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sTNF receptor 1 could rescue intestinal epithelial damage in the TAK1 mutant mice. Conclu-sion: TAK1 signaling is essential for preventing TNF-induced enterocyte apoptosis and formaintaining intestinal epithelial integrity. We propose that aberration in TAK1 signaling mightdisrupt intestinal homeostasis and favor the development of inflammatory bowel disease.

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Mechanism of Interferon-Gamma Induced Increase in Intestinal EpithelialTight Junction Permeability: Role of JNK Pathway and Cross-Talk with the PI-3 Kinase PathwayPraveen K. Roy, Jing Chen, Farzana Rashid, Suzanne Ridenhour, Jamal A. Ibdah

Background: Interferon-gamma (IFN-γ) induced increase in intestinal epithelial tight junction(TJ) permeability has been proposed as an important pathogenic mechanism contributingto intestinal inflammation. However, the intracellular pathways that mediate IFN-γ inducedincrease in intestinal permeability have only recently been studied. Recent studies havedemonstrated that IFN-γ induced increase in TJ permeability is mediated by NF-κB and PI-3 kinase pathways. However, the role of MAP kinase pathways have not been well studied.Thus, we investigated the possible role of the JNK pathway (a member of the MAP kinasefamily) in mediating the IFN-γ induced increase in intestinal permeability by using filter-grown T84 intestinal epithelial cells. We also explored the cross-talk between JNK and PI-3 kinase pathways. Methods: T84 TJ permeability was assessed by measuring trans-epithelialresistance (TER). JNK activation was assessed by western blot, FACE assay, and in-vitrokinase assay. Tight junction protein expression was assessed by western blot. Results: IFN-γ produced a concentration and time-dependent decrease in TER. The drop in TER wasalso associated with a decrease in occludin and claudin-2 protein expression. IFN-γ treatmentresulted in phosphorylation of JNK with 15 minutes of treatment with IFN-γ by westernblot analysis. FACE assay and JNK in-vitro kinase assay confirmed these findings. SP 600125(a JNK inhibitor) prevented the IFN-γ induced decrease in TER and down-regulation ofoccludin and claudin-2 protein expression, suggesting that JNK pathway was involved inIFN-γ induced increase in TJ permeability. We also explored possible cross-talk betweenJNK and PI-3 kinase pathways. JNK inhibitor (SP 600125) inhibited p-AKT expression.However, PI-3 kinase inhibitor (LY 294002) did not prevent expression of p-JNK, suggestingthat JNK activation occurred upstream of PI-3 kinase pathway. Conclusion: Our resultssuggest that the JNK pathway mediates IFN-γ induced increase in intestinal permeability.Additionally, IFN-γ induced activation of JNK occurs upstream of PI-3 kinase pathwayactivation. Therefore, the JNK pathway could be an important and potential therapeutictarget in inflammatory bowel disease.

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Effect of Chronic Oral Exposure to the Xenoestrogen Bisphenol a (BPA) OnEpithelial Paracellular Permeability and Inflammatory Response in the RatColonViorica Braniste, Mathilde Leveque, Lionel Bueno, Jean Fioramonti, Eric Houdeau

Background & aim: Bisphenol A (BPA) is an estrogen-mimetic chemical used as plasticizerin food-packaging industry. Human exposition is attested by its presence in plasma andurine. Since estrogens affect inflammatory responses in the gut, we evaluated in the femalerat colon the effects of BPA for low dose regimens (No Observed Adverse Effect Level-NOAEL and Tolerable Dose Intake-TDI) on i/ epithelial paracellular permeability, ii/ theseverity of experimental colitis, particularly through the expression of macrophage migrationinhibitory factor (MIF), an estrogen targeted proinflammatory cytokine1. Methods: First,BPA [NOAEL: 5 mg/kg/d; TDI: 50 mg/kg/d], estradiol benzoate [EB: 0.6 mg/kg/d], or vehicle[Ve, corn oil] were administered orally for 15 days to ovariectomized (OVX) rats, with orwithout estrogen receptor (ER) antagonist ICI 182,780 (2 mg/kg/d s.c.). Epithelial paracellularpermeability was assessed by measuring FITC-dextran flux of colon strips in Ussing chambers.Second, colitis was induced by intrarectal instillation of trinitrobenzene sulfonic acid (TNBS;80 mg/kg) to OVX rats treated orally with BPA or Ve for 15 days, with or without ICI182,780. After 24h, macroscopic damage scores (MDS) in the colon and myeloperoxidase(MPO) activity were used as index for colitis severity. MIF content was evaluated by Westernblotting and IL-1b by ELISA. Results: EB-treated OVX rats showed a 48% decrease ofdextran flux in comparison with OVX controls (0.33±0.03 vs 0.64±0.09 nmol.cm-2.h-1;P<.01). Similarly, BPA treatments at NOAEL and TDI decreased dextran flux by 50% and41%, respectively, compared to controls (0.32±0,02 and 0.38±0,04 vs 0.64±0.09 nmol.cm-2.h-1; P<.001 and P<.01). The co-treatment with ICI 182,780 reversed the effects of EBand BPA. In colitis, TDI for BPA did not affect inflammatory response, while NOAEL dosageimproved MDS (-28%, P<.001) and decreased MPO activity (-44%, P<.01), IL-1b (-31%,P<.01) and MIF colonic contents (-32% ; P<.05) compared to controls (Ve+TNBS). Treatmentwith ICI 182,780 blocked the effects of NOAEL on MPO activity and MIF level. Conclusions:This study shows that chronic exposure to low doses of BPA decreases colonic paracellularpermeability similarly to estradiol. Furthermore, NOAEL for BPA displays antiinflammatoryactivity on colitis, by reducing MIF and IL-1b production, as reported for estradiol1. Thesechanges were blocked by the ICI 182,780, thus were ER-mediated. Consequently, theestrogenic activity of BPA at NOAEL and TDI reinforces colonic paracellular permeability,while only NOAEL for BPA improves inflammatory response in the female rat colon. 1Hou-deau et al Gastroenterology (2007)

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Prevention of DSS-Induced Colitis in Mice By a Probiotic-Derived SolubleProteinFang Yan, Hanwei Cao, M. Kay Washington, D. Brent Polk

Background. Lactobacillus rhamnosus GG (LGG) has been shown to reduce the severity ofseveral intestinal inflammatory disorders. We have purified and cloned a LGG-derived solubleprotein, p40, which prevents TNF-induced intestinal epithelial apoptosis and injury throughactivation of EGF receptor (EGFR) and its down-stream target, Akt, In Vitro and in a colon

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organ culture model. This study was designed to test the hypothesis that p40 prevents colitisIn Vivo in an EGFR-dependent manner. Methods. For colon-specific delivery of His-p40fusion protein, C57BL/6 (wild-type, wt) and EGFR kinase defective (C57BL/6Wa2) mice weregavaged with pectin/zein beads containing His-p40 and sacrificed after 4 h treatment. His-p40 recovery and EGFR and Akt activation were detected using immunostaining of tissuesections and Western blot analysis of mucosal lysates with antibodies against p40, EGFR-Tyr1068 and Akt-Ser473. Wt and C57BL/6Wa2 mice were administered 3% DSS in drinkingwater, or water only, for 4 or 13 days. His-p40 beads (10 µg/bead/day), or beads only, weregavaged once a day beginning at the same day of DSS treatment until mice were sacrificed.Colon sections were prepared for H&E staining to determine intestinal tissue injury score(0: normal, 15: severe colitis). Body weight and colon length were assessed. Results. Immuno-staining showed that His-p40 was recovered in colon epithelium of both wt and C57BL/6Wa2 mice with limited amounts found in small intestine and stomach. Immunostainingand Western blot analysis showed that His-p40 beads stimulated EGFR and Akt activationin colon epithelial cells of wt, but not C57BL/6Wa2 mice, indicating that His-p40 specificallydelivered to the colon is biologically active. In wt mice, DSS-induced colitis (score: 9.5 for4 d and 15 for 13 d treatment) was significantly reduced by His-p40 treatment (score: 6.6for 4 d and 8.5 for 13 d treatment), by decreasing colon ulceration, crypt damage, andseverity and extent of inflammation. In addition, His-p40 beads significantly reduced DSS-induced body weight loss and colon shortening at 13 d. In contrast, DSS-induced colonpathology and clinical activity in C57BL/6Wa2 mice were not relieved by His-p40 treatment.Conclusion. p40 protects the intestinal epithelium from DSS-induced tissue damage andcolitis in an EGFR-dependent manner. Delivery of biologically active soluble bacterial proteinsto the colon epithelium provides an important proof-of-principle for this therapeuticapproach. Furthermore, our novel findings provide a clinical basis and mechanism of actionfor therapeutic application of soluble probiotic proteins in ulcerative intestinal inflammat-ory disorders.

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Epigenetic Regulation of FOXP3+ Regulatory T Cells (Treg) By Small MoleculeTargeting of a Single HDAC, Hdac6, Enhances Treg Function and AmelioratesDevelopment of IBD in Murine ModelsEdwin F. deZoeten, James E. Bradner, Ralph Mazitschek, Wayne W. Hancock

We are interested in determining the role of naturally occurring CD4+CD25+ Tregs thatexpress the transcription factor, Foxp3, in regulating development of experimental colitis,with the background considerations of, is Treg function impaired in patients with IBD, andhow can the therapeutic potential of Tregs best be exploited? We have recently shown thatbroadly acting inhibitors of histone deacetylase (HDACi) have beneficial effects on T cell-dependent diseases by promoting the acetylation and function of Foxp3, as well as enhancingthe production of Foxp3+ Tregs. However, HDAC expression is ubiquitous and globalinhibition may conceivably have deleterious effects, leading us to test the effects of targetinga specific HDAC. We now report the beneficial effects of using the HDAC6-specific inhibitor,tubacin, which promotes the acetylation of alpha-tubulin and HSP90. We found that TCR-activated murine Tregs had 5-6 fold more HDAC6 mRNA than resting CD4+CD25- non-Treg or Treg cells. Interestingly, use of the HDAC6-specific inhibitor, tubacin (but not thecontrol compound, niltubacin), increased Treg cell suppressive function In Vitro, in associ-ation with enhanced expression of CTLA, IL-10, GITR, PD-1 and other Treg-associatedgenes. Tubacin also enhanced the conversion of CD4+CD25- cells into CD4+ Foxp3+ TregIn Vitro, and globally decreased cytokine production, with the exception of IL-10 and IL-17 mRNA. Use of tubacin In Vivo significantly decreased the severity of colitis in two murineIBD models, dextran sodium sulfate-induced colitis and the CD4+CD25- adoptive transfermodel of colitis, as assessed by standard clinical and histologic criteria. We have previouslyshown that inhibition of a specific HDAC, e.g. HDAC9, can affect the development andprogression of colitis, as seen using HDAC9-deficient experimental animals. However, thecurrent study shows that use of the first known HDAC-specific small molecule inhibitorcan also have significant therapeutic effects in normal animals, including in models of IBD.Ongoing studies are directed towards unraveling the interactions of HDAC6-dependentpathways and Treg functions. Dissecting the complex interactions of histone acetyltransferasesand HDACs is but one of several potential ways to pharmacologically manipulate Treg cellsIn Vitro and In Vivo, with likely future beneficial consequences for the therapy of IBD.

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Deletion of the Chloride Transporter Slc26a9 Causes Loss of Tubulovesicles inGastric Parietal Cells and Impairs Acid SecretionJie Xu, Jordi Ehrenfeld, Marian L. Miller, Zhaohui Wang, Sharon Barone, Seth Alper, GaryE. Shull, John G. Forte, Frank Borgese, Manoocher Soleimani

Slc26a9 is a recently identified member of a conserved family of anion transporters whichis abundantly expressed in gastric epithelial cells. To study its role in stomach physiology,gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant Slc26a9-/-mice appeared healthy and displayed normal growth. The pH of the gastric secretions wassignificantly more alkaline in Slc26a9-/- mice, with values of 3.1 +/- 0.2 in Slc26a9+/+ miceand 6.4 +/- 0.2 in Slc26a9-/- mice (p<0.001). The quantitation of gastric acid secretionrevealed almost no acid secretion in Slc26a9-/- mice, with values of 75 +/- 6 mEq/g wetweight in Slc26a9+/+ mice and 3 +/- 1 in Slc26a9-/- mice (p<0.0001). These results indicatethe complete inhibition of gastric acid secretion in Slc26a9 null mice. There was a moderatereduction in the number of parietal cells in Slc26a9 null mice at 5 weeks of age. Immunofluo-rescence labeling demonstrated that gastric H-K-ATPase is detected exclusively on the apicalpole of gastric parietal cells at resting state in Slc26a9-/- mice, in contrast to the predominantcytoplasmic localization in Slc26a9+/+ mice. Light microscopy indicated that the gastricglands were dilated in Slc26a9 mutant mice. Electron micrographs displayed a distinct andstriking absence of tubulovesicles in parietal cells in Slc26a9-/- stomach. At 19 days afterbirth, Slc26a9 null mice demonstrated significant inhibition of acid secretion (61 +/- 4 mEq/g wet weight in Slc26a9+/+ mice and 39 +/- 3 in Slc26a9-/- mice (p<0.05), with nearlynormal looking parietal cell morphology on light microscopy. Expression studies in oocytesand cultured cells indicated that Slc26a9 can function as an chloride channel as well as a