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Saccharomyces Boulardii Inhibits VEGFR Signaling and Angiogenesis in Intestinal Inflammation
controlling gastroenteric disease in SAMP1/Yit mice enable distinct pathogenic mechanisms to cause inflammation in separate sites within the digestive tract.
AGA Abstracts
Tu1840 Toll-Like Receptor 5 is Significantly Associated With Canine Inflammatory Bowel Disease: Genetical and Functional Analysis Aarti Kathrani, Angela Holder, Brian Catchpole, Dirk Werling, Karin Allenspach TLR5 has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (IBD) and animal models of this disease. A polymorphism in the TLR5 gene which terminates TLR5 signaling has shown to be protective for Crohn's disease (CD) in a Jewish cohort. Similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (SNPs) in the canine TLR5 gene (G22A, C100T and T1844C) and inflammatory bowel disease (IBD) in German shepherd dogs (GSDs). Therefore the aim of this study was to determine the functional significance of the canine TLR5 SNPs by transfecting wild-type and mutant receptors into human embryonic kidney cells (HEK) and carrying out nuclear factor-kappa B (NF-κB) luciferase assay and CXCL8 ELISA. The TLR5 gene containing the risk haplotype for IBD (ACC) and wild-type haplotype (GTT) as determined by the case-control analysis in GSDs with IBD were cloned into plasmids expressing yellow-fluorescent protein (YFP). These were then stably transfected into HEK cells. Transfection was confirmed by assessing the expression of YFP by confocal microscopy and fluorescence-activated cell sorting analysis. NF-κB activity was measured by transiently transfecting the cells with NF-κB promoter firefly and HSV-thymidine kinase promoter renilla plasmids. The cells were then stimulated with various ligands (0.1μg/ml flagellin, 0.01μg/ml flagellin, 1μg/ml LPS, 1μg/ml PAM3CSK and media control). Firefly and renilla luciferase activities were measured using the DualGlo Luciferase Assay System (Promega, UK). The supernatants were harvested and used in a CXCL8 ELISA (R&D Systems, UK). Human TLR5 transfected HEK cells (Invivogen, UK) served as positive controls in all experiments. Independent T-test was used to determine the significance of relative luciferase activity and CXCL8 concentration between wild-type and mutated TLR5 cells. There was a significant increase in NF-κB activity when the cells with mutated TLR5 were stimulated with 0.1μg/ml of flagellin compared to the cells expressing wild-type TLR5 (p=0.027), which correlated with CXCL8 expression in the supernatant measured by ELISA (p=0.025). We show for the first time that polymorphisms associated with canine IBD are functionally hyper-responsive to flagellin compared to the wild-type receptor. This suggests that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease and may therefore serve as a model for human IBD. Further In-Vivo functional analysis of TLR5, especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions.
Tu1842 Secreted Bioactive Effectors of Lactobacillus Acidophilus Attenuate the Exaggerated Inflammatory Response Responsible for Necrotizing Enterocolitis Kriston A. Ganguli, W. Allan Walker, N. Nanda Nanthakumar Background & Aims: Necrotizing enterocolitis (NEC), a severe intestinal inflammatory disease of premature infants, results from an inappropriate inflammatory (IL-6) response during the initial stages of bacterial colonization. Randomized placebo-controlled trials have shown a significant reduction in the incidence of NEC when very low birth weight infants were supplemented with combination probiotics (Lactobacillus acidophilus and Bifidobacterium infantis). The goal of this study was to determine whether soluble factors secreted by these bacteria, grown individually, attenuate the inflammatory response of immature enterocytes In Vitro and to characterize these factors. Methods: Immature primary enterocyte cultures of NEC and fetal (IEC) tissue were assayed for IL-6 secretion after stimulation with an endogenous inflammatory stimulus, IL-1-beta, while concurrently being exposed to probiotic conditioned media (PCM) from either L. acidophilus or B. infantis. The IL-6 assays were repeated using L.acidophilus conditioned media which was exposed to size-fractionation, heat stress or Ulex europaeus agglutinin-1 (UEA-1) treatment. Results: In NEC-IEC and fetal IEC cell culture, a significant decrease (p<0.01) in IL-6 secretion was shown with L.acidophilus but not with B.infantis-conditioned media. The significant reduction in IL-6 secretion by NEC-IEC cells with L.acidophilus-conditioned media (p <0.001) was lost when the conditioned media was treated with a fucose-specific lectin, UEA-1. L.acidophilus conditioned media retained its anti-inflammatory properties after exposure to heat stress and fractionation < 30kDa (p<0.05). Conclusions: We conclude that PCM with L. acidophilus provides superior inflammatory attenuation over that of B. infantis in NEC-IEC and fetal IEC cells. The data suggest that the bioactive effector(s) secreted by L. acidophilus is a heat stable molecule less than 30kDa in size. The activity of the effector(s) is modified after exposure to a fucose-specific lectin, UEA-1, suggesting a glycoconjugate. However, additional studies are necessary for further characterization of L.acidophilus secreted effectors.
Tu1841 Profile of Anti-Microbiota Antibody Responses in Crohn's Disease and Ulcerative Colitis Nicola Patuto, Emma Slack, Bruno Strebel, Maria L. Balmer, Philipp Schuetz, Frank Seibold, Andrew J. Macpherson Background: Crohn's disease and ulcerative colitis are both diseases which manifest as a failed mutualism with the intestinal microbiota, clearly demonstrated by the amelioration of symptoms on diversion of the fecal stream. In Crohn's disease, a large number of reports document the presence of elevated serum antibody responses specific for bacterial and yeast products. We have recently demonstrated in murine models that elevated anti-microbiota immunity is a common sequalae of innate immune deficiency. We therefore compared the spectrum of anti-microbiota immunity in patients with Crohn's disease or ulcerative colitis to functional measurements of innate immune functionality. Methods: Anti-microbiota immunity was quantified using a technique that we have developed based on high-affinity binding of serum antibodies to the surface of live bacteria (Figure 1). This binding is then quantified per bacterium by flow cytometry. By carrying out live bacterial staining with dose-titrations of plasma, titration curves can be plotted in a similar manner to classical ELISA titres. To functionally characterise innate immunity, concentrations of innate immune stimuli required to give 50% activation of granulocyte and monocyte CD62L-shedding and oxidative burst production were calculated for each patient. The IBD patient cohort was collected from the outpatient clinic of the Inselspital and Tiefenauspital in Bern and each patient diagnosis was re-confirmed. Conclusions: Crohn's disease patients show dramatically elevated IgG and IgA titres specific for gamma-proteobacteria, in particular Klebsiella pneumoniae (Figure 1) whilst Ulcerative colitis patients show highly elevated IgG titres specific for predominantly unculturable fecal bacteria. Innate immune sensitivity of patients varied considerably between individuals with up to 10000-fold differences in sensitivity to TLR1 ligands observed, but no obvious disease-associated trends (Figure 2). Further analysis of reactivity to bacterial species representative of the intestinal microbiota will reveal whether positive associations exist between innate immune sensitivity, anti-microbiota immunity and disease phenotype.
AGA Abstracts
Tu1843 Saccharomyces Boulardii Inhibits VEGFR Signaling and Angiogenesis in Intestinal Inflammation Xinhua Chen, Guoxun Yang, Joo Hye Song, Hans-Christian Reinecker, Huiyan Zeng, Ciaran P. Kelly, Hua Xu BACKGROUND AND AIM: Saccharomyces boulardii (Sb), a probiotic yeast, has been used for decades to protect against intestinal injury and inflammation. We previously reported Sb protects against Clostridium difficile colitis and intestinal tumor formation through modulation of host signaling including the EGFR/Erk pathway. Angiogenesis is increasingly recognized as an important component of intestinal inflammation. We found that Sb inhibits VEGFR (vascular endothelial growth factor receptor) signaling, a central pathway regulating angiogenesis. Using In Vitro assays and In Vivo models, we aim to examine whether Sb inhibits angiogenesis. METHODS: HUVEC cells were treated with VEGF with and without Sb supernatant (SbS). Antibodies against p-VEGFR2, p-Erk1/2, or p-PLCγ were used in Western analyses. The ECMatrixTM In Vitro angiogenesis assay was used to study the effects of SbS on HUVEC capillary tube formation. To assess the effects of SbS on VEGF-induced angiogenesis In Vivo, we used an adenovirus expressing VEGF-A(164) in the ears of adult nude mice (NuNu). The DSS-colitis mouse model was used to assess the effect of Sb on neo-vascularization in acute colitis. Blood vessels were stained with Alexa647 WGA injected retro-orbitally and tissue specimens were imaged with a Bio-Rad Radiance 2000 confocal microscope and three-dimensional analysis of vascular density and volume carried out after processing in Volocity software. RESULTS: 1) SbS inhibited the phosphorylation of VEGFR2 in response to VEGF and also reduced activation of the downstream kinases PLCγ and Erk1/2. 2) SbS significantly inhibited HUVEC capillary tube formation in a dose-dependent
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increased expression of MOR receptors with L-NCFM. In addition, we demonstrated post receptor signaling effect. Our data provides the first evidence for a probiotic effect on opioidmediated pain pathways in humans. The physiological (sensation threshold) and clinical (pain experience) implications of these findings requires further investigation. Tu1847 Exploring the Mechanism of a Probiotic Combination VSL#3 in Irritable Bowel Syndrome (IBS): A Randomized Double-Blind Placebo Controlled Study Reuben 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 mechanism remains obscure. One hypothesis is the mediation of melatonin levels, leading to changes in 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 melatonin levels. Methods: 42 IBS patients were randomly assigned to receive 4 capsules of either VSL#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 sensitivity study as well as saliva and fecal melatonin assays. Results: VSL#3 and placebo decreased the 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 compared with 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#3 patients (38.4 to 42.5 mmHg) compared to controls. A correlation between increase in pain tolerance threshold and improvement in abdominal pain scores (r=0.51,p=0.02) was seen with VSL#3 but not placebo. No significant changes following treatment were observed in psychological indices nor sleep parameters. There was an increase in salivary morning melatonin levels in males (5.43pg/ml to 9.74pg/ml,p=0.03) treated with VSL#3, which correlated (r=0.61,p=0.058) with improved satisfaction in bowel habits. When subjects were grouped into normal vs. abnormal baseline diurnal melatonin levels, the former showed an increase 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 severity scores (-123.8 vs. -45) and abdominal pain duration (-27.8 vs. -12.9) when treated with VSL#3 vs. placebo. They also had significantly improved satisfaction with bowel movements and quality of life. Conclusions: VSL#3 reduced abdominal pain duration and distension intensity in IBS subjects. Rectal pain thresholds were improved, correlating with an improved abdominal pain scores. The improvement in symptoms correlated with a rise in morning systemic melatonin, which was significant in males and subjects with normal circadian rhythm. We postulate that the probiotic acts by influencing melatonin production, hence modulating IBS symptoms, in individuals with a “normal” diurnal melatonin levels but not in those with a baseline disordered circadian rhythm.
Tu1844 Lactobacillus Casei Prevents Osmotic Stress-Induced Disruption of Tight Junctions (TJ) and Adherens Junctions (AJ) in CACO-2 Cell Monolayers by a PKC-Dependent Mechanism, but Independent of EGF Receptor Tyrosine Kinase Activity Rupa 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 osmotic stress (OS). We investigated the effect of L. casei on OS-induced TJ disruption in Caco-2 cell monolayers. Methods: Cell monolayers were exposed to OS (650 mOsM, adjusted with mannitol) for 0.5-2 hrs in the presence or absence of L. casei (live or UV-killed). Barrier function was evaluated by measuring transepithelial electrical resistance (TER) and FITCinulin 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 was determined by immunoprecipitation of p-Tyr and immunoblot analysis. Association of TJ proteins with detergent-insoluble fraction was assessed by immunoblot analysis. Role of PKC, EGFR kinase and MAP kinase activities in the mechanism of L. casei effect was determined by evaluating the effects of inhibitors, Ro-32-0432 (for PKC), AG1478 (for EGFR), and U0126 (for MEK). L. casei-mediated PKC activation was analyzed by immunoblot analysis in plasma membrane fractions. Results: OS reduced TER and increased inulin flux in a time-dependent manner. Pretreatment of cell monolayers with L. casei (live or UVkilled) significantly attenuated the OS-induced changes in TER and inulin permeability. OSinduced redistribution of occludin, ZO-1, E-cadherin, β-catenin and claudin-3 from the intercellular junction into the intracellular compartments was attenuated by L. casei. L. casei induced a significant enhancement of actin organization at the mid region perijunctional area and effectively attenuated OS-induced reorganization of actin cytoskeleton. L. casei attenuated 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-cadherin and β-catenin, which was abrogated by L. casei. L. casei-mediated prevention of OS-induced decrease in TER, increase in inulin permeability and redistribution of TJ and AJ proteins was prevented by Ro-32-0432, but not by AG1478 or U0126. These inhibitors by themselves showed no significant influence on TER or inulin permeability in control or OS-treated cell monolayers. L. casei induced a transient increase in the levels of plasma membrane associated PKCε, but not PKCβI. Conclusion: These studies demonstrate that L. casei ameliorates OSinduced disruption of apical junctional complexes by a mechanism dependent on PKC, but independent of EGF receptor and MAP kinases. Supported by DK55532 and AA12307.
Tu1848 The Role of Intestinal Bacteria and Bifidobacterium Breve NCC2950 in Barrier Homeostasis and Intestinal Injury in NOD1 and 2 Knockout Mice Jane M. Natividad, XianXi Huang, Jennifer Jury, Giada de Palma, Yolanda Sanz, Dana Philpott, Clara L. Garcia Rodenas, Kathy McCoy, Elena F. Verdu Background and Aim: The precise mechanisms by which (NOD)-like receptors contribute to inflammatory bowel disease (IBD) are not completely understood. Emerging data support a role of NOD1 and 2 receptors in the modulation of intestinal barrier homeostasis and intestinal microbiota composition. We hypothesized that intestinal microbiota composition and probiotic supplementation affects the intestinal barrier and host responses to intestinal injury in mice with defective NOD 1 and 2 signaling. Methods: Fecal microbiota composition was 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+/+) bred from a different facility. Intestinal permeability was assessed by In Vitro and In Vivo techniques and expression of epithelial apical junction proteins determined by RT-PCR and immunoflourescence. Acute intestinal injury was induced by Dextran Sodium Sulfate (DSS, 3.5%) in the drinking water for 5 days. Mice were sacrificed 3 days post DSS and intestinal injury was assessed by disease severity index, histological scores, myeloperoxidase (MPO) activity, and cytokine production in the colon. Bifidobacterium breve NCC2950 was administered by gavage (109 CFU/ 100 μl/mouse) 14 days before colitis induction. Results: NOD1/2-/- mice had microbiota profiles different to NOD1/2+/+ controls bred from a different facility but had similar microbiota profiles compared to heterozygous littermate NOD1/2+/- controls. Despite this, naïve NOD1/2-/- mice had increased paracellular permeability compared to NOD1/2+/- controls. This was paralleled by decreased expression of E-cadherin, but no spontaneous development of colonic inflammation. After induction of intestinal injury, disease severity index, histological score, MPO activity, colonic IL-12p70 and TNF-α levels were significantly higher by 50%, 27%, 71%, 60% and 45% respectively, in NOD1/2-/- mice compared 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 dysfunction and increase susceptibility to intestinal injury regardless of microbiota composition. Bifidobacterium breve NCC2950 prevents DSS-induced inflammation, and may be of potential benefit to decrease IBD risk in patients with NOD mutations. This work is supported by the Crohn's and Colitis Foundation of Canada
Tu1846 The Probiotic Bacteria Lactobacillus Acidophilus NCFM® (L-NCFM) and Bifidobacterium Lactis Bi-07 (B-Lbi07) Increase Expression of Intestinal Opioid Receptors - First Evidence in Humans Yehuda 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-NCFM has been shown to increase expression of opioid (MOR) receptors in the intestinal mucosa and decrease intestinal pain sensation in animal studies [Rousseaux 2007]. A recent study with the combination of L-NCFM and B-LBi07 suggested a beneficial effect on GI symptoms in humans [Ringel 2010]. Intestinal STAT3 activation has recently been linked with opioid signaling [Goldsmith 2010]. Aim: To investigate the effect of L-NCFM and B-LBi07 on colonic mu opioid receptors (MOR) expression in patients with functional abdominal pain. Methods: Caucasian women (n=17) 18-70 years with mild to moderate abdominal pain were investigated. Colonic biopsies were collected during un-sedated, un-prepped flexible sigmoidoscopy pre- and post- 21 days consumption of the probiotics at a total dose of 1.0 x 1010 CFU bid. Cellular RNA was extracted from the mucosal biopsies and reverse transcribed to cDNA. mRNA expression of mucosal MOR receptors was determined by RT-PCR on an ABI Prism HT7700 Thermocycler (Applied Biosystems) via SybrGreen. Relative fold-changes were determined using the ΔΔCT calculation method. Immunohistochemistry (IHC) for pSTAT3 was performed on paraffin-embedded sections of mucosal biopsies with monocloncal antibodies for pSTAT3(Y705) (Cell Signaling Technology Inc) were used at a 1:50 dilution. Slides were developed using horseradish peroxidase-conjugated secondary antibody and a VectaStain Elite ABC Kit (Vector Laboratories Inc). Results: Pre- to post-intervention with L-NCFM and B-LBi07 or L-NCFM alone was associated with a 5.5-fold increase in MOR expression (8.2E-6 vs 1.5E-6 fold-over-β-actin, P=0.014; n=13). The increase in MOR expression was significantly (~10-fold) greater with L-NCFM alone compared to the combination 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 treated with L-NCFM alone had a 39.9-fold pre- to post-intervention increase in the levels of the receptor (P =0.0313). IHC for pStat3 showed increase in mucosal biopsies only in the LNCFM group. Conclusions: We replicated in humans the animal model observation of
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AGA Abstracts
AGA Abstracts
manner (p<0.01). 3) To assess the effects of SbS on VEGF-induced angiogensis In Vivo the right ears of mice (N=5) were injected with SbS and the left ears were injected with vehicle as control. The first treatment was administered at 1 hour after 5x10^6 pfu Ad-VEGF-A164 was injected into both ears. The second SbS/vehicle injections were administered 24 hours later. An inhibitory effect of SbS was clearly evident by day 7 with reduced new vessel formation in the right ear compared to the left. The effect of SbS remained evident until the end of the experiment on day 21. 4) DSS (4% for 5 days) was administered to mice to induce colitis. Daily gavage of Sb significantly attenuated weight-loss (N=5,p<0.01). DSS treated mice had significantly increased blood vessel density and volume compared to healthy mice. Sb treatment significantly reduced the neo-vascularization associated with inflammation in the colon. CONCLUSIONS: Sb inhibits VEGFR signaling and reduces neo-vascularization In Vitro and in two separate In Vivo models. Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis in intestinal injury and repair, which provides a novel mechanism for its beneficial effects.
AGA Abstracts
Tu1840 Toll-Like Receptor 5 is Significantly Associated With Canine Inflammatory Bowel Disease: Genetical and Functional Analysis Aarti Kathrani, Angela Holder, Brian Catchpole, Dirk Werling, Karin Allenspach TLR5 has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (IBD) and animal models of this disease. A polymorphism in the TLR5 gene which terminates TLR5 signaling has shown to be protective for Crohn's disease (CD) in a Jewish cohort. Similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (SNPs) in the canine TLR5 gene (G22A, C100T and T1844C) and inflammatory bowel disease (IBD) in German shepherd dogs (GSDs). Therefore the aim of this study was to determine the functional significance of the canine TLR5 SNPs by transfecting wild-type and mutant receptors into human embryonic kidney cells (HEK) and carrying out nuclear factor-kappa B (NF-κB) luciferase assay and CXCL8 ELISA. The TLR5 gene containing the risk haplotype for IBD (ACC) and wild-type haplotype (GTT) as determined by the case-control analysis in GSDs with IBD were cloned into plasmids expressing yellow-fluorescent protein (YFP). These were then stably transfected into HEK cells. Transfection was confirmed by assessing the expression of YFP by confocal microscopy and fluorescence-activated cell sorting analysis. NF-κB activity was measured by transiently transfecting the cells with NF-κB promoter firefly and HSV-thymidine kinase promoter renilla plasmids. The cells were then stimulated with various ligands (0.1μg/ml flagellin, 0.01μg/ml flagellin, 1μg/ml LPS, 1μg/ml PAM3CSK and media control). Firefly and renilla luciferase activities were measured using the DualGlo Luciferase Assay System (Promega, UK). The supernatants were harvested and used in a CXCL8 ELISA (R&D Systems, UK). Human TLR5 transfected HEK cells (Invivogen, UK) served as positive controls in all experiments. Independent T-test was used to determine the significance of relative luciferase activity and CXCL8 concentration between wild-type and mutated TLR5 cells. There was a significant increase in NF-κB activity when the cells with mutated TLR5 were stimulated with 0.1μg/ml of flagellin compared to the cells expressing wild-type TLR5 (p=0.027), which correlated with CXCL8 expression in the supernatant measured by ELISA (p=0.025). We show for the first time that polymorphisms associated with canine IBD are functionally hyper-responsive to flagellin compared to the wild-type receptor. This suggests that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease and may therefore serve as a model for human IBD. Further In-Vivo functional analysis of TLR5, especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions.
Tu1842 Secreted Bioactive Effectors of Lactobacillus Acidophilus Attenuate the Exaggerated Inflammatory Response Responsible for Necrotizing Enterocolitis Kriston A. Ganguli, W. Allan Walker, N. Nanda Nanthakumar Background & Aims: Necrotizing enterocolitis (NEC), a severe intestinal inflammatory disease of premature infants, results from an inappropriate inflammatory (IL-6) response during the initial stages of bacterial colonization. Randomized placebo-controlled trials have shown a significant reduction in the incidence of NEC when very low birth weight infants were supplemented with combination probiotics (Lactobacillus acidophilus and Bifidobacterium infantis). The goal of this study was to determine whether soluble factors secreted by these bacteria, grown individually, attenuate the inflammatory response of immature enterocytes In Vitro and to characterize these factors. Methods: Immature primary enterocyte cultures of NEC and fetal (IEC) tissue were assayed for IL-6 secretion after stimulation with an endogenous inflammatory stimulus, IL-1-beta, while concurrently being exposed to probiotic conditioned media (PCM) from either L. acidophilus or B. infantis. The IL-6 assays were repeated using L.acidophilus conditioned media which was exposed to size-fractionation, heat stress or Ulex europaeus agglutinin-1 (UEA-1) treatment. Results: In NEC-IEC and fetal IEC cell culture, a significant decrease (p<0.01) in IL-6 secretion was shown with L.acidophilus but not with B.infantis-conditioned media. The significant reduction in IL-6 secretion by NEC-IEC cells with L.acidophilus-conditioned media (p <0.001) was lost when the conditioned media was treated with a fucose-specific lectin, UEA-1. L.acidophilus conditioned media retained its anti-inflammatory properties after exposure to heat stress and fractionation < 30kDa (p<0.05). Conclusions: We conclude that PCM with L. acidophilus provides superior inflammatory attenuation over that of B. infantis in NEC-IEC and fetal IEC cells. The data suggest that the bioactive effector(s) secreted by L. acidophilus is a heat stable molecule less than 30kDa in size. The activity of the effector(s) is modified after exposure to a fucose-specific lectin, UEA-1, suggesting a glycoconjugate. However, additional studies are necessary for further characterization of L.acidophilus secreted effectors.
Tu1841 Profile of Anti-Microbiota Antibody Responses in Crohn's Disease and Ulcerative Colitis Nicola Patuto, Emma Slack, Bruno Strebel, Maria L. Balmer, Philipp Schuetz, Frank Seibold, Andrew J. Macpherson Background: Crohn's disease and ulcerative colitis are both diseases which manifest as a failed mutualism with the intestinal microbiota, clearly demonstrated by the amelioration of symptoms on diversion of the fecal stream. In Crohn's disease, a large number of reports document the presence of elevated serum antibody responses specific for bacterial and yeast products. We have recently demonstrated in murine models that elevated anti-microbiota immunity is a common sequalae of innate immune deficiency. We therefore compared the spectrum of anti-microbiota immunity in patients with Crohn's disease or ulcerative colitis to functional measurements of innate immune functionality. Methods: Anti-microbiota immunity was quantified using a technique that we have developed based on high-affinity binding of serum antibodies to the surface of live bacteria (Figure 1). This binding is then quantified per bacterium by flow cytometry. By carrying out live bacterial staining with dose-titrations of plasma, titration curves can be plotted in a similar manner to classical ELISA titres. To functionally characterise innate immunity, concentrations of innate immune stimuli required to give 50% activation of granulocyte and monocyte CD62L-shedding and oxidative burst production were calculated for each patient. The IBD patient cohort was collected from the outpatient clinic of the Inselspital and Tiefenauspital in Bern and each patient diagnosis was re-confirmed. Conclusions: Crohn's disease patients show dramatically elevated IgG and IgA titres specific for gamma-proteobacteria, in particular Klebsiella pneumoniae (Figure 1) whilst Ulcerative colitis patients show highly elevated IgG titres specific for predominantly unculturable fecal bacteria. Innate immune sensitivity of patients varied considerably between individuals with up to 10000-fold differences in sensitivity to TLR1 ligands observed, but no obvious disease-associated trends (Figure 2). Further analysis of reactivity to bacterial species representative of the intestinal microbiota will reveal whether positive associations exist between innate immune sensitivity, anti-microbiota immunity and disease phenotype.
AGA Abstracts
Tu1843 Saccharomyces Boulardii Inhibits VEGFR Signaling and Angiogenesis in Intestinal Inflammation Xinhua Chen, Guoxun Yang, Joo Hye Song, Hans-Christian Reinecker, Huiyan Zeng, Ciaran P. Kelly, Hua Xu BACKGROUND AND AIM: Saccharomyces boulardii (Sb), a probiotic yeast, has been used for decades to protect against intestinal injury and inflammation. We previously reported Sb protects against Clostridium difficile colitis and intestinal tumor formation through modulation of host signaling including the EGFR/Erk pathway. Angiogenesis is increasingly recognized as an important component of intestinal inflammation. We found that Sb inhibits VEGFR (vascular endothelial growth factor receptor) signaling, a central pathway regulating angiogenesis. Using In Vitro assays and In Vivo models, we aim to examine whether Sb inhibits angiogenesis. METHODS: HUVEC cells were treated with VEGF with and without Sb supernatant (SbS). Antibodies against p-VEGFR2, p-Erk1/2, or p-PLCγ were used in Western analyses. The ECMatrixTM In Vitro angiogenesis assay was used to study the effects of SbS on HUVEC capillary tube formation. To assess the effects of SbS on VEGF-induced angiogenesis In Vivo, we used an adenovirus expressing VEGF-A(164) in the ears of adult nude mice (NuNu). The DSS-colitis mouse model was used to assess the effect of Sb on neo-vascularization in acute colitis. Blood vessels were stained with Alexa647 WGA injected retro-orbitally and tissue specimens were imaged with a Bio-Rad Radiance 2000 confocal microscope and three-dimensional analysis of vascular density and volume carried out after processing in Volocity software. RESULTS: 1) SbS inhibited the phosphorylation of VEGFR2 in response to VEGF and also reduced activation of the downstream kinases PLCγ and Erk1/2. 2) SbS significantly inhibited HUVEC capillary tube formation in a dose-dependent
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increased expression of MOR receptors with L-NCFM. In addition, we demonstrated post receptor signaling effect. Our data provides the first evidence for a probiotic effect on opioidmediated pain pathways in humans. The physiological (sensation threshold) and clinical (pain experience) implications of these findings requires further investigation. Tu1847 Exploring the Mechanism of a Probiotic Combination VSL#3 in Irritable Bowel Syndrome (IBS): A Randomized Double-Blind Placebo Controlled Study Reuben 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 mechanism remains obscure. One hypothesis is the mediation of melatonin levels, leading to changes in 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 melatonin levels. Methods: 42 IBS patients were randomly assigned to receive 4 capsules of either VSL#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 sensitivity study as well as saliva and fecal melatonin assays. Results: VSL#3 and placebo decreased the 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 compared with 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#3 patients (38.4 to 42.5 mmHg) compared to controls. A correlation between increase in pain tolerance threshold and improvement in abdominal pain scores (r=0.51,p=0.02) was seen with VSL#3 but not placebo. No significant changes following treatment were observed in psychological indices nor sleep parameters. There was an increase in salivary morning melatonin levels in males (5.43pg/ml to 9.74pg/ml,p=0.03) treated with VSL#3, which correlated (r=0.61,p=0.058) with improved satisfaction in bowel habits. When subjects were grouped into normal vs. abnormal baseline diurnal melatonin levels, the former showed an increase 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 severity scores (-123.8 vs. -45) and abdominal pain duration (-27.8 vs. -12.9) when treated with VSL#3 vs. placebo. They also had significantly improved satisfaction with bowel movements and quality of life. Conclusions: VSL#3 reduced abdominal pain duration and distension intensity in IBS subjects. Rectal pain thresholds were improved, correlating with an improved abdominal pain scores. The improvement in symptoms correlated with a rise in morning systemic melatonin, which was significant in males and subjects with normal circadian rhythm. We postulate that the probiotic acts by influencing melatonin production, hence modulating IBS symptoms, in individuals with a “normal” diurnal melatonin levels but not in those with a baseline disordered circadian rhythm.
Tu1844 Lactobacillus Casei Prevents Osmotic Stress-Induced Disruption of Tight Junctions (TJ) and Adherens Junctions (AJ) in CACO-2 Cell Monolayers by a PKC-Dependent Mechanism, but Independent of EGF Receptor Tyrosine Kinase Activity Rupa 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 osmotic stress (OS). We investigated the effect of L. casei on OS-induced TJ disruption in Caco-2 cell monolayers. Methods: Cell monolayers were exposed to OS (650 mOsM, adjusted with mannitol) for 0.5-2 hrs in the presence or absence of L. casei (live or UV-killed). Barrier function was evaluated by measuring transepithelial electrical resistance (TER) and FITCinulin 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 was determined by immunoprecipitation of p-Tyr and immunoblot analysis. Association of TJ proteins with detergent-insoluble fraction was assessed by immunoblot analysis. Role of PKC, EGFR kinase and MAP kinase activities in the mechanism of L. casei effect was determined by evaluating the effects of inhibitors, Ro-32-0432 (for PKC), AG1478 (for EGFR), and U0126 (for MEK). L. casei-mediated PKC activation was analyzed by immunoblot analysis in plasma membrane fractions. Results: OS reduced TER and increased inulin flux in a time-dependent manner. Pretreatment of cell monolayers with L. casei (live or UVkilled) significantly attenuated the OS-induced changes in TER and inulin permeability. OSinduced redistribution of occludin, ZO-1, E-cadherin, β-catenin and claudin-3 from the intercellular junction into the intracellular compartments was attenuated by L. casei. L. casei induced a significant enhancement of actin organization at the mid region perijunctional area and effectively attenuated OS-induced reorganization of actin cytoskeleton. L. casei attenuated 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-cadherin and β-catenin, which was abrogated by L. casei. L. casei-mediated prevention of OS-induced decrease in TER, increase in inulin permeability and redistribution of TJ and AJ proteins was prevented by Ro-32-0432, but not by AG1478 or U0126. These inhibitors by themselves showed no significant influence on TER or inulin permeability in control or OS-treated cell monolayers. L. casei induced a transient increase in the levels of plasma membrane associated PKCε, but not PKCβI. Conclusion: These studies demonstrate that L. casei ameliorates OSinduced disruption of apical junctional complexes by a mechanism dependent on PKC, but independent of EGF receptor and MAP kinases. Supported by DK55532 and AA12307.
Tu1848 The Role of Intestinal Bacteria and Bifidobacterium Breve NCC2950 in Barrier Homeostasis and Intestinal Injury in NOD1 and 2 Knockout Mice Jane M. Natividad, XianXi Huang, Jennifer Jury, Giada de Palma, Yolanda Sanz, Dana Philpott, Clara L. Garcia Rodenas, Kathy McCoy, Elena F. Verdu Background and Aim: The precise mechanisms by which (NOD)-like receptors contribute to inflammatory bowel disease (IBD) are not completely understood. Emerging data support a role of NOD1 and 2 receptors in the modulation of intestinal barrier homeostasis and intestinal microbiota composition. We hypothesized that intestinal microbiota composition and probiotic supplementation affects the intestinal barrier and host responses to intestinal injury in mice with defective NOD 1 and 2 signaling. Methods: Fecal microbiota composition was 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+/+) bred from a different facility. Intestinal permeability was assessed by In Vitro and In Vivo techniques and expression of epithelial apical junction proteins determined by RT-PCR and immunoflourescence. Acute intestinal injury was induced by Dextran Sodium Sulfate (DSS, 3.5%) in the drinking water for 5 days. Mice were sacrificed 3 days post DSS and intestinal injury was assessed by disease severity index, histological scores, myeloperoxidase (MPO) activity, and cytokine production in the colon. Bifidobacterium breve NCC2950 was administered by gavage (109 CFU/ 100 μl/mouse) 14 days before colitis induction. Results: NOD1/2-/- mice had microbiota profiles different to NOD1/2+/+ controls bred from a different facility but had similar microbiota profiles compared to heterozygous littermate NOD1/2+/- controls. Despite this, naïve NOD1/2-/- mice had increased paracellular permeability compared to NOD1/2+/- controls. This was paralleled by decreased expression of E-cadherin, but no spontaneous development of colonic inflammation. After induction of intestinal injury, disease severity index, histological score, MPO activity, colonic IL-12p70 and TNF-α levels were significantly higher by 50%, 27%, 71%, 60% and 45% respectively, in NOD1/2-/- mice compared 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 dysfunction and increase susceptibility to intestinal injury regardless of microbiota composition. Bifidobacterium breve NCC2950 prevents DSS-induced inflammation, and may be of potential benefit to decrease IBD risk in patients with NOD mutations. This work is supported by the Crohn's and Colitis Foundation of Canada
Tu1846 The Probiotic Bacteria Lactobacillus Acidophilus NCFM® (L-NCFM) and Bifidobacterium Lactis Bi-07 (B-Lbi07) Increase Expression of Intestinal Opioid Receptors - First Evidence in Humans Yehuda 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-NCFM has been shown to increase expression of opioid (MOR) receptors in the intestinal mucosa and decrease intestinal pain sensation in animal studies [Rousseaux 2007]. A recent study with the combination of L-NCFM and B-LBi07 suggested a beneficial effect on GI symptoms in humans [Ringel 2010]. Intestinal STAT3 activation has recently been linked with opioid signaling [Goldsmith 2010]. Aim: To investigate the effect of L-NCFM and B-LBi07 on colonic mu opioid receptors (MOR) expression in patients with functional abdominal pain. Methods: Caucasian women (n=17) 18-70 years with mild to moderate abdominal pain were investigated. Colonic biopsies were collected during un-sedated, un-prepped flexible sigmoidoscopy pre- and post- 21 days consumption of the probiotics at a total dose of 1.0 x 1010 CFU bid. Cellular RNA was extracted from the mucosal biopsies and reverse transcribed to cDNA. mRNA expression of mucosal MOR receptors was determined by RT-PCR on an ABI Prism HT7700 Thermocycler (Applied Biosystems) via SybrGreen. Relative fold-changes were determined using the ΔΔCT calculation method. Immunohistochemistry (IHC) for pSTAT3 was performed on paraffin-embedded sections of mucosal biopsies with monocloncal antibodies for pSTAT3(Y705) (Cell Signaling Technology Inc) were used at a 1:50 dilution. Slides were developed using horseradish peroxidase-conjugated secondary antibody and a VectaStain Elite ABC Kit (Vector Laboratories Inc). Results: Pre- to post-intervention with L-NCFM and B-LBi07 or L-NCFM alone was associated with a 5.5-fold increase in MOR expression (8.2E-6 vs 1.5E-6 fold-over-β-actin, P=0.014; n=13). The increase in MOR expression was significantly (~10-fold) greater with L-NCFM alone compared to the combination 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 treated with L-NCFM alone had a 39.9-fold pre- to post-intervention increase in the levels of the receptor (P =0.0313). IHC for pStat3 showed increase in mucosal biopsies only in the LNCFM group. Conclusions: We replicated in humans the animal model observation of
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AGA Abstracts
AGA Abstracts
manner (p<0.01). 3) To assess the effects of SbS on VEGF-induced angiogensis In Vivo the right ears of mice (N=5) were injected with SbS and the left ears were injected with vehicle as control. The first treatment was administered at 1 hour after 5x10^6 pfu Ad-VEGF-A164 was injected into both ears. The second SbS/vehicle injections were administered 24 hours later. An inhibitory effect of SbS was clearly evident by day 7 with reduced new vessel formation in the right ear compared to the left. The effect of SbS remained evident until the end of the experiment on day 21. 4) DSS (4% for 5 days) was administered to mice to induce colitis. Daily gavage of Sb significantly attenuated weight-loss (N=5,p<0.01). DSS treated mice had significantly increased blood vessel density and volume compared to healthy mice. Sb treatment significantly reduced the neo-vascularization associated with inflammation in the colon. CONCLUSIONS: Sb inhibits VEGFR signaling and reduces neo-vascularization In Vitro and in two separate In Vivo models. Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis in intestinal injury and repair, which provides a novel mechanism for its beneficial effects.