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manner (p<0.01). 3) To assess the effects of SbS on VEGF-induced angiogensis In Vivo theright ears of mice (N=5) were injected with SbS and the left ears were injected with vehicleas control. The first treatment was administered at 1 hour after 5x10^6 pfu Ad-VEGF-A164was injected into both ears. The second SbS/vehicle injections were administered 24 hourslater. An inhibitory effect of SbS was clearly evident by day 7 with reduced new vesselformation in the right ear compared to the left. The effect of SbS remained evident untilthe end of the experiment on day 21. 4) DSS (4% for 5 days) was administered to mice toinduce colitis. Daily gavage of Sb significantly attenuated weight-loss (N=5,p<0.01). DSStreated mice had significantly increased blood vessel density and volume compared to healthymice. Sb treatment significantly reduced the neo-vascularization associated with inflammationin the colon. CONCLUSIONS: Sb inhibits VEGFR signaling and reduces neo-vascularizationIn Vitro and in two separate In Vivo models. Our findings indicate that the probiotic yeastS boulardii can modulate angiogenesis in intestinal injury and repair, which provides a novelmechanism for its beneficial effects.

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Lactobacillus Casei Prevents Osmotic Stress-Induced Disruption of TightJunctions (TJ) and Adherens Junctions (AJ) in CACO-2 Cell Monolayers by aPKC-Dependent Mechanism, but Independent of EGF Receptor TyrosineKinase ActivityRupa Rao, Geetha Samak, Radhakrishna (RK) Rao

Evidence indicates that probiotics play a crucial role in preserving the gastrointestinal (GI)mucosal homeostasis. GI epithelium is exposed to various types of stress, including osmoticstress (OS). We investigated the effect of L. casei on OS-induced TJ disruption in Caco-2cell monolayers. Methods: Cell monolayers were exposed to OS (650 mOsM, adjusted withmannitol) for 0.5-2 hrs in the presence or absence of L. casei (live or UV-killed). Barrierfunction was evaluated by measuring transepithelial electrical resistance (TER) and FITC-inulin flux. TJ and AJ integrity was assessed by confocal microscopy for TJ and AJ proteins,and for F-actin to examine actin organization. Tyr-phosphorylation of TJ proteins wasdetermined by immunoprecipitation of p-Tyr and immunoblot analysis. Association of TJproteins with detergent-insoluble fraction was assessed by immunoblot analysis. Role ofPKC, EGFR kinase and MAP kinase activities in the mechanism of L. casei effect wasdetermined by evaluating the effects of inhibitors, Ro-32-0432 (for PKC), AG1478 (forEGFR), and U0126 (for MEK). L. casei-mediated PKC activation was analyzed by immunoblotanalysis in plasma membrane fractions. Results: OS reduced TER and increased inulin fluxin a time-dependent manner. Pretreatment of cell monolayers with L. casei (live or UV-killed) significantly attenuated the OS-induced changes in TER and inulin permeability. OS-induced redistribution of occludin, ZO-1, E-cadherin, β-catenin and claudin-3 from theintercellular junction into the intracellular compartments was attenuated by L. casei. L. caseiinduced a significant enhancement of actin organization at the mid region perijunctionalarea and effectively attenuated OS-induced reorganization of actin cytoskeleton. L. caseiattenuated OS-induced reduction in the levels of detergent-insoluble fractions of occludin,ZO-1 and claudin-3. OS caused tyrosine phosphorylation of occludin, ZO-1, E-cadherinand β-catenin, which was abrogated by L. casei. L. casei-mediated prevention of OS-induceddecrease in TER, increase in inulin permeability and redistribution of TJ and AJ proteinswas prevented by Ro-32-0432, but not by AG1478 or U0126. These inhibitors by themselvesshowed no significant influence on TER or inulin permeability in control or OS-treated cellmonolayers. L. casei induced a transient increase in the levels of plasma membrane associatedPKCε, but not PKCβI. Conclusion: These studies demonstrate that L. casei ameliorates OS-induced disruption of apical junctional complexes by a mechanism dependent on PKC, butindependent of EGF receptor and MAP kinases. Supported by DK55532 and AA12307.

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The Probiotic Bacteria Lactobacillus Acidophilus NCFM® (L-NCFM) andBifidobacterium Lactis Bi-07 (B-Lbi07) Increase Expression of Intestinal OpioidReceptors - First Evidence in HumansYehuda Ringel, Jason R. Goldsmith, Ian M. Carroll, Jennica P. Siddle, Christian Jobin,Silvana P. Barros, Tamar Ringel-Kulka

Probiotics have been shown to be beneficial in alleviating various functional GI symptoms,however, the mechanisms of these effect are unknown. The probiotic bacterium L-NCFMhas been shown to increase expression of opioid (MOR) receptors in the intestinal mucosaand decrease intestinal pain sensation in animal studies [Rousseaux 2007]. A recent studywith the combination of L-NCFM and B-LBi07 suggested a beneficial effect on GI symptomsin humans [Ringel 2010]. Intestinal STAT3 activation has recently been linked with opioidsignaling [Goldsmith 2010]. Aim: To investigate the effect of L-NCFM and B-LBi07 oncolonic mu opioid receptors (MOR) expression in patients with functional abdominal pain.Methods: Caucasian women (n=17) 18-70 years with mild to moderate abdominal painwere investigated. Colonic biopsies were collected during un-sedated, un-prepped flexiblesigmoidoscopy pre- and post- 21 days consumption of the probiotics at a total dose of 1.0x 1010 CFU bid. Cellular RNA was extracted from the mucosal biopsies and reverse transcribedto cDNA. mRNA expression of mucosal MOR receptors was determined by RT-PCR on anABI Prism HT7700 Thermocycler (Applied Biosystems) via SybrGreen. Relative fold-changeswere determined using the ΔΔCT calculation method. Immunohistochemistry (IHC) forpSTAT3 was performed on paraffin-embedded sections of mucosal biopsies with monocloncalantibodies for pSTAT3(Y705) (Cell Signaling Technology Inc) were used at a 1:50 dilution.Slides were developed using horseradish peroxidase-conjugated secondary antibody and aVectaStain Elite ABC Kit (Vector Laboratories Inc). Results: Pre- to post-intervention withL-NCFM and B-LBi07 or L-NCFM alone was associated with a 5.5-fold increase in MORexpression (8.2E-6 vs 1.5E-6 fold-over-β-actin, P=0.014; n=13). The increase in MORexpression was significantly (~10-fold) greater with L-NCFM alone compared to the combina-tion of B-LBi07 and L-NCFM (1.1E-5 vs1.8E-6 fold-over-β-actin post-treatment increase,P=0.022). Analysis of MOR expression on a per-patient-basis showed that patients treatedwith L-NCFM alone had a 39.9-fold pre- to post-intervention increase in the levels of thereceptor (P =0.0313). IHC for pStat3 showed increase in mucosal biopsies only in the L-NCFM group. Conclusions: We replicated in humans the animal model observation of

S-847 AGA Abstracts

increased expression of MOR receptors with L-NCFM. In addition, we demonstrated postreceptor signaling effect. Our data provides the first evidence for a probiotic effect on opioid-mediated pain pathways in humans. The physiological (sensation threshold) and clinical(pain experience) implications of these findings requires further investigation.

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Exploring the Mechanism of a Probiotic Combination VSL#3 in IrritableBowel Syndrome (IBS): A Randomized Double-Blind Placebo Controlled StudyReuben K. Wong, Cao Yang, Claudio De Simone, Guanghui Song, Jennie Y. Wong,Shyam Prakash, Khek Yu Ho

Background & Aims: Probiotics have treatment efficacy in IBS, but the exact mechanismremains obscure. One hypothesis is the mediation of melatonin levels, leading to changesin IBS symptoms. This study aims to evaluate the effects of a probiotic, VSL#3, on symptoms,sleep parameters and pain sensitivity in IBS, and relate these parameters to In-Vivo melatoninlevels. Methods: 42 IBS patients were randomly assigned to receive 4 capsules of eitherVSL#3 (n=20) or identical placebos (n=22), twice daily, for 6 weeks. Pre and post-treatment,subjects completed bowel and psychological questionnaires, and underwent rectal sensitivitystudy as well as saliva and fecal melatonin assays. Results: VSL#3 and placebo decreasedthe mean IBS Severity Score from 224.5 to 158.0 (p<0.05), and 226.4 to 183.5 (p<0.05),respectively. The VSL#3 subjects had a larger improvement (-66.5) than the placebo group(-42.9), and the difference was statistically significant amongst males but not females.Abdominal pain duration decreased (-18.5 vs. -7.3,p<0.05) in the VSL#3 arm comparedwith the placebo controls, as did abdominal distension intensity (-14.5 vs. -12.3, p<0.05).Rectal distension pressures needed to induce pain significantly increased in the VSL#3patients (38.4 to 42.5 mmHg) compared to controls. A correlation between increase in paintolerance threshold and improvement in abdominal pain scores (r=0.51,p=0.02) was seenwith VSL#3 but not placebo. No significant changes following treatment were observed inpsychological indices nor sleep parameters. There was an increase in salivary morningmelatonin levels in males (5.43pg/ml to 9.74pg/ml,p=0.03) treated with VSL#3, whichcorrelated (r=0.61,p=0.058) with improved satisfaction in bowel habits. When subjects weregrouped into normal vs. abnormal baseline diurnal melatonin levels, the former showed anincrease in morning melatonin levels with VSL#3 treatment (3.19pg/ml to 6.58pg/ml,p=0.07), which significantly correlated with improved satisfaction in bowel habits (r=0.68,p=0.04). Similarly, subjects with a normal circadian melatonin had reduced symptom severityscores (-123.8 vs. -45) and abdominal pain duration (-27.8 vs. -12.9) when treated withVSL#3 vs. placebo. They also had significantly improved satisfaction with bowel movementsand quality of life. Conclusions: VSL#3 reduced abdominal pain duration and distensionintensity in IBS subjects. Rectal pain thresholds were improved, correlating with an improvedabdominal pain scores. The improvement in symptoms correlated with a rise in morningsystemic melatonin, which was significant in males and subjects with normal circadianrhythm. We postulate that the probiotic acts by influencing melatonin production, hencemodulating IBS symptoms, in individuals with a “normal” diurnal melatonin levels but notin those with a baseline disordered circadian rhythm.

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The Role of Intestinal Bacteria and Bifidobacterium Breve NCC2950 in BarrierHomeostasis and Intestinal Injury in NOD1 and 2 Knockout MiceJane M. Natividad, XianXi Huang, Jennifer Jury, Giada de Palma, Yolanda Sanz, DanaPhilpott, Clara L. Garcia Rodenas, Kathy McCoy, Elena F. Verdu

Background and Aim: The precise mechanisms by which (NOD)-like receptors contributeto inflammatory bowel disease (IBD) are not completely understood. Emerging data supporta role of NOD1 and 2 receptors in the modulation of intestinal barrier homeostasis andintestinal microbiota composition. We hypothesized that intestinal microbiota compositionand probiotic supplementation affects the intestinal barrier and host responses to intestinalinjury in mice with defective NOD 1 and 2 signaling.Methods: Fecal microbiota compositionwas assessed by DGGE and RT-PCR in NOD1 and NOD2 double-deficient mice (NOD1/2-/-), heterozygous littermate controls (NOD1/2+/-), and wild type controls (NOD1/2+/+) bredfrom a different facility. Intestinal permeability was assessed by In Vitro and In Vivo techniquesand expression of epithelial apical junction proteins determined by RT-PCR and immunoflou-rescence. Acute intestinal injury was induced by Dextran Sodium Sulfate (DSS, 3.5%) inthe drinking water for 5 days. Mice were sacrificed 3 days post DSS and intestinal injurywas assessed by disease severity index, histological scores, myeloperoxidase (MPO) activity,and cytokine production in the colon. Bifidobacterium breve NCC2950 was administered bygavage (109 CFU/ 100 μl/mouse) 14 days before colitis induction. Results: NOD1/2-/- micehad microbiota profiles different to NOD1/2+/+ controls bred from a different facility buthad similar microbiota profiles compared to heterozygous littermate NOD1/2+/- controls.Despite this, naïve NOD1/2-/- mice had increased paracellular permeability compared toNOD1/2+/- controls. This was paralleled by decreased expression of E-cadherin, but nospontaneous development of colonic inflammation. After induction of intestinal injury,disease severity index, histological score, MPO activity, colonic IL-12p70 and TNF-α levelswere significantly higher by 50%, 27%, 71%, 60% and 45% respectively, in NOD1/2-/- micecompared to NOD1/2+/- controls. Bifidobacterium breve did not normalize altered permeability,but prevented the increased susceptibility to DSS injury in NOD1/2-/- mice. Conclusion:The results suggest that NOD1 and NOD2 signaling is associated with barrier dysfunctionand increase susceptibility to intestinal injury regardless of microbiota composition. Bifidobac-terium breve NCC2950 prevents DSS-induced inflammation, and may be of potential benefitto decrease IBD risk in patients with NOD mutations. This work is supported by the Crohn'sand Colitis Foundation of Canada

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