- Email: [email protected]
562 Microbiota-Induced Serum Amyloid A is an Important Mediator of Innate Immune Response in Zebrafish
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of AIEC reference strain LF82 was screened to search for putative virulence factors allowing bacteria to target PP. Wild-type LF82 bacteria and mutants were tested for their abilities to interact with murine and human isolated PP, and M cells. Results: Numerous AIEC LF82 bacteria were observed within PP as shown by ex vivo assays using murine and human PP. The AIEC LF82 bacteria translocated at a very high level through M cell monolayers, but not as the result of the loss of monolayer integrity. A functional operon encoding long polar fimbriae (LPF) is present in AIEC strain LF82. The LF82-delta lpfA mutant was impaired in its ability to interact with murine and human isolated PP and to translocate across M cell monolayers. In addition, increased numbers of E. coli are associated with PP from CD patients compared to those from controls. Increased numbers of AIEC LF82 bacteria associated with PP were also observed in Nod2-/- mice due to the increased numbers of M cells and confocal analysis confirmed that AIEC LF82 bacteria target M cells at the surface of PP dependently on the expression of LPF. The prevalence of subjects positive for ileum-associated AIEC strains harboring lpf operon was 21.8% in CD patients as against only 3.5 % in controls (P=0.027). Conclusions: As a key factor for AIEC to target M cells in PP, LPF encoding gene should be screened among ileal-associated E. coli strains to define patients at high risk of developing CD or a recurrence of the disease.
AGA Abstracts
Intestine-Specific Deletion of the Mouse KrüPpel-Like Factor 4 Gene Results in Altered Homeostasis of Intestinal Epithelial Cells Amr Ghaleb, Beth B. McConnell, Jonathan P. Katz, Klaus H. Kaestner, Vincent W. Yang BACKGROUND: The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is normally expressed in the differentiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. In Vitro, KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein. In Vivo, KLF4 exhibits a tumor suppressive effect on intestinal tumorigenesis. Klf4-null mice die by postnatal day 1 and show changes in differentiation of epithelial tissues including the conjunctiva, epidermis and gastrointestinal tract. AIM: To characterize the intestinal phenotype in mice following conditional deletion of the Klf4 gene from the intestine. METHODS: Conditional gene ablation was used to generate mice lacking Klf4 in their intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre) mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and control (Klf4fl/fl) mice were sacrificed at 3 weeks of age for histological and immunuohistochemical characterization of the intestinal tract. Cell migration was measured by 5-bromo2-deoxyuridine (BrdU) pulse labeling experiments. RESULTS: Vil-Cre;Klf4fl/fl mice were born in a Mendelian ratio and remained viable at 4 months. At 3 weeks of age, the colon of the mutant mice displayed distorted architecture characterized by dilated crypt glands. Both the small and large intestine of the mutant mice exhibited reduced number and size of mature goblet cells compared to controls as demonstrated by Alcian blue staining. Brush-border alkaline phosphatase staining in the villus epithelial cells was also significantly reduced in the small intestine of mutant mice. In addition, Paneth cells were not restricted to the base of the crypts and were mispositioned in the upper crypt region in the small intestine of mutant mice. In contrast, the number of enteroendocrine cells was not affected by Klf4 deletion. Although the number of Ki67 or cyclin D1-positive cells per crypt in the small intestine was not significantly different between mutant and control mice, mutant mice had a statistically significant increase in both the number and migratory rate of BrdU-positive cells following a 24-hour pulse when compared to controls. CONCLUSION: The results of this study provide new insights into the function of KLF4 in postnatal maturation, proliferation, migration and positioning of intestinal epithelial cells, demonstrating an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis.
561 The Stress Response Significantly Changes Microbial Populations in the Intestines and Increases Susceptibility to Enteric Infection Michael T. Bailey, Jeffrey D. Galley, Scot E. Dowd, Mark Lyte The ability of the stress response to influence susceptibility to enteric disease through the direct modulation of the commensal microbiota is unknown. The commensal microbiota have many beneficial effects on health and participate in host defense against enteric pathogens. Previous studies using culture-based methods have demonstrated that stressor exposure can change microbial populations in the intestines, but the extent, and biological importance, of these changes are not well known. We have been using next generation pyrosequencing to characterize the microbiota of mice exposed to an overnight restraint stressor. The diversity of the microbiota within the luminal contents, as well as adhered to colonic tissue, was significantly reduced by exposure to the stressor. Double clustering analysis of the microbiota indicated that although a single night of stressor exposure was not enough to significantly affect community structure, repeated nights of stressor exposure significantly changed community structure at the class, family, and genus level of analysis. This clustering was primarily due to a stressor-induced reduction in the relative abundance of bacteria in the genus Tannerella in the luminal contents, and a reduction of bacteria in the genus Lactobacillus associated with the colonic tissue. To begin testing whether stressor-induced changes in the microbiota impact susceptibility to enteric infection, mice were restrained overnight for 7 consecutive nights prior to oral challenge with the murine pathogen Citrobacter rodentium, which causes a disease that is similar to human infection with enteropathogenic Escherichia coli. Exposure to the restraint stressor increased C. rodentium colonization by approximately 4 log units. This increased colonization was associated with increased gene expression for TNF-α, iNOS, and IL-1β, and decreased gene expression for IL-10 in the colonic tissue. Gene expression for mouse β-defensin 1 and colonic levels of IgA were unaffected by stressor exposure. Thus, the data are consistent with the view that stressor-induced changes in the microbiota result in increased susceptibility to C. rodentium; higher levels of C. rodentium in turn lead to increased colonic inflammation. Thus, the ability of stressors to directly modulate the composition of the enteric microbiota may represent a new mechanism governing susceptibility to enteric infection. This model may also be a useful tool for studying post-infectious irritable bowel syndrome, where alterations of the microbiota, prolonged inflammation, and psychological distress are all known to be predictive of this clinically important disease.
559 The Human Variant D299G of the TLR4 Gene Links Inflammation and Cancer Progression in the Intestinal Epithelium Annette Eyking, Guido Gerken, Daniel K. Podolsky, Elke Cario Background: Chronic recurrent intestinal inflammation in inflammatory bowel diseases (IBD) may result from aberrant stimulation of the mucosal immune system by the resident microflora. TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC) in IBD colitis. The variant D299G of the TLR4 gene has been associated with increased IBD susceptibility. However, the phenotypic consequences of this gene variant in IEC have not been resolved yet. The aim of the study was to investigate how the IBD-associated variant TLR4-D299G may affect IEC biology and molecular function. Methods: IEC lines (Caco2) stably overexpressing HA-tagged full-length (FL) TLR4 or mutant TLR4-D299G or TLR4T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed and network data were generated through Ingenuity Pathways Analysis. Results were confirmed by qRT-PCR and validated by western/ELISA. Mitosis was examined by confocal immunofluorescence. Functional studies included assessment of proliferation (BrdU), metabolic activity (MTT) and invasive potential (matrigel). InVivo tumorigenicity was tested using the CD-1 nu/nu mouse xenograft model. Results: Functional clustering of the microarray datasets revealed that the major TLR4-D299G targets were dominated by genes involved with promotion of inflammation and tumorigenesis, and these genes were markedly downregulated in IEC expressing TLR4-FL or TLR4-T399I. Three distinct pro-inflammatory systems (acute phase response, coagulation and complement) were the most significant canonical pathways modulated by the differentially regulated genes in TLR4-D299G. In contrast to TLR4-FL, TLR4-D299G IEC constitutively secreted large amounts of complement proteins (C3a; C5a) and coagulation factors (TFPI; A2M). Functionally, IEC expressing TLR4-D299G revealed central features of tumorigenicity - which were not evident in TLR4-FL: TLR4-D299G IEC 1) were multinucleated with multipolar mitotic spindles, 2) exhibited increased proliferation associated with elevated metabolic activity and repressed differentiation, and 3) demonstrated pronounced invasion via STAT3 activation. Compared with TLR4-FL-xenografts, In-Vivo tumor growth was significantly stimulated in TLR4-D299G-xenografts. Conclusions: Our results suggest that the TLR4-D299G variant represents a gain-of-function mutation with enhanced oncogenic potential. The TLR4-D299G variant establishes a pro-inflammatory microenvironment which may drive cancer progression in IEC. Thus, IBD patients with TLR4-D299G may be at increased risk to develop a more aggressive phenotype of colitis-associated neoplasia.
562 Microbiota-Induced Serum Amyloid A is an Important Mediator of Innate Immune Response in Zebrafish Ja Seol Koo, Michelle Kanther, John F. Rawls, Christian Jobin Background: The serum amyloid A family of acute phase protein (SAA1-4) have been traditionally viewed as markers of inflammation and their role in modulating innate host response is currently unknown. Since the zebrafish encodes only a single SAA homolog, this vertebrate animal represents an ideal system to investigate SAA function in host-microbe interaction. Methods: Germ-free (GF) transgenic NFKB:EGFP reporter zebrafish (Tg(NFKB:EGFP)) were conventionalized (CONVD) and pattern of EGFP expression was determined by fluorescent microscopy. SAA expression in GF and CONVD zebrafish was determined by wholemount in situ hybridization (WISH). Conventionally-raised Tg(NFKB:EGFP) zebrafish were injected with SAA morpholino (MO) at the 1-cell stage (0.9 pg/embryo) to block SAA expression. Impact of SAA on NF-κB signaling and gene expression In Vitro was determined using zebrafish PAC2 embryonic fibroblasts. NF-κB signaling was determined using immunofluorescent staining (RelA), western blot (phospho-IκBa), and transcriptional activity (luciferase reporter gene, NFKB:EGFP reporter plasmid). Transcription of NF-κB target genes In Vitro and In Vivo was determined using ABI Prism 7700HT detection system. Results: Recombinant human SAA (1μM) stimulated IκBα serine 32/36 phosphorylation, RelA nuclear translocation, and luciferase activity (relative fold change 3.5±0.07 vs ctl, p<0.01) in PAC2 cells. SAA induced EGFP expression in PAC2 cells transfected with pNFKB:EGFP. In addition, SAA induced NF-κB target genes IκBαa and MMP9 (relative fold change, 25.8±1.70 and 19.9±0.69 vs ctl respectively, p<0.01). Colonization of GF Tg(NFKB:EGFP) zebrafish with a normal microbiota resulted in elevated EGFP expression in the liver, dorsal root ganglia, the neuromasts of the lateral line, and a distinct population of intestinal cells. WISH and quantitative RT-PCR showed that CONVD Tg(NFKB:EGFP) zebrafish strongly up-regulated SAA gene expression in the liver and intestine compared to GF zebrafish. Importantly, although global cellular EGFP expression was not significantly inhibited in SAA-MO injected Tg(NFKB:EGFP) zebrafish, a strong decrease in endogenous mRNA accumulation of NF-κB targets IκBαa (40%) and MMP9 (90%) was observed in these fish compared to control MO injected zebrafish. Conclusion: Our data indicate that microbial colonization enhances SAA expression, which then impacts on NF-κB
560 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Target Peyer's Patches via Long Polar Fimbriae Benoit Chassaing, Nathalie Rolhion, Amelie De Vallee, Sa'ad Salim, Maelle Prorok-Hamon, Christel Neut, Barry J. Campbell, Jean-Pierre Hugot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud Background & Aims: Ileal lesions of patients with Crohn's disease (CD) are colonized by adherent-invasive Escherichia coli (AIEC) and the earliest observable lesions of CD are microscopic erosions of the follicle-associated epithelium (FAE) lining the Peyer's patches(PP). We aimed in the present study to investigate the ability of AIEC to interact with PP and M cells of the FAE, to quantify the PP-associated E. coli in CD patients compared to controls, and to search for the presence of a virulence factor in AIEC that could be involved in the targeting of PP by these invasive bacteria. Methods: PP from CD patients and controls were analyzed by q-PCR for the presence of E. coli 16S r-RNA encoding gene, and the genome
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563 Colonization-Dependent Colonic Fucosylation is Mediated by Gram Negative Bacteria via Activation of a Novel Fucosylated TLR4 Di Meng, Weishu Zhu, W. Allan Walker, N. Nanda Nanthakumar Adult colonic mucosa is highly fucosylated relative to immature mucosa, and this fucosylation is dependent upon bacterial colonization. Toll-like receptor-4 (TLR4) was hypothesized to mediate this colonization-dependent fucosylation. Germ-free (GF) and bacteria-depleted (BD) mice were compared with conventional and mutant mice with regard to induction of epithelial fut2 mRNA levels and α1,2-fucosyltransferase (FUT2) activity. Induction of these activities by recolonization were compared with induction by LPS (TLR4 ligand) and monocolonization by bacteriodes and E. coli strains. Cultured human erythroleukemia (Hel) cells were used to study fucosylated TLR4 In Vitro. Colonization-dependent induction of fucosylation occurs only in TLR2-/- and wild-type mice but not in TLR4-/- and MyD88-/- mice, suggesting TLR4-MyD88 dependent signaling is important for fucosylation. TLR4 is fucosylated in the colonocytes of only GF and BD mice. LPS activate fut2 mRNA and FUT2 activity in the absence of bacterial colonization. Fucose-utilizing Bacteroides fragilis induce colonic fucosylation, but not a fucose-non-utilizing mutant. In Hel cells, which express fucosylated TLR4 activates fut2 mRNA and fucosylation. TLR4-specific siRNA inhibits this activation, and fucosidase treatment abolished TLR4-dependent activation of fut2 mRNA, confirming the central role of fucosylated-TLR4 in activating fut2 and cell surface fucosylation. Fucosylated-TLR4 expressed on uncolonized colonocytes mediates bacterially-induced activation of fut2 mRNA and FUT2 activity without activating inflammation, and is a necessary initial step in the communication between the colonizing bacteria and the host epithelium.
566 Eicosapentaenoic Acid (EPA) Free Fatty Acid Reduces Polyp Burden in Familial Adenomatous Polyposis (FAP): Results of a Randomised, PlaceboControlled Trial Nicholas J. West, Susan K. Clark, Andrea Belluzzi, John M. Hutchinson, Mark Hull, Roger J. Leicester, Robin K. Phillips
564 Genes Encoding E. coli Small Heat Shock Proteins IbpA/B Have a Protective Role in Experimental Colitis in E. coli Monoassociated Il10-Deficient Mice Laura G. Patwa, R. Balfour Sartor, Jonathan J. Hansen
INTRODUCTION: EPA, as the free fatty acid (EPA-FFA), is an efficient means of increasing tissue levels. EPA-FFA dramatically reduces adenoma formation in the ApcMin/+ mouse and decreases cell proliferation in colon mucosal crypts in subjects with a history of sporadic colorectal polyps, effects consistent with a chemopreventative action. We report here the effects of EPA-FFA on polyp burden in subjects with FAP in a single-centre, randomised, placebo-controlled trial. METHODS: Subjects ≥18 years with FAP, having had a previous colectomy and ileo-rectal anastomosis, were randomised 1:1 to EPA-FFA (S.L.A. Pharma AG, Switzerland) as 2x500mg, enteric coated capsules, bd, or placebo capsules (PLA; capric/ caprylic acid triglycerides) for 6 months. A focal area of rectum (≥3 polyps ≥2mm) was tattooed at baseline endoscopy. Photographic records of this area at 0 and 6 months were used to determine numbers of polyps and their diameters. Video records of baseline and 6 month endoscopies were compared blind, in randomised order, independently, by each of a 5-member expert review panel and scored as Worse ( 1), Same as (0), or Better (+1). Fatty acid content of rectal biopsies was determined by GC-MS. RESULTS: Fifty eight subjects were randomised (EPA-FFA 29; PLA 29). Of these, efficacy data were available for 55 (EPAFFA 28; PLA 27). There was a statistically significant difference between treatment groups for change in polyp numbers (0-6 months) in the tattooed area (EPA-FFA mean [±SD] number of polyps 4.13 [2.47] at baseline decreased to 3.61 [2.31] at 6 months; PLA 4.50 [2.63] at baseline increased to 5.05 [3.30]; ANCOVA p=0.0046), a net difference of -22.4% [95%CI -39.6 to 5.1] (ANCOVA within-subject comparison p=0.0122). There were corresponding net differences in total polyp diameters (reduction of -29.8% [95%CI -56.1 to -3.6] for EPA-FFA vs PLA; p=0.027). These findings were corroborated by mean scores from the video review panel (EPA-FFA +0.09 [95%CI -0.14 to +0.32] vs PLA -0.34 [95%CI 0.56 to -0.11]; p=0.011). Rectal mucosal levels of EPA increased significantly (+159%) in the EPAFFA group compared to PLA (+78%; p=0.0179) as did docosapentaenoic acid (DPA) (+82.8% vs +1.3%; p=0.0103). EPA-FFA was well-tolerated. CONCLUSION: This formulation of EPA, as the free fatty acid, exhibited anti-neoplastic activity in subjects with FAP, evidenced by reductions in rectal polyp number and size and improvements in endoscopic appearance. These changes were paralleled by increased mucosal EPA levels. EPA-FFA has potential as a safe and effective chemopreventative agent.
Inflammatory bowel diseases (IBD) are thought to result from dysregulated immune responses to the intestinal microbiota. While host immune responses to microbial products have been extensively studied, relatively little is known about commensal microbial responses to intestinal inflammation. We have previously shown that bacterial stress response genes, ibpA and ibpB, are upregulated in luminal E. coli from colitic IL10-deficient (IL10-/-), but not healthy wild-type (wt), mice selectively colonized (monoassociated) with a non-pathogenic murine isolate of E. coli (NC101). The aim of this study is to investigate the influence of ibpA/B on experimental colitis. Methods: We monoassociated germ-free wt and IL10-/- mice with NC101 or an isogenic strain from which we deleted ibpA/B (ΔibpAB NC101). Intestinal inflammation was quantified using histological scoring and spontaneous secretion of IL12/ 23p40 from colonic explant cultures. Bacterial antigen-specific T-cell responses were assessed by measuring IFN-γ secretion in cultures of unseparated mesenteric lymph node (MLN) cells stimulated with NC101 lysate. Luminal concentrations of viable bacteria in the cecum were quantified by culturing serial dilutions on agar plates. In Vitro survival of bacteria in J774 murine macrophages was measured using gentamicin protection assays. Results: Histological inflammation scores, IL12/23p40 secretion in colon explant cultures, IFN-γ secretion in NC101 lysate-stimulated MLN cultures, and cecal bacterial concentrations were significantly greater in IL10-/- mice monoassociated with ΔibpAB NC101 compared to NC101 at both 3 and 5 wks. Similar results were obtained in wt mice monoassociated with ΔibpAB NC101 or NC101 for 9 wks, although the values were significantly lower compared to IL10-/- mice. Interestingly, survival of ΔibpAB NC101 within J774 cells was decreased compared to NC101. This difference was abrogated when cells were treated with a synthetic inhibitor of iNOS. Conclusions: Expression of E. coli ibpA/B attenuates experimental colitis and is associated with decreased luminal bacterial concentrations and decreased pro-inflammatory cytokine secretion, indicating that these bacterial stress response genes have a protective role in this model. These findings have implications in the development of treatment strategies for IBD that target host-microbial interactions.
567 Abusing Oncogenic RAS to Selectively Target Colorectal Cancer Cells Inna Naumov, Diana Kazanov, Shiran Shapira, Sarah Kraus, Nadir Arber Background: RAS mutations are present in 40-50% of human colorectal cancer (CRC) cases leading to the activation of downstream target genes. The PUMA (p53 up-regulated modulator of apoptosis) protein interacts with Bcl-XL and Bcl-2 to induce the release of mitochondrial cytochrome c and apoptosis thus can serve as a lethal gene. We have previously shown that recombinant adenovirus, carrying the PUMA gene (gift from Dr. B. Vogelstein) under the control of β-catenin- or Ras- responsive elements (TOP or Py2-SV40-PUMA, respectively) inhibit human cancer cell growth [1-3]. Aim: To selectively kill RAS transformed cells by overexpressing the pro-apoptotic PUMA gene under the control of RAS-responsive-elements (RRE). Materials and methods: SW480, HCT116 and DLD1 KRAS-mutated cells, HT29 cells harboring WT-KRAS were used, as well as normal enterocytes; IEC18/RIE and their KRAS/ HRAS transformed counterparts, R1/RIE-RAS, respectively. Two adenoviruses were constructed: PY4-PUMA, containing RRE as a promoter and a control virus, SV40 minimal promoter-PUMA, lacking RRE. Prior to infection, RAS activity was determined using GSTRBD pull-down assay. Inhibition of proliferation, cell viability, and protein expression were determined. In Vivo, HCT116 (5,000,000) cells were injected s.c. into athymic mice (n= 30). PY4-PUMA/ SV40-PUMA was injected i.p. twice, for a period of 4 weeks. All experiments were repeated 3 times. Results: KRAS mutated cells showed increased RAS activity compared to cells with WT-KRAS-PY4-PUMA. A marked inhibition in cell growth (up to 50%) and spontaneous apoptosis (~50%) was seen in cells exhibiting high RAS activity following
mean±SEM, p<0.05 for all (NC101 vs. ΔibpAB NC101) 565 Ind-Enabling Studies for CEQ508 Targeting β-Catenin of GI Polyps: First Oral RNAI Drug Natalya Bodyak, Alex Borrelli, Johannes Fruehauf, Jens Harborth, Moreshwar B. Vaze, Catherine Grillot-Courvalin, Alison Silva Targeted delivery remains one of the biggest challenges in the development of RNAi-based therapeutics. Cequent has developed a proprietary delivery technique for RNA interference, transkingdom RNAi (tkRNAi), in which non-pathogenic bacteria are engineered to invade specific target cells, produce and release short hairpin RNA (shRNA). We have previously shown tkRNAi to be successful in cell culture assays, in a mouse model for human colon
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cancer (APCmin), and in non-human primates (cynomolgus monkeys), by suppressing the oncogene, β-catenin. β-catenin is the key oncogene implicated in Colorectal Cancer (CRC) and Familial Adenomatous Polyposis (FAP); the latter being an orphan hereditary disease resulting in the formation of hundreds of polyps in the gastrointestinal tract and ultimately leads to the development of colon cancer without surgical intervention. No pharmaceutical treatment is available for patients with FAP. The non-human primate studies designed to evaluate on-target toxicities were conducted with a precursor (CEQ501) to our clinical candidate, CEQ508. Treatment significantly reduced mRNA levels of β-catenin in the mucosa of the gastrointestinal tract, an effect that was reversible after termination of dosing. The target gene was found reduced by 58-64% in treated compared to control animals. Following optimization, which most importantly included platform engineering to improve hairpin production and processing, we conducted a large GLP mouse bridging toxicity study comparing off-target effects of CEQ508 and CEQ501. No CEQ508 or CEQ501-related adverse responses were identified in the following study parameters analyzed: clinical observations, body weights, serum chemistry, hematology, cytokines, gross or histopathology. As no test article-related changes were identified under the conditions of this study at the highest dose evaluated, the No Observed Effect Level (NOEL) for daily oral administration of either CEQ508 or CEQ501 was 5x109 cfu/day, or 2x1011 cfu/kg/day. (CEQ501 in non-human primates showed a similar safety profile with the No Observed Adverse Effect Level (NOAEL) determined to be 1x1012 cfu/day, or 2x1011 cfu/kg/day). Together with multiple internally conducted pharmacology and pharmacokinetic experiments, Cequent used this data in the nonclinical section of an IND filed in late 2009. In addition to presenting detailed In Vivo data from our filing, an update on further development will be presented on CEQ508 for the proposed daily dosing of FAP patients with this oral tkRNAi therapeutic targeting β-catenin.
activity and down-stream gene expression in zebrafish. Therefore, we suggest that microbiotainduced SAA expression contributes to shape innate immune response in zebrafish and could represent an important component of host homeostasis.
of AIEC reference strain LF82 was screened to search for putative virulence factors allowing bacteria to target PP. Wild-type LF82 bacteria and mutants were tested for their abilities to interact with murine and human isolated PP, and M cells. Results: Numerous AIEC LF82 bacteria were observed within PP as shown by ex vivo assays using murine and human PP. The AIEC LF82 bacteria translocated at a very high level through M cell monolayers, but not as the result of the loss of monolayer integrity. A functional operon encoding long polar fimbriae (LPF) is present in AIEC strain LF82. The LF82-delta lpfA mutant was impaired in its ability to interact with murine and human isolated PP and to translocate across M cell monolayers. In addition, increased numbers of E. coli are associated with PP from CD patients compared to those from controls. Increased numbers of AIEC LF82 bacteria associated with PP were also observed in Nod2-/- mice due to the increased numbers of M cells and confocal analysis confirmed that AIEC LF82 bacteria target M cells at the surface of PP dependently on the expression of LPF. The prevalence of subjects positive for ileum-associated AIEC strains harboring lpf operon was 21.8% in CD patients as against only 3.5 % in controls (P=0.027). Conclusions: As a key factor for AIEC to target M cells in PP, LPF encoding gene should be screened among ileal-associated E. coli strains to define patients at high risk of developing CD or a recurrence of the disease.
AGA Abstracts
Intestine-Specific Deletion of the Mouse KrüPpel-Like Factor 4 Gene Results in Altered Homeostasis of Intestinal Epithelial Cells Amr Ghaleb, Beth B. McConnell, Jonathan P. Katz, Klaus H. Kaestner, Vincent W. Yang BACKGROUND: The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is normally expressed in the differentiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. In Vitro, KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein. In Vivo, KLF4 exhibits a tumor suppressive effect on intestinal tumorigenesis. Klf4-null mice die by postnatal day 1 and show changes in differentiation of epithelial tissues including the conjunctiva, epidermis and gastrointestinal tract. AIM: To characterize the intestinal phenotype in mice following conditional deletion of the Klf4 gene from the intestine. METHODS: Conditional gene ablation was used to generate mice lacking Klf4 in their intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre) mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and control (Klf4fl/fl) mice were sacrificed at 3 weeks of age for histological and immunuohistochemical characterization of the intestinal tract. Cell migration was measured by 5-bromo2-deoxyuridine (BrdU) pulse labeling experiments. RESULTS: Vil-Cre;Klf4fl/fl mice were born in a Mendelian ratio and remained viable at 4 months. At 3 weeks of age, the colon of the mutant mice displayed distorted architecture characterized by dilated crypt glands. Both the small and large intestine of the mutant mice exhibited reduced number and size of mature goblet cells compared to controls as demonstrated by Alcian blue staining. Brush-border alkaline phosphatase staining in the villus epithelial cells was also significantly reduced in the small intestine of mutant mice. In addition, Paneth cells were not restricted to the base of the crypts and were mispositioned in the upper crypt region in the small intestine of mutant mice. In contrast, the number of enteroendocrine cells was not affected by Klf4 deletion. Although the number of Ki67 or cyclin D1-positive cells per crypt in the small intestine was not significantly different between mutant and control mice, mutant mice had a statistically significant increase in both the number and migratory rate of BrdU-positive cells following a 24-hour pulse when compared to controls. CONCLUSION: The results of this study provide new insights into the function of KLF4 in postnatal maturation, proliferation, migration and positioning of intestinal epithelial cells, demonstrating an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis.
561 The Stress Response Significantly Changes Microbial Populations in the Intestines and Increases Susceptibility to Enteric Infection Michael T. Bailey, Jeffrey D. Galley, Scot E. Dowd, Mark Lyte The ability of the stress response to influence susceptibility to enteric disease through the direct modulation of the commensal microbiota is unknown. The commensal microbiota have many beneficial effects on health and participate in host defense against enteric pathogens. Previous studies using culture-based methods have demonstrated that stressor exposure can change microbial populations in the intestines, but the extent, and biological importance, of these changes are not well known. We have been using next generation pyrosequencing to characterize the microbiota of mice exposed to an overnight restraint stressor. The diversity of the microbiota within the luminal contents, as well as adhered to colonic tissue, was significantly reduced by exposure to the stressor. Double clustering analysis of the microbiota indicated that although a single night of stressor exposure was not enough to significantly affect community structure, repeated nights of stressor exposure significantly changed community structure at the class, family, and genus level of analysis. This clustering was primarily due to a stressor-induced reduction in the relative abundance of bacteria in the genus Tannerella in the luminal contents, and a reduction of bacteria in the genus Lactobacillus associated with the colonic tissue. To begin testing whether stressor-induced changes in the microbiota impact susceptibility to enteric infection, mice were restrained overnight for 7 consecutive nights prior to oral challenge with the murine pathogen Citrobacter rodentium, which causes a disease that is similar to human infection with enteropathogenic Escherichia coli. Exposure to the restraint stressor increased C. rodentium colonization by approximately 4 log units. This increased colonization was associated with increased gene expression for TNF-α, iNOS, and IL-1β, and decreased gene expression for IL-10 in the colonic tissue. Gene expression for mouse β-defensin 1 and colonic levels of IgA were unaffected by stressor exposure. Thus, the data are consistent with the view that stressor-induced changes in the microbiota result in increased susceptibility to C. rodentium; higher levels of C. rodentium in turn lead to increased colonic inflammation. Thus, the ability of stressors to directly modulate the composition of the enteric microbiota may represent a new mechanism governing susceptibility to enteric infection. This model may also be a useful tool for studying post-infectious irritable bowel syndrome, where alterations of the microbiota, prolonged inflammation, and psychological distress are all known to be predictive of this clinically important disease.
559 The Human Variant D299G of the TLR4 Gene Links Inflammation and Cancer Progression in the Intestinal Epithelium Annette Eyking, Guido Gerken, Daniel K. Podolsky, Elke Cario Background: Chronic recurrent intestinal inflammation in inflammatory bowel diseases (IBD) may result from aberrant stimulation of the mucosal immune system by the resident microflora. TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC) in IBD colitis. The variant D299G of the TLR4 gene has been associated with increased IBD susceptibility. However, the phenotypic consequences of this gene variant in IEC have not been resolved yet. The aim of the study was to investigate how the IBD-associated variant TLR4-D299G may affect IEC biology and molecular function. Methods: IEC lines (Caco2) stably overexpressing HA-tagged full-length (FL) TLR4 or mutant TLR4-D299G or TLR4T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed and network data were generated through Ingenuity Pathways Analysis. Results were confirmed by qRT-PCR and validated by western/ELISA. Mitosis was examined by confocal immunofluorescence. Functional studies included assessment of proliferation (BrdU), metabolic activity (MTT) and invasive potential (matrigel). InVivo tumorigenicity was tested using the CD-1 nu/nu mouse xenograft model. Results: Functional clustering of the microarray datasets revealed that the major TLR4-D299G targets were dominated by genes involved with promotion of inflammation and tumorigenesis, and these genes were markedly downregulated in IEC expressing TLR4-FL or TLR4-T399I. Three distinct pro-inflammatory systems (acute phase response, coagulation and complement) were the most significant canonical pathways modulated by the differentially regulated genes in TLR4-D299G. In contrast to TLR4-FL, TLR4-D299G IEC constitutively secreted large amounts of complement proteins (C3a; C5a) and coagulation factors (TFPI; A2M). Functionally, IEC expressing TLR4-D299G revealed central features of tumorigenicity - which were not evident in TLR4-FL: TLR4-D299G IEC 1) were multinucleated with multipolar mitotic spindles, 2) exhibited increased proliferation associated with elevated metabolic activity and repressed differentiation, and 3) demonstrated pronounced invasion via STAT3 activation. Compared with TLR4-FL-xenografts, In-Vivo tumor growth was significantly stimulated in TLR4-D299G-xenografts. Conclusions: Our results suggest that the TLR4-D299G variant represents a gain-of-function mutation with enhanced oncogenic potential. The TLR4-D299G variant establishes a pro-inflammatory microenvironment which may drive cancer progression in IEC. Thus, IBD patients with TLR4-D299G may be at increased risk to develop a more aggressive phenotype of colitis-associated neoplasia.
562 Microbiota-Induced Serum Amyloid A is an Important Mediator of Innate Immune Response in Zebrafish Ja Seol Koo, Michelle Kanther, John F. Rawls, Christian Jobin Background: The serum amyloid A family of acute phase protein (SAA1-4) have been traditionally viewed as markers of inflammation and their role in modulating innate host response is currently unknown. Since the zebrafish encodes only a single SAA homolog, this vertebrate animal represents an ideal system to investigate SAA function in host-microbe interaction. Methods: Germ-free (GF) transgenic NFKB:EGFP reporter zebrafish (Tg(NFKB:EGFP)) were conventionalized (CONVD) and pattern of EGFP expression was determined by fluorescent microscopy. SAA expression in GF and CONVD zebrafish was determined by wholemount in situ hybridization (WISH). Conventionally-raised Tg(NFKB:EGFP) zebrafish were injected with SAA morpholino (MO) at the 1-cell stage (0.9 pg/embryo) to block SAA expression. Impact of SAA on NF-κB signaling and gene expression In Vitro was determined using zebrafish PAC2 embryonic fibroblasts. NF-κB signaling was determined using immunofluorescent staining (RelA), western blot (phospho-IκBa), and transcriptional activity (luciferase reporter gene, NFKB:EGFP reporter plasmid). Transcription of NF-κB target genes In Vitro and In Vivo was determined using ABI Prism 7700HT detection system. Results: Recombinant human SAA (1μM) stimulated IκBα serine 32/36 phosphorylation, RelA nuclear translocation, and luciferase activity (relative fold change 3.5±0.07 vs ctl, p<0.01) in PAC2 cells. SAA induced EGFP expression in PAC2 cells transfected with pNFKB:EGFP. In addition, SAA induced NF-κB target genes IκBαa and MMP9 (relative fold change, 25.8±1.70 and 19.9±0.69 vs ctl respectively, p<0.01). Colonization of GF Tg(NFKB:EGFP) zebrafish with a normal microbiota resulted in elevated EGFP expression in the liver, dorsal root ganglia, the neuromasts of the lateral line, and a distinct population of intestinal cells. WISH and quantitative RT-PCR showed that CONVD Tg(NFKB:EGFP) zebrafish strongly up-regulated SAA gene expression in the liver and intestine compared to GF zebrafish. Importantly, although global cellular EGFP expression was not significantly inhibited in SAA-MO injected Tg(NFKB:EGFP) zebrafish, a strong decrease in endogenous mRNA accumulation of NF-κB targets IκBαa (40%) and MMP9 (90%) was observed in these fish compared to control MO injected zebrafish. Conclusion: Our data indicate that microbial colonization enhances SAA expression, which then impacts on NF-κB
560 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Target Peyer's Patches via Long Polar Fimbriae Benoit Chassaing, Nathalie Rolhion, Amelie De Vallee, Sa'ad Salim, Maelle Prorok-Hamon, Christel Neut, Barry J. Campbell, Jean-Pierre Hugot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud Background & Aims: Ileal lesions of patients with Crohn's disease (CD) are colonized by adherent-invasive Escherichia coli (AIEC) and the earliest observable lesions of CD are microscopic erosions of the follicle-associated epithelium (FAE) lining the Peyer's patches(PP). We aimed in the present study to investigate the ability of AIEC to interact with PP and M cells of the FAE, to quantify the PP-associated E. coli in CD patients compared to controls, and to search for the presence of a virulence factor in AIEC that could be involved in the targeting of PP by these invasive bacteria. Methods: PP from CD patients and controls were analyzed by q-PCR for the presence of E. coli 16S r-RNA encoding gene, and the genome
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563 Colonization-Dependent Colonic Fucosylation is Mediated by Gram Negative Bacteria via Activation of a Novel Fucosylated TLR4 Di Meng, Weishu Zhu, W. Allan Walker, N. Nanda Nanthakumar Adult colonic mucosa is highly fucosylated relative to immature mucosa, and this fucosylation is dependent upon bacterial colonization. Toll-like receptor-4 (TLR4) was hypothesized to mediate this colonization-dependent fucosylation. Germ-free (GF) and bacteria-depleted (BD) mice were compared with conventional and mutant mice with regard to induction of epithelial fut2 mRNA levels and α1,2-fucosyltransferase (FUT2) activity. Induction of these activities by recolonization were compared with induction by LPS (TLR4 ligand) and monocolonization by bacteriodes and E. coli strains. Cultured human erythroleukemia (Hel) cells were used to study fucosylated TLR4 In Vitro. Colonization-dependent induction of fucosylation occurs only in TLR2-/- and wild-type mice but not in TLR4-/- and MyD88-/- mice, suggesting TLR4-MyD88 dependent signaling is important for fucosylation. TLR4 is fucosylated in the colonocytes of only GF and BD mice. LPS activate fut2 mRNA and FUT2 activity in the absence of bacterial colonization. Fucose-utilizing Bacteroides fragilis induce colonic fucosylation, but not a fucose-non-utilizing mutant. In Hel cells, which express fucosylated TLR4 activates fut2 mRNA and fucosylation. TLR4-specific siRNA inhibits this activation, and fucosidase treatment abolished TLR4-dependent activation of fut2 mRNA, confirming the central role of fucosylated-TLR4 in activating fut2 and cell surface fucosylation. Fucosylated-TLR4 expressed on uncolonized colonocytes mediates bacterially-induced activation of fut2 mRNA and FUT2 activity without activating inflammation, and is a necessary initial step in the communication between the colonizing bacteria and the host epithelium.
566 Eicosapentaenoic Acid (EPA) Free Fatty Acid Reduces Polyp Burden in Familial Adenomatous Polyposis (FAP): Results of a Randomised, PlaceboControlled Trial Nicholas J. West, Susan K. Clark, Andrea Belluzzi, John M. Hutchinson, Mark Hull, Roger J. Leicester, Robin K. Phillips
564 Genes Encoding E. coli Small Heat Shock Proteins IbpA/B Have a Protective Role in Experimental Colitis in E. coli Monoassociated Il10-Deficient Mice Laura G. Patwa, R. Balfour Sartor, Jonathan J. Hansen
INTRODUCTION: EPA, as the free fatty acid (EPA-FFA), is an efficient means of increasing tissue levels. EPA-FFA dramatically reduces adenoma formation in the ApcMin/+ mouse and decreases cell proliferation in colon mucosal crypts in subjects with a history of sporadic colorectal polyps, effects consistent with a chemopreventative action. We report here the effects of EPA-FFA on polyp burden in subjects with FAP in a single-centre, randomised, placebo-controlled trial. METHODS: Subjects ≥18 years with FAP, having had a previous colectomy and ileo-rectal anastomosis, were randomised 1:1 to EPA-FFA (S.L.A. Pharma AG, Switzerland) as 2x500mg, enteric coated capsules, bd, or placebo capsules (PLA; capric/ caprylic acid triglycerides) for 6 months. A focal area of rectum (≥3 polyps ≥2mm) was tattooed at baseline endoscopy. Photographic records of this area at 0 and 6 months were used to determine numbers of polyps and their diameters. Video records of baseline and 6 month endoscopies were compared blind, in randomised order, independently, by each of a 5-member expert review panel and scored as Worse ( 1), Same as (0), or Better (+1). Fatty acid content of rectal biopsies was determined by GC-MS. RESULTS: Fifty eight subjects were randomised (EPA-FFA 29; PLA 29). Of these, efficacy data were available for 55 (EPAFFA 28; PLA 27). There was a statistically significant difference between treatment groups for change in polyp numbers (0-6 months) in the tattooed area (EPA-FFA mean [±SD] number of polyps 4.13 [2.47] at baseline decreased to 3.61 [2.31] at 6 months; PLA 4.50 [2.63] at baseline increased to 5.05 [3.30]; ANCOVA p=0.0046), a net difference of -22.4% [95%CI -39.6 to 5.1] (ANCOVA within-subject comparison p=0.0122). There were corresponding net differences in total polyp diameters (reduction of -29.8% [95%CI -56.1 to -3.6] for EPA-FFA vs PLA; p=0.027). These findings were corroborated by mean scores from the video review panel (EPA-FFA +0.09 [95%CI -0.14 to +0.32] vs PLA -0.34 [95%CI 0.56 to -0.11]; p=0.011). Rectal mucosal levels of EPA increased significantly (+159%) in the EPAFFA group compared to PLA (+78%; p=0.0179) as did docosapentaenoic acid (DPA) (+82.8% vs +1.3%; p=0.0103). EPA-FFA was well-tolerated. CONCLUSION: This formulation of EPA, as the free fatty acid, exhibited anti-neoplastic activity in subjects with FAP, evidenced by reductions in rectal polyp number and size and improvements in endoscopic appearance. These changes were paralleled by increased mucosal EPA levels. EPA-FFA has potential as a safe and effective chemopreventative agent.
Inflammatory bowel diseases (IBD) are thought to result from dysregulated immune responses to the intestinal microbiota. While host immune responses to microbial products have been extensively studied, relatively little is known about commensal microbial responses to intestinal inflammation. We have previously shown that bacterial stress response genes, ibpA and ibpB, are upregulated in luminal E. coli from colitic IL10-deficient (IL10-/-), but not healthy wild-type (wt), mice selectively colonized (monoassociated) with a non-pathogenic murine isolate of E. coli (NC101). The aim of this study is to investigate the influence of ibpA/B on experimental colitis. Methods: We monoassociated germ-free wt and IL10-/- mice with NC101 or an isogenic strain from which we deleted ibpA/B (ΔibpAB NC101). Intestinal inflammation was quantified using histological scoring and spontaneous secretion of IL12/ 23p40 from colonic explant cultures. Bacterial antigen-specific T-cell responses were assessed by measuring IFN-γ secretion in cultures of unseparated mesenteric lymph node (MLN) cells stimulated with NC101 lysate. Luminal concentrations of viable bacteria in the cecum were quantified by culturing serial dilutions on agar plates. In Vitro survival of bacteria in J774 murine macrophages was measured using gentamicin protection assays. Results: Histological inflammation scores, IL12/23p40 secretion in colon explant cultures, IFN-γ secretion in NC101 lysate-stimulated MLN cultures, and cecal bacterial concentrations were significantly greater in IL10-/- mice monoassociated with ΔibpAB NC101 compared to NC101 at both 3 and 5 wks. Similar results were obtained in wt mice monoassociated with ΔibpAB NC101 or NC101 for 9 wks, although the values were significantly lower compared to IL10-/- mice. Interestingly, survival of ΔibpAB NC101 within J774 cells was decreased compared to NC101. This difference was abrogated when cells were treated with a synthetic inhibitor of iNOS. Conclusions: Expression of E. coli ibpA/B attenuates experimental colitis and is associated with decreased luminal bacterial concentrations and decreased pro-inflammatory cytokine secretion, indicating that these bacterial stress response genes have a protective role in this model. These findings have implications in the development of treatment strategies for IBD that target host-microbial interactions.
567 Abusing Oncogenic RAS to Selectively Target Colorectal Cancer Cells Inna Naumov, Diana Kazanov, Shiran Shapira, Sarah Kraus, Nadir Arber Background: RAS mutations are present in 40-50% of human colorectal cancer (CRC) cases leading to the activation of downstream target genes. The PUMA (p53 up-regulated modulator of apoptosis) protein interacts with Bcl-XL and Bcl-2 to induce the release of mitochondrial cytochrome c and apoptosis thus can serve as a lethal gene. We have previously shown that recombinant adenovirus, carrying the PUMA gene (gift from Dr. B. Vogelstein) under the control of β-catenin- or Ras- responsive elements (TOP or Py2-SV40-PUMA, respectively) inhibit human cancer cell growth [1-3]. Aim: To selectively kill RAS transformed cells by overexpressing the pro-apoptotic PUMA gene under the control of RAS-responsive-elements (RRE). Materials and methods: SW480, HCT116 and DLD1 KRAS-mutated cells, HT29 cells harboring WT-KRAS were used, as well as normal enterocytes; IEC18/RIE and their KRAS/ HRAS transformed counterparts, R1/RIE-RAS, respectively. Two adenoviruses were constructed: PY4-PUMA, containing RRE as a promoter and a control virus, SV40 minimal promoter-PUMA, lacking RRE. Prior to infection, RAS activity was determined using GSTRBD pull-down assay. Inhibition of proliferation, cell viability, and protein expression were determined. In Vivo, HCT116 (5,000,000) cells were injected s.c. into athymic mice (n= 30). PY4-PUMA/ SV40-PUMA was injected i.p. twice, for a period of 4 weeks. All experiments were repeated 3 times. Results: KRAS mutated cells showed increased RAS activity compared to cells with WT-KRAS-PY4-PUMA. A marked inhibition in cell growth (up to 50%) and spontaneous apoptosis (~50%) was seen in cells exhibiting high RAS activity following
mean±SEM, p<0.05 for all (NC101 vs. ΔibpAB NC101) 565 Ind-Enabling Studies for CEQ508 Targeting β-Catenin of GI Polyps: First Oral RNAI Drug Natalya Bodyak, Alex Borrelli, Johannes Fruehauf, Jens Harborth, Moreshwar B. Vaze, Catherine Grillot-Courvalin, Alison Silva Targeted delivery remains one of the biggest challenges in the development of RNAi-based therapeutics. Cequent has developed a proprietary delivery technique for RNA interference, transkingdom RNAi (tkRNAi), in which non-pathogenic bacteria are engineered to invade specific target cells, produce and release short hairpin RNA (shRNA). We have previously shown tkRNAi to be successful in cell culture assays, in a mouse model for human colon
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cancer (APCmin), and in non-human primates (cynomolgus monkeys), by suppressing the oncogene, β-catenin. β-catenin is the key oncogene implicated in Colorectal Cancer (CRC) and Familial Adenomatous Polyposis (FAP); the latter being an orphan hereditary disease resulting in the formation of hundreds of polyps in the gastrointestinal tract and ultimately leads to the development of colon cancer without surgical intervention. No pharmaceutical treatment is available for patients with FAP. The non-human primate studies designed to evaluate on-target toxicities were conducted with a precursor (CEQ501) to our clinical candidate, CEQ508. Treatment significantly reduced mRNA levels of β-catenin in the mucosa of the gastrointestinal tract, an effect that was reversible after termination of dosing. The target gene was found reduced by 58-64% in treated compared to control animals. Following optimization, which most importantly included platform engineering to improve hairpin production and processing, we conducted a large GLP mouse bridging toxicity study comparing off-target effects of CEQ508 and CEQ501. No CEQ508 or CEQ501-related adverse responses were identified in the following study parameters analyzed: clinical observations, body weights, serum chemistry, hematology, cytokines, gross or histopathology. As no test article-related changes were identified under the conditions of this study at the highest dose evaluated, the No Observed Effect Level (NOEL) for daily oral administration of either CEQ508 or CEQ501 was 5x109 cfu/day, or 2x1011 cfu/kg/day. (CEQ501 in non-human primates showed a similar safety profile with the No Observed Adverse Effect Level (NOAEL) determined to be 1x1012 cfu/day, or 2x1011 cfu/kg/day). Together with multiple internally conducted pharmacology and pharmacokinetic experiments, Cequent used this data in the nonclinical section of an IND filed in late 2009. In addition to presenting detailed In Vivo data from our filing, an update on further development will be presented on CEQ508 for the proposed daily dosing of FAP patients with this oral tkRNAi therapeutic targeting β-catenin.
activity and down-stream gene expression in zebrafish. Therefore, we suggest that microbiotainduced SAA expression contributes to shape innate immune response in zebrafish and could represent an important component of host homeostasis.