Cecal Tissue Research Articles

The local immune response in a mouse model of acute Clostridium difficile infection (67.8)

Abstract The current report presents our initial findings on the immune response to C. difficile infection (CDI) in an acute mouse model. 5-8 week old male C57BL/6 mice were either left untreated or received a cocktail of 5 antibiotics in drinking water for 3 days followed by a single intra-peritoneal dose of clindamycin. The antibiotic-treated mice were then infected with 105 CFUs of the C. difficile strain VPI10463 and sacrificed 42 hours later. Flow-cytometric study of the spleens, mesenteric lymph nodes (MLN), colons and ceca of the infected mice showed a significant increase in the numbers of B cells, CD4 and CD8 T cells, FOXP3+ CD4 cells, NK cells and neutrophils in the colons and ceca, as well as cells of the monocyte/macrophage lineage and dendritic cells in the ceca of the infected mice. The spleens and MLN of the infected mice did not display such an increase. A substantial fraction of the recruited neutrophils in the colons and ceca of the infected mice displayed upregulated levels of CD11b, whereas a similar fraction of CD4 and CD8 cells in the colons and ceca of uninfected and infected mice showed an upregulated level of CD69. Multiplex qPCR analysis of colonic and cecal tissue from the infected mice showed a significant increase in the mRNA levels of numerous neutrophil-attracting chemokines and a number of cytokines produced in an innate immune response, but no indication of T cell polarization, thereby collectively underscoring the innate, local nature of the response.

Intestinal bacterial overgrowth includes potential pathogens in the carbohydrate overload models of equine acute laminitis

Carbohydrate overload models of equine acute laminitis are used to study the development of lameness. It is hypothesized that a diet-induced shift in cecal bacterial communities contributes to the development of the pro-inflammatory state that progresses to laminar failure. It is proposed that vasoactive amines, protease activators and endotoxin, all bacterial derived bioactive metabolites, play a role in disease development. Questions regarding the oral bioavailability of many of the bacterial derived bioactive metabolites remain. This study evaluates the possibility that a carbohydrate-induced overgrowth of potentially pathogenic cecal bacteria occurs and that bacterial translocation contributes toward the development of the pro-inflammatory state. Two groups of mixed-breed horses were used, those with laminitis induced by cornstarch (n=6) or oligofructan (n=6) and non-laminitic controls (n=8). Cecal fluid and tissue homogenates of extra-intestinal sites including the laminae were used to enumerate Gram-negative and -positive bacteria. Horses that developed Obel grade2 lameness, revealed a significant overgrowth of potentially pathogenic Gram-positive and Gram-negative intestinal bacteria within the cecal fluid. Although colonization of extra-intestinal sites with potentially pathogenic bacteria was not detected, results of this study indicate that cecal/colonic lymphadenopathy and eosinophilia develop in horses progressing to lameness. It is hypothesized that the pro-inflammatory state in carbohydrate overload models of equine acute laminitis is driven by an immune response to the rapid overgrowth of Gram-positive and Gram-negative cecal bacterial communities in the gut. Further equine research is indicated to study the immunological response, involving the lymphatic system that develops in the model.

Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Abstract Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+/+ mice and ran Real Time-PCR or Western Blot for different Toll-like Receptor Pathway components. Results: qRT-PCR showed that Smad3−/-− mice have a significantly lower quantity of Bacteroides sp; the introduction of Helicobacter hepaticus results in a significant increase in B. distasonis and a significant decrease in B. thetaiotaomicron in Smad3−/− mice. These data suggest that loss of SMAD3 and the addition of Helicobacter hepaticus alters the colonic microflora. Previous studies illustrated the importance of epithelial signaling in microbial induced inflammation. To examine if Smad3 is altering the Toll-like receptor pathway we examined RT-PCR of the cecum and found a significant increase in Tlr4, Irak4, Cd14, Myd88, Nfkb, Cox2, and Nos2 mRNA in the Smad3−/−− mice compared to Smad3+/+ mice. Western blots confirmed these data by showing that cecal tissue from Smad3−/− mice has increased IRAK4 and pNF-κB. Conclusion: Smad3−/− mice show heightened Toll-like receptor pathway activity which leads to a hyper-inflammatory response of the mucosa to endogenous microbes. Further, loss of SMAD3 results in dysbiosis of the gut microbiota. These findings suggest that SMAD3 plays a role in microbial-host homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 407. doi:1538-7445.AM2012-407

Lactocepin Secreted By Lactobacillus Exerts Anti-Inflammatory Effects By Selectively Degrading Proinflammatory Chemokines

The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L.casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, Tcell infiltration andinflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD.

Pseudomonas aeruginosa Virulence Expression Is Directly Activated by Morphine and Is Capable of Causing Lethal Gut-Derived Sepsis in Mice During Chronic Morphine Administration

This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a μ opioid receptor antagonist. Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

To establish Helicobacter hepaticus infected models in different mice strains and to analyze pathological characteristics

Objective To obtain stable animal models and observe Helicobacter hepaticas (Hh)colonization and pathological claracteristics,through infecting different mice strains with Hh.Methods SPF-class male BABL/c Cr,SCID/Cr and C57BL/6 Cr mice were inoculated 0.2 ml Hh standard strain ATCC51450 bacterial suspension (1 × 108CUF/ml),inoculated for 3 times with 48 hours intervals,the control group was fed with the same volume of PBS.Mice were executed at 4 weeks,8weeks and 16 weeks since last Hh inoculation,and mice esophagus,stomach,jejunum,ileum,cecum,colon,liver and pancreas tissue were taken for histopathology examination,Micro-aerobic bacteria isolation,culture and identification and Hh specific 16S rRNA gene amplification.Results The colonization rates of Hh in cecum after inoculated in BALB/c Cr mice and SCID/Cr mice at 4 weeks,8 weeks and 16 weeks were all 8/8,colonization rates in colon at 4 weeks,8 weeks and 16 weeks were 4/8,5/8,5/8 and 3/8,6/8,5/8 respectively,colonization rates in ileum and jejunum at 16 weeks were 1/8,colonization rates in liver at 8 weeks and 16 weeks were 2/8,3/8 and 2/8,2/8respectively.The colonization rates of Hh in cecum after inoculated in C57BL/6 Cr mice at 4 weeks,8weeks and 16 weeks were 1/8,2/8 and 2/8 respectively,colonization rates in colon at 8 weeks and 16weeks were 1/8,2/8 respectively.Compared with C57BL/6 Cr mice,the inflammatory changes in liver,cecum and colon were more significant in Hh infected BALB/c Cr and SCID/Cr mice (P＜0.01),and histological scores gradually increased as infection time extended (P＜0.05,P＜ 0.01 ).The histological scores were significantly higher in those with colon and liver Hh bacterial colonization than those without Hh bacterial colonization (P＜0.05).The histopathological score of cecal tissue was positively correlated with the density of Hh colonization.Conclusion Different mice strains are with different susceptibility to Hh,and better Hh infection model can be obtained in Hh inoculated BALB/c Cr and SCID/Cr mice. Key words: Liver: Helicobacter infections; Disease models; Mice, SCIO; Mice, inbred BALB/c

Hypolipidemic effect of Smallanthus sonchifolius (yacon) roots on diabetic rats: Biochemical approach

Fructooligosaccharides (FOS) are sugars found naturally at high concentrations in the storage roots of yacon. This study was designed to analyze the beneficial effects of subchronic oral consumption of yacon root flour as a diet supplement in Streptozotocin-induced diabetic Wistar rats. The experiments were carried out using yacon flour tablets containing the desired level of FOS (340 or 6800 mg FOS/kg body weight/day). Yacon flour is a natural product obtained by a simple process of dehydration of yacon roots without added preservatives or chemicals. The administration of FOS-rich yacon flour to diabetic rats for 90 days did not significantly alter the body weight of animals throughout the experimental period. Interestingly, a significant decrease in fasting plasma triacylglycerol and very low-density lipoprotein levels were observed. In addition, the treatment was able to protect the diabetic rats of the postprandial peak of plasma triacylglycerol. Yacon-supplemented rats showed an increased insulin-positive pancreatic cell mass distributed in small cell clusters within the exocrine parenchyma, but can only observe a slight increase in fasting plasma insulin levels. Glucagon like peptide-1 content in the cecum was significantly higher in diabetic rats treated with a diet supplemented with yacon flour compared with untreated diabetic animals, accompanied by an important cecal tissue enlargement. All these findings lead us to suggest that this incretin could be an effective mediator of the lipid lowering effects of FOS present in yacon flour. In conclusion, yacon root flour is a natural product rich in FOS that could be well positioned as a nutraceutical product since the present results demonstrate its beneficial effects on diabetes-associated hyperlipidemia.

Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids. Part I: Effects on growth performance, microbial populations, and immune status1

Pigs (n = 88) weaned at 19 ± 2 d of age were used in a 14-d study to evaluate the effects of water-delivered direct-fed microbials (DFM) or organic acids on growth, immune status, Salmonella infection and shedding, and intestinal microbial populations after intranasal inoculation of Salmonella Typhimurium (10(10) cfu/pig). Pigs were challenged with Salmonella 6 d after commencement of water treatments. Treatments were 1) control diet; 2) control diet + DFM (Enterococcus faecium, Bacillus subtilis, and Bacillus licheniformis) in drinking water at 10(9) cfu/L for each strain of bacteria; 3) control diet + an organic acid-based blend (predominantly propionic, acetic, and benzoic acid) in drinking water at 2.58 mL/L; and 4) control diet + 55 mg/kg of carbadox. Serum samples were taken on d 6, 8, 10, and 14 for determination of tumor necrosis factor α (TNFα) concentrations. Fecal samples were taken on d 0, 5, 7, and 11 for determination of Salmonella shedding and enumeration of coliforms. Pigs were euthanized on d 6, 8, 10, and 14. Intestinal and cecal tissue and digesta and mesenteric lymph nodes were sampled and analyzed for Salmonella. Duodenal, jejunal, and ileal mucosal scrapings were sampled for measurement of mucosal TNFα concentrations. Water delivery of DFM prevented a decline in ADG on d 2 to 6 postchallenge compared with the negative control (P < 0.05). Coliform counts tended to be greater (P = 0.09) in the cecum of the DFM treatment group on d 2 postinfection compared with the negative control and acid treatment groups. However, Salmonella prevalence in the feces, gastrointestinal tract, or lymph nodes was not affected by water delivery of acids or DFM. Serum and mucosal TNFα concentrations were not affected by treatment throughout the study with the exception of ileal concentrations on d 4 postchallenge, which were greater in the negative control group compared with all other treatments (P < 0.05). The in-feed antibiotic was the only treatment that reduced Salmonella prevalence and this was localized to the cecum on d 8 postinfection. In conclusion, the DFM and organic acid treatments used in this study offered little or no benefits to pigs infected with Salmonella and should not be considered under the constraints of this study as viable alternatives to in-feed antibiotics in a pathogen challenge situation.

Soy Protein Diet, but Not Lactobacillus rhamnosus GG, Decreases Mucin-1, Trefoil Factor-3, and Tumor Necrosis Factor-α in Colon of Dextran Sodium Sulfate-Treated C57BL/6 Mice ,

The incidence of inflammatory bowel diseases has increased during recent decades. Within the colon, the families of mucins (MUC) and trefoil factors (TFF) facilitate mucosal protection. Probiotic administration influences the intestinal MUC layer. Additionally, food components may affect gut microflora or have direct effects on the MUC barrier. Our objective was to determine whether diet and/or Lactobacillus rhamnosus GG (LGG) would mediate dextran sodium sulfate (DSS)-induced colitis by altering expression of the MUC and TFF genes. C57BL/6 mice were fed diets containing 20% (wt:wt) casein, soy, or whey proteins with or without LGG for 12 d. Seven days after starting LGG diets, the mice were given 2% DSS in drinking water for 4 d. Two additional casein groups with or without LGG were given tap water, for a total of 8 groups. One day after the DSS treatment, the mice were killed and the colon and cecum tissues and cecum contents were collected and analyzed by qRT-PCR. Whey protein significantly increased cecal LGG content compared with the other diets. In the casein diet groups, MUC1 and TFF-3 expression in colon was significantly induced by DSS independent of LGG. Compared with other DSS-treated groups, soy protein decreased MUC-1 and TFF-3 in the colon. Similarly, soy protein decreased the impact of DSS on inflammatory scores, TNFα gene expression, and colon shortening. There was no overall effect of LGG on these measurements. In conclusion, soy protein suppressed the DSS-induced inflammatory stimulation of MUC, TFF, and TNFα gene expression independently of LGG.

Magnetization transfer helps detect intestinal fibrosis in an animal model of Crohn disease.

To determine the utility of magnetization transfer (MT) in the identification and quantification of intestinal fibrosis in a rat model of Crohn disease. The university committee on the use and care of animals approved this study (UCUCA 08592). Lewis rats injected subserosally with peptidoglycan-polysaccharide (PG-PS) develop bowel inflammation 1 day after laparotomy (early phase) and fibrosis starting 14 days after laparotomy (late phase). The authors performed 2.0-T magnetic resonance (MR) imaging in 25 rats injected with PG-PS and 13 injected with human serum albumin (HSA) (control animals). Imaging was performed before laparotomy and on a weekly basis thereafter for up to 28 days. The MT ratio in the bowel wall was calculated. Resected cecal tissue was scored for inflammation and fibrosis. Tissue fibrosis was determined with colorimetric analysis of trichrome-stained specimens. Collagen content was measured with Western blot analysis. Statistical analyses were performed with the Student t test for continuous bivariate comparisons, the Pearson correlation for continuous variables, and the Spearman correlation for ordinal variables. All rats developed early inflammation, which subsided over time. Rats injected with PG-PS developed increased fibrosis in the late phase, whereas control rats did not. The mean MT ratio of rats injected with PG-PS with late-phase fibrosis was higher than that in rats with early phase inflammation (P = .017). In addition, the MT ratio of rats injected with PG-PS with late-phase fibrosis was higher than that of control animals that did not develop fibrosis in the late phase (P = .0001). The MT ratio of control animals remained unchanged over time as inflammation subsided. The MT ratio in rats injected with PG-PS showed correlation with tissue fibrosis (ρ = 0.63). The MT ratio showed correlation with tissue collagen (R = 0.74). The positive and negative predictive values of the MT ratio in the prediction of fibrosis were 92% (12 of 13 rats) and 83% (five of six rats), respectively. These results indicate that MT is sensitive to bowel wall fibrosis as occurs in Crohn strictures.

Cronobacter sakazakiiInfection Induced Fatal Clinical Sequels Including Meningitis in Neonatal ICR Mice

Cronobacter sakazakii (C. sakazakii), formerly Enterobacter sakazakii, is an emerging pathogen associated with the ingestion of contaminated reconstituted formula that causes serious illnesses such as bacteremia, septicemia, necrotizing enterocolitis, meningitis and death in low-birth-weight preterm neonatal infants. The objective of this study was to develop an animal model for human neonatal C. sakazakii infections. We acquired timed-pregnant ICR mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered orally a total dose of approximately 107 CFU C. sakazakii strain 3439. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from cecum and other tissues in inoculated mice. In the tissues of C. sakazakii infected mice, meningitis and gliosis were detected in brain. In this study, we confirmed the neonatal ICR mice may be used a very effective animal model for human neonatal C. sakazakii infections.

PPARγ Ligands Switched High Fat Diet-Induced Macrophage M2b Polarization toward M2a Thereby Improving Intestinal Candida Elimination

Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-αhigh, IL-10high, MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-αlow, IL-10low, MRhigh, Dectin-1high) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level.

Ingestion of Epilactose, a Non-digestible Disaccharide, Improves Postgastrectomy Osteopenia and Anemia in Rats through the Promotion of Intestinal Calcium and Iron Absorption

Gastrectomy often results in osteopenia and anemia because of calcium (Ca) and iron (Fe) malabsorption. Here, we investigated the effects of feeding epilactose, a non-digestible disaccharide, on gastrectomy-induced osteopenia, anemia, and Ca and Fe malabsorption in male Sprague-Dawley rats. Totally gastrectomized or sham-operated rats were fed the control or epilactose (50 g/kg) diets for 30 days. Gastrectomy severely decreased intestinal Ca and Fe absorption, femoral bone strength, Ca content, hemoglobin concentration, and hematocrit. These decreases were partly or totally restored by feeding epilactose. Feeding epilactose increased the cecal tissue weight and the soluble Ca concentration and short-chain fatty acid pools of the cecal contents. Collectively, the increases in cecal mucosal area and/or soluble Ca concentration of the cecal contents, resulting from short-chain fatty acid production by intestinal microbes, are thought to be responsible for the epilactose-mediated promotion of Ca and Fe absorption in the gastrectomized rats.

A melon pulp concentrate rich in superoxide dismutase reduces stress proteins along the gastrointestinal tract of pigs

Objective A melon ( Cucumis melo LC.) pulp concentrate (MPC) rich in superoxide dismutase (SOD) activity was tested for its ability to decrease stress protein expressions along the gastrointestinal tract in a swine model. Methods Pig sextuplets weaned at 21 d of age were selected from among six litters ( n = 36). After a 2-d fasting period, the pigs were fed at similar levels of intake of the control, MPC1, and MPC2 diets, which provided 0, 12.5, and 50 IU of added SOD per kilogram of food, respectively. One triplet of pigs per litter was slaughtered at 7 d and the second triplet at 14 d after weaning. SOD, catalase, and digestive enzymes were determined enzymatically and stress protein expressions by western blotting. Results Plasma SOD increased with MPC dose at day 14 ( P < 0.05). Mucosal weights in the proximal and mid small intestine were lower at day 14 ( P < 0.05), cecum tissue weight was greater ( P < 0.05), and sucrase-specific activity in mid and distal small intestine mucosa was lower ( P = 0.05) in the MPC2 group than in the control group. MPC supplementation essentially decreased ( P < 0.05 to P < 0.001) stress proteins in the stomach (all), the mid small intestine (heat-shock protein-27, neuronal nitric oxide synthase) and the colon (heat-shock protein-70, neuronal nitric oxide synthase). Conclusion A SOD-rich MPC provided at the dose of 50 IU/kg of food for up to 12 d was effective in lowering the level of stress proteins along the gastrointestinal tract of pigs after weaning.

Comparison of virulence of three strains of Cronobacter sakazakii in neonatal CD-1 mice.

Cronobacter sakazakii (Enterobacter sakazakii) is an emerging pathogen that has been isolated from powdered infant formula and associated with outbreaks of infection in infants in neonatal intensive care units. In a previous study, we observed that neonatal CD-1 mice are susceptible to C. sakazakii infection and that the pathogen invades brain, liver, and cecum tissues. The study objective was to compare the virulence of three strains of C. sakazakii in neonatal CD-1 mice. The strains tested were MNW2 (a food isolate), SK81 (a clinical isolate), and 3290 (a clinical isolate). Timed-pregnant CD-1 mice were allowed to give birth on gestation day 19 or 20. Neonatal mice were sexed and culled to 10 per litter, each having five males and five females. Neonates were orally gavaged with C. sakazakii strains MNW2, SK81, or 3290 at doses ranging from 10(2.8) to 10(10.5) CFU on postnatal day 3.5. Pups surviving to postnatal day 10.5 were euthanized, and brain, liver, and cecum tissues were excised. C. sakazakii was isolated from all three tissues in mice treated with C. sakazakii, regardless of strain. C. sakazakii strain 3290 was significantly more invasive in brains (42.1% of mice) than were strains MNW2 (6.7%) and SK81 (15.9%). Mortality was observed for all strains of C. sakazakii tested, with SK81 being significantly more lethal (5.6%) than MNW2 (1.2%) or 3290 (0.6%). Our findings suggest that invasiveness does not necessarily correlate with mortality among different strains of C. sakazakii, and the clinical isolates are more virulent than the food isolate.

M1718 Fructo-Oligosaccharide (FOS) Supplementation Increases Enteroendocrine L Cells Containing GLP-1 and SCFA Receptor GPR43 (Ffa2) in the Large Intestine

Background and aim: Glucagon-like peptide-1 (GLP-1) is known as an incretin and a satiety hormone released by enteroendocrine L-cells, and its plasma concentration is reported to increase by dietary non-digestible carbohydrates such as certain dietary fibers and oligosaccharides. However, the triggers and modulators of the GLP-1 release remain unclear. We have previously reported that the L-cells containing peptide YY (PYY) express short-chain fatty acid (SCFA) receptor GPR43 in human and rat colon. PYY is reported to be contained in the same granules together with GLP-1. In the present study, we therefore investigated 1) the coexpression of GLP-1 and GPR43 in human and rat lower intestine, and 2) the effects of a long-term feeding of fructo-oligosaccharide (FOS) supplemented diet on these expressions in rats. Methods: 1) The expression of GLP-1, 5-hydroxytriptamine (5-HT), and GPR43 in the enteroendocrine cells were quantified by immunohistochemistry in the colon and terminal ileum isolated from rat and surgical specimens of humans. 2) Control and FOS-supplemented rats were fed a non-digestible carbohydrate-free and 5% (w/w) FOScontaining diet (16 g/day), respectively. On the 28th day, the rats were killed. The tissue and content weights of cecum, and GLP-1-, 5-HT-, and GPR43-positive cells in the colon, cecum and terminal ileum were measured. Results: Immunohistochemical analysis confirmed that GPR43-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HTcontaining enterochromaffin (EC) cells, both in human and rat colon and terminal ileum. Furthermore, compared to control, 4week FOS (5%) supplementation in rats significantly increased cecal tissue weight (but not its content weight) and the densities (cell number/ mm2 mucosa) of both GLP-1and GPR43-containing L-cells in particular the proximal colon. On the other hand, 5-HT-expressing EC cells were not affected by the FOS supplementation. Conclusion: The results showed that FOS supplementation increased the expression of GLP1and GPR43-containing enteroendocrine L-cells in the large intestine, suggesting that the prebiotic effects of FOS, such as improvement of the colonic environment to protect against pathogen and inflammation, is associated with the expression of the SCFA receptor GPR43. Therefore, it is possible that the long-term FOS ingestion contributes to the prevention of obesity and diabetes through PYY/GLP-1 release mediated by SCFA-stimulation of GPR43 in the large intestine.

M1719 Umami Receptor Activation Increases Duodenal Bicarbonate Secretion via GLP-2 Release in Rats

Background and aim: Glucagon-like peptide-1 (GLP-1) is known as an incretin and a satiety hormone released by enteroendocrine L-cells, and its plasma concentration is reported to increase by dietary non-digestible carbohydrates such as certain dietary fibers and oligosaccharides. However, the triggers and modulators of the GLP-1 release remain unclear. We have previously reported that the L-cells containing peptide YY (PYY) express short-chain fatty acid (SCFA) receptor GPR43 in human and rat colon. PYY is reported to be contained in the same granules together with GLP-1. In the present study, we therefore investigated 1) the coexpression of GLP-1 and GPR43 in human and rat lower intestine, and 2) the effects of a long-term feeding of fructo-oligosaccharide (FOS) supplemented diet on these expressions in rats. Methods: 1) The expression of GLP-1, 5-hydroxytriptamine (5-HT), and GPR43 in the enteroendocrine cells were quantified by immunohistochemistry in the colon and terminal ileum isolated from rat and surgical specimens of humans. 2) Control and FOS-supplemented rats were fed a non-digestible carbohydrate-free and 5% (w/w) FOScontaining diet (16 g/day), respectively. On the 28th day, the rats were killed. The tissue and content weights of cecum, and GLP-1-, 5-HT-, and GPR43-positive cells in the colon, cecum and terminal ileum were measured. Results: Immunohistochemical analysis confirmed that GPR43-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HTcontaining enterochromaffin (EC) cells, both in human and rat colon and terminal ileum. Furthermore, compared to control, 4week FOS (5%) supplementation in rats significantly increased cecal tissue weight (but not its content weight) and the densities (cell number/ mm2 mucosa) of both GLP-1and GPR43-containing L-cells in particular the proximal colon. On the other hand, 5-HT-expressing EC cells were not affected by the FOS supplementation. Conclusion: The results showed that FOS supplementation increased the expression of GLP1and GPR43-containing enteroendocrine L-cells in the large intestine, suggesting that the prebiotic effects of FOS, such as improvement of the colonic environment to protect against pathogen and inflammation, is associated with the expression of the SCFA receptor GPR43. Therefore, it is possible that the long-term FOS ingestion contributes to the prevention of obesity and diabetes through PYY/GLP-1 release mediated by SCFA-stimulation of GPR43 in the large intestine.

DHA Exacerbates Experimentally Induced Colitis in SMAD3−/− Mice

Inflammatory bowel disease (IBD) increases the risk of developing colorectal cancer. It is hypothesized that dietary interventions can reduce inflammation and associated cancer risk. The long chain omega‐3 fatty acid, docosahexaenoic acid (DHA) has potent anti‐inflammatory properties. The objective of this study was to determine whether dietary DHA could reduce experimentally induced colitis and subsequent colon cancer risk. We utilized a mouse model of colitis and colon adenocarcinoma formation (SMAD3−/−). When SMAD3−/− mice are exposed to H. hepaticus, colitis is observed 4 wks post infection. Mice (10 per group) were fed AIN‐93G powdered diets supplemented with corn oil, safflower oil, or DHA‐rich fish oil (doses ranging from 0.75–6%) for 8 wks. Mice were then gavaged with H. hepaticus, continued on their diets and sacrificed 4 weeks post‐infection. Colon and cecal tissue were collected for histopathology and scored for inflammation and dysplasia. Spleen, peyers patch and mesenteric lymph nodes were collected for CD3+ cell populations. Contrary to expectations, DHA (2.25–6%) induced severe colitis and adenocarcinoma formation. Increased severity of colitis in DHA fed mice was associated with a CD8+ driven response to bacterial infection. These results suggest that DHA supplementation in immune‐associated diseases like IBD should be approached with caution.

The NF-κB p50 Subunit Is Protective during Intestinal Entamoeba histolytica Infection of 129 and C57BL/6 Mice

Entamoeba histolytica is the agent of amebic colitis. In this work we examined the intestinal NF-kappaB response to this parasite. Using an enzyme-linked immunosorbent assay (ELISA) and an electrophoretic mobility shift assay, we found that the NF-kappaB subunit p50 predominated in nuclear extracts of whole cecal tissue and of isolated crypts from mice inoculated with E. histolytica. p50 was protective, since C57BL/6 and 129 mice in which there was targeted deletion of this subunit were more susceptible to E. histolytica infection as measured by culture results, cecal parasite ELISA results, and/or histologic scores. The transepithelial electrical resistance of cecal explants from C57BL/6 and 129 p50 knockout mice decreased markedly in response to the parasite compared with the transepithelial electrical resistance of their wild-type counterparts, suggesting that a protective function of p50 was present in the epithelium itself. This work shows that NF-kappaB activity, particularly activity of the p50 subunit, is one factor that contributes to resistance of the gut to E. histolytica infection.

Cross-Linked Hyaluronic Acid Films to Reduce Intra-Abdominal Postsurgical Adhesions in an Experimental Model

Background/Aims: Intra-abdominal adhesions typically occur after surgically damaged tissues are situated in apposition, leading to fibrotic connections. The goal of this study was to demonstrate the in vivo efficacy of a cross-linked and insoluble hyaluronic acid (HA) film to reduce postsurgical adhesion in a rat model. Methods: To measure in vitro adhesion resistance, porcine monocytes were deposited on the surface of films and their attachment was monitored by scanning electron microscopy. A rat cecum abrasion and abdominal insult model was utilized to demonstrate in vivo efficacy. Briefly, an HA film was deployed as a barrier between the damaged cecal and abdominal tissue surfaces for 21 days; control animals did not receive treatment. At the study conclusion, the rats were sacrificed and the degree of adhesion was determined using a scale from 0 to 3, where 0 = no adhesion and 3 = severe fibrosis. Results: HA films resisted monocyte adhesion in vitro. The in vivo study results demonstrated a significantly lower mean adhesion score (0.625 ± 0.517) with HA film treatment compared to the controls (2.09 ± 1.22). Conclusion: Placement of HA films between injured tissues significantly decreases the severity of abdominal adhesions. Furthermore, the HA film’s resistance to monocyte adhesion could be contributory to lowering in vivo adhesion scores.