-TNFYasuo Suzuki, Akihiro Yamada

BACKGROUND: Infliximab (IFX) is an anti-tumour necrosis factor (TNF)-α antibody usedin the treatment of patients with Crohn's disease (CD). However, antibodies to IFX (ATI)emerge, which potentially can impair its efficacy as well as invoking hypersensitivity reactions,having a negative impact on the safety of IFX therapy. We have developed a new assay formeasuring ATI. METHODS: In this study, we developed a fluid-phase enzyme immunoassay(FP-EIA) by immunizing rabbits with IFX Fab fragments to produce ATI, which we usedto measure serum functional IFX (f-IFX) in patients with CD on maintenance IFX therapy.Thirty-one patients, 16 had maintained response to IFX (group I) and 15 had lost responsein spite of good initial response to IFX (group II) were included. Serum f-IFX was measuredjust before and immediately after IFX infusion, and the values together with CD activity index(CDAI) and C-reactive protein (CRP) were compared between the two groups. RESULTS: Theimmunized rabbits produced high levels of anti-idiotypic antibody titer as measured at avery finite dilution of 1:4000. The new FP-EIA showed a smooth dose-response curve forincreasing amounts of IFX vs TNF-α. The binding capacity of IFX for TNF-α was almostabolished by the addition of 1% serum from patients who had lost response to IFX (hadthe lowest f-IFX level even immediately after an IFX-infusion) or by the addition of 1%serum from an immunized rabbit. The assay could measure antibodies that had emergedagainst IFX. Further, the duration of IFX therapy in groups I and II were 1.8±1.2 and2.7±1.5 years, respectively, while the median dose frequency was 56 days in group I and29 days in group II. On the infusion day, CRP and CDAI in group II were significantlyhigher than in group I, while the median trough f-IFX for groups I and II were 4.7μg/mLand 6.3μg/mL, respectively. The median f-IFX immediately after IFX infusion for groups Iand II were 149.5μg/mL and 126.3μg/mL respectively (P=0.0488), showing negative correla-tion with CRP (r=-0.516, P=0.0035) and CDAI (r=-0.468, P=0.0092). In contrast troughIFX level did not show significant correlation with either CRP or CDAI. CONCLUSIONS:This new FP-EIA could accurately measure f-IFX. High serum ATI strongly impacted f-IFXlevels even immediately after IFX infusion. The f-IFX level immediately after an infusionwas associated with clinical response. f-IFX level should be valuable in decision making tooptimize treatment efficacy with this biologic.

Tu1823

Co-Administration of Anthocyanine Extracts Ameliorate ProinflammatoryEffects of TNF and IFNγ in Human Intestinal Epithelial Cells and MonocytesMichael Scharl, Heidi Piberger, Isabelle Müller, Joba M. Arikkat, Theresa Pesch, SilviaKellermeier, Florian Obermeier, Juergen Schoelmerich, Simone Peschke, Gerhard E.Krammer, Michael Fried, Gerhard Rogler

Background: Patients with Crohn's disease (CD) exhibit lower frequencies and less painfulcourses of acute flares after ingestion of bilberries that are composed of approximately 10%of anthocyanins (AC).We have previously shown that oral administration of dried bilberriesduring acute and chronic dextrane sulphate sodium (DSS)-induced colitis ameliorates theextent of inflammation. Ingestion of AC reduced intestinal inflammation in acute and chronicDSS-colitis, as shown by decreased histological scores and cytokine secretion as well asattenuated reduction of colon length. In chronic DSS-colitis, both bilberries and AC preventedapoptosis in colonic epithelial cells. Here, we studied the molecular mechanisms of the anti-inflammatory effects of anthocyanins In Vitro. Methods: Human THP-1 monocytes, MM6macrophages and T84 intestinal epithelial cells (IEC) were treated with TNF (10 ng/ml),IFNγ (10 ng/ml) and/or anthocyane extract (ACE; 10 μg/ml), respectively. Protein levelswere analyzed by Western blotting, cytokine secretion by ELISA. Apoptosis was assessedby propidium-iodide staining and subsequent FACS analysis. Results: Co-administration of

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