Cecal Tissue Research Articles

Steroid Therapy Exacerbates Clostridium difficile Infection in a Mouse Model

Purpose: The importance of IClostridium difficile infection (CDI) amongst patients with inflammatory bowel disease (IBD) is increasingly recognized. CDI is now the leading recognized cause of infectious colitis in IBD patients. Recent studies indicate increased morbidity, mortality, and health costs in IBD patients with CDI, and guidelines now promote testing for C. difficile in IBD patients experiencing a relapse. Similar to colitis in IBD, CDI is associated with acute inflammatory injury to the colon, leading us to hypothesize that colitis in CDI might respond to anti-inflammatory agents used as adjuncts to antibiotic treatment. The objective of this study is to determine whether adjunct treatment with mesalamine (5-ASA) or corticosteroids (systemic hydrocortisone or enteric budesonide) changes outcomes in CDI-induced colitis, in comparison to fidaxomicin treatment alone. Methods: We used a mouse model of antibiotic-associated CDI, as previously reported. Twenty-four hours after C. difficile challenge (0.5x10E5 cfu), mice received oral fidaxomcin (20 mg/kg) either alone or in combination with 5-ASA (200 or 400 mg/kg) or budesonide (0.2, 1, or 10 mg/kg) for 5 days. For systemic steroid experiments, mice were given a single dose of hydrocortisone (50mg/kg) 48 hours prior to C. difficile challenge. Mice survival and weight were recorded. Colonic and cecal tissues were harvested for histologic scoring of colitis severity. Results: Fidaxomicin completely protected against CDI for all parameters (weight, survival, and histopathology). Adjunctive administration of 5-ASA (200 or 400 mg/kg) did not significantly alter any of the study parameters. Budesonide, given with fidaxomicin, worsened disease outcomes in terms of weight loss, colitis severity, and survival. Similarly, hydrocortisone administration prior to C. difficile challenge significantly increased CDI-related mortality. Conclusion: Fidaxomicin prevented the effects of CDI in mice. No additional benefit was evident using 5-ASA as an adjuvant, but this negative finding is limited by the fact that fidaxomicin alone was completely protective. Conversely, corticosteroid therapy, either enteric budesonide or systemic hydrocortisone, exacerbated CDI and lead to more severe disease outcomes. These studies may have important clinical implications in terms of limiting steroid therapy in patients with IBD, until acute CDI is brought under control using antibiotic therapy. Disclosure - Xinhua Chen: No conflict of interest Lei Lu: No conflict of interest Hua Xu: No conflict of interst Pam Sears - Employee, Optimer Pharmaceuticals, Inc. Ciaran P Kelly: No conflict of interst. This research was supported by an industry grant from This project was partially sponsored by Optimer Pharmaceuticals, Inc.

Campylobacter jejuni Colonization Is Associated with a Dysbiosis in the Cecal Microbiota of Mice in the Absence of Prominent Inflammation

Background Campylobacter jejuni causes enterocolitis in humans, but does not incite disease in asymptomatic carrier animals. To survive in the intestine, C. jejuni must successfully compete with the microbiota and overcome the host immune defense. Campylobacter jejuni colonization success varies considerably amongst individual mice, and we examined the degree to which the intestinal microbiota was affected in mice (i.e. a model carrier animal) colonized by C. jejuni at high relative to low densities.MethodsMice were inoculated with C. jejuni or buffer, and pathogen shedding and intestinal colonization were measured. Histopathologic scoring and quantification of mRNA expression for α-defensins, toll-like receptors, and cytokine genes were conducted. Mucosa-associated bacterial communities were characterized by two approaches: multiplexed barcoded pyrosequencing and terminal restriction fragment length polymorphism analysis.ResultsTwo C. jejuni treatments were established based on the degree of cecal and colonic colonization; C. jejuni Group A animals were colonized at high cell densities, and C. jejuni Group B animals were colonized at lower cell densities. Histological examination of cecal and colonic tissues indicated that C. jejuni did not incite visible pathologic changes. Although there was no significant difference among treatments in expression of mRNA for α-defensins, toll-like receptors, or cytokine genes, a trend for increased expression of toll-like receptors and cytokine genes was observed for C. jejuni Group A. The results of the two methods to characterize bacterial communities indicated that the composition of the cecal microbiota of C. jejuni Group A mice differed significantly from C. jejuni Group B and Control mice. This difference was due to a reduction in load, diversity and richness of bacteria associated with the cecal mucosa of C. jejuni Group A mice.ConclusionsHigh density colonization by C. jejuni is associated with a dysbiosis in the cecal microbiota independent of prominent inflammation.

Commensal Akkermansia muciniphila Exacerbates Gut Inflammation in Salmonella Typhimurium-Infected Gnotobiotic Mice

Excessive mucin degradation by intestinal bacteria may contribute to inflammatory bowel diseases because access of luminal antigens to the intestinal immune system is facilitated. This study investigated how the presence of a mucin degrading commensal bacterium affects the severity of an intestinal Salmonella enterica Typhimurium-induced gut inflammation. Using a gnotobiotic C3H mouse model with a background microbiota of eight bacterial species (SIHUMI) the impact of the mucin-degrading commensal bacterium Akkermansia muciniphila (SIHUMI-A) on inflammatory and infectious symptoms caused by S. Typhimurium was investigated. Presence of A. muciniphila in S. Typhimurium-infected SIHUMI mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, TNF-α, IL-12, IL-17 and IL-6 in cecal and colonic tissue. The increase in pro-inflammatory cytokines was accompanied by 10-fold higher S. Typhimurium cell numbers in mesenteric lymph nodes of SIHUMI mice associated with A. muciniphila and S. Typhimurium (SIHUMI-AS) compared to SIHUMI mice with S. Typhimurium only (SIHUMI-S). The number of mucin filled goblet cells was 2- to 3- fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI-S, SIHUMI-A or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ mRNA levels (r2 = −0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI mice containing both A. muciniphila and S. Typhimurium compared to other mouse groups. Concomitant presence of A. muciniphila and S. Typhimurium resulted in a drastic change in microbiota composition of SIHUMI mice: the proportion of B. thetaiotaomicron in SIHUMI-AS mice was 0.02% of total bacteria compared to 78% – 88% in the other mouse groups and the proportion of S. Typhimurium was 94% in SIHUMI-AS mice but only 2.2% in the SIHUMI-S mice. These results indicate that A. muciniphila exacerbates S. Typhimurium-induced intestinal inflammation by its ability to disturb host mucus homeostasis.

Atg16l1 is Required for Autophagy in Intestinal Epithelial Cells and Protection of Mice From Salmonella Infection

Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

Modulation of Chicken Intestinal Immune Gene Expression by Small Cationic Peptides as Feed Additives during the First Week Posthatch

We have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium, Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activities in vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization by Salmonella enterica serovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and without S. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged with S. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P ≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection with S. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response to S. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection against Salmonella infections.

The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model

BackgroundOral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention.MethodsRecombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay.ResultsAfter the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups.ConclusionRecombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

Comparison of the pathogenicity among Cronobacter species in a neonatal mouse model

Neonatal infection caused by Cronobacter species can result in serious illnesses such as bacteremia, septicemia, meningitis, and death in at-risk infants who are orally fed contaminated reconstituted powdered infant formulas. The objective of this study was to compare the virulence among three Cronobacter species strains by using an animal model for human neonatal Cronobacter species infections. We acquired timed-pregnant ICR mice and all owed them to give birth naturally. On postnatal day 3, each pup was administered orally a total dose of <TEX>$1{\times}10^7$</TEX> CFU Cronobacter species strain 3439, CDC 1123-79, and 3231. Mice were observed twice daily for morbidity and mortality. At postnatal day 10, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of Cronobacter species. Cronobacter species were isolated from cecum and other tissues in inoculated mice. In the tissues of Cronobacter species infected mice, meningitis and gliosis were detected in the brain. In this study, we identified the virulence among Cronobacter species strains by using a neonatal mice model which was a very effective animal model for human neonatal Cronobacter species infections.

Localization and (semi-)quantification of fluorescent beads of 2 sizes in chickens over time after simultaneous intratracheal and cloacal administration

Environmental particles enter the chicken via several routes. Entry via the respiratory and cloacal routes likely activates immune responses. We studied the localization of simultaneous intratracheally and cloacally applied beads of 2 sizes in the chicken body in time, and when possible, semiquantified the amount of beads. Ten broiler hens, 3.5 wk of age, received 1.25 × 109 1.0-µm beads and 1.05 × 107 10-µm fluorescein isothiocyanate (green) labeled cloacally, and simultaneously the same number and same sizes of tetramethylrhodamine isothiocyanate (red) labeled beads intratracheally. The bursa of Fabricius, lung, liver, kidney, gallbladder, spleen, thymus, small intestine (upper ileum), cecum, intestinal luminal contents, aerated bones, feces, and blood, from 2 chickens per moment were sampled at 1 h, 6 h, 24 h, 48 h, and 1 wk after challenge and studied for the presence of beads using fluorescence microscopy. The highest amount of beads was found in organs closest to the application site after 1 h (i.e., the lungs for red beads, and the bursa for green beads). All tissue samples showed all 4 types of beads at all time moments, most of them within 1 h. Lower levels of beads were found in lungs and bursa after 6 h and in all other organs after 24 h, except for the kidneys where levels declined after 48 h. Surprisingly, beads were found in thymus tissue and only relatively few beads were found in the spleen. At 1 h, 1-µm intratracheally applied red beads were also found in the cecal luminal content and cecal tissue, but not in the small intestinal luminal content, suggesting that ceca are capable of excreting small particles entering the body via the respiratory route. The presence of nondegradable and nonimmunogenic beads of different sizes in all sampled organs throughout the whole chicken body for 7 d suggested potentially negative chronic health and welfare risks for the chicken of environmental particles.

Sa1795 Changes in the Cytokine Milieu Over Time in the Peptidoglycan-Polysaccharide Lewis Rat Model: Possible Insights Into the Loss of Efficacy of Anti-TNFα Agents in Late Crohn's Disease

The peptidoglycan-polysaccharide (PGPS) model shares many etiologic and pathologic features with Crohn's disease (CD). Anti-TNFα therapy reduces both inflammation and fibrosis in the PGPS model if begun early but, as in CD, has diminished efficacy when instituted late in the course of the model. To begin to tease out the mechanism of diminished antiTNFα efficacy in advanced disease, we examined changes in mRNA levels of a broad panel of proinflammatory and profibrotic factors over time in the PGPS model. Methods: PGPS or human serum albumin (HSA, control) was injected into the bowel wall of Lewis rats at laparotomy. Rats (1 HSA, 3 PGPS) were euthanized at each time point (1, 4, 7, 11, 14, 21, and 28 d post injection). Non-operated rats (3) served as d0 controls. In separate experiments, rats received Ab501 (CA156_00501 Fab'-PEG; a pegylated murinized rabbit anti-mouse TNF) beginning on d1 or d13. Rats receiving anti-TNF were studied on d12 or d28. Gross abdominal score (sum of adhesions, mesenteric thickening, cecal wall thickening, cecal nodules, and liver nodules on 0-4 scales) was determined. A pathologist scored cecal sections. Quantitative real-time PCR was performed on cecal tissue. Results: IL1 β, IL6, TNFα, and TGFβ1 mRNAs showed a biphasic response, peaking at d1 and again at d11 post PGPS injection (previously shown). The second peak at d11 was accompanied by maximal mRNA levels of Th1 (IFNγ, IL2) and Th17 (IL17A) cytokines, IL23, granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein-1, arginase-1, tissue transglutaminase 2, and tissue inhibitor of metalloproteinase 1. Osteopontin mRNA was maximally elevated at d1 (.500-fold), then progressively declined to ~40-fold control at d21-28. IL13 mRNA was not elevated. Anti-TNF begun on d1 decreased expression of IL1 β (5.5±1.8 v 12.8±1.2, p=0.014) and IL6 (11.2±6.6 v 51.5±7.6, p=0.008) when assessed on day 12. Anti-TNF begun on d13 did not change expression of TNFα, IL1β, IL6 mRNAs; procollagens I and III trended higher on d21 (col I: 23.1±6.2 v 11.6±1.9, p=0.07; col III: 14.7±2.1 v 9.14±1.7, p=0.06). Conclusions: The robust Th17 response and increased GM-CSF near the midpoint of the PGPS model time course may render the ongoing immune response no longer controllable by targeting TNFα alone. Blockade of TNFα activity initiated at times in the course of disease when TGF β1 mRNA expression is maximal could exaggerate the results of loss of TNFα inhibitory effects on TGFβ profibrotic activity. This could explain why anti-TNFα begun on d13 results in increased collagen mRNA at d21. Decreased tissue compliance, suggested by increased collagen deposition at d7, may play a role in triggering the d7-11 wave of increased profibrotic and proinflammatory factors. Cytokine milleu and tissue factors likely determine the effect of anti-TNF on tissue inflammation and fibrosis.

Effects of dehydrated Opuntia ficus indica (Nopal) consumption on adiposity and gut physiology in Sprague Dawley rats fed a high fat diet

We have previously shown that Opuntia ficus indica (Nopal), a plant rich in dietary fiber and polyphenols is able to ameliorate hepatic steatosis in obese Zucker rats. However, its effect in high fat diet‐induced obese models has not been evaluated. Six weeks old male Sprague‐Dawley rats (n = 42) were fed with a low fat diet (LF) or a high fat diet (HF) for 4 weeks, The groups were then divided; one half of each group was assigned to a LF + Nopal (LF‐N) or HF + Nopal (HF‐N) and fed for a further 6 weeks.ResultsNopal consumption significantly decreased adiposity and adipocyte hypertrophy and decreased serum leptin. Nopal consumption significantly increased the weight of cecal contents and cecal tissue regardless of the diet. Finally Nopal consumption decreased reactive oxygen species in the cecum and colon of HF rats. This study shows that Nopal consumption prevents HF diet‐derived adiposity. The significant association between adiposity, adipocyte size and cecum weight may imply that fermentation of the dietary fibers present in Nopal could be partly responsible for the changes observed in the adipose tissue. Supported by NIH DDK 41004 grant (HR)

Comparison of the effects of Mg–6Zn and titanium on intestinal tract in vivo

To evaluate the ability of Mg-6Zn to replace titanium nails in the reconstruction of the intestinal tract in general surgery, we compared the Mg-6Zn and titanium implants with respect to their effects on rat's intestinal tract by biochemical, radiological, pathological and immunohistochemical methods. The results indicated that Mg-6Zn implants started to degrade at the third week and disintegrate at the fourth week. No bubbles appeared, which may be associated with intestinal absorption of the Mg-6Zn implants. Pathological analyses (containing liver, kidney and cecum tissues) and biochemical measurements, including serum magnesium, creatinine, blood urea nitrogen, glutamic-pyruvic-transaminase and glutamic-oxaloacetic-transaminase proved that degradation of Mg-6Zn did not harm the important organs, which is an improvement over titanium implants. Immunohistochemical results showed that Mg-6Zn could enhance the expression of transforming growth factor-β1. Mg-6Zn reduced the expression of tumor necrosis factor at different stages. In general, our study demonstrates that the Mg-6Zn alloy had good biocompatibility in vivo and performed better than titanium at promoting healing and reducing inflammation. It may be a promising candidate for stapler pins in intestinal reconstruction.

Identification of a Novel Human Polyomavirus in Organs of the Gastrointestinal Tract

Polyomaviruses are small, non-enveloped viruses with a circular double-stranded DNA genome. Using a generic polyomavirus PCR targeting the VP1 major structural protein gene, a novel polyomavirus was initially identified in resected human liver tissue and provisionally named Human Polyomavirus 12 (HPyV12). Its 5033 bp genome is predicted to encode large and small T antigens and the 3 structural proteins VP1, VP2 and VP3. Phylogenetic analyses did not reveal a close relationship to any known human or animal polyomavirus. Investigation of organs, body fluids and excretions of diseased individuals and healthy subjects with both HPyV12-specific nested PCR and quantitative real-time PCR revealed additional virus-positive samples of resected liver, cecum and rectum tissues and a positive fecal sample. A capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV12. Seroprevalences of 23% and 17%, respectively, were determined in sera from healthy adults and adolescents and a pediatric group of children. These data indicate that the virus naturally infects humans and that primary infection may already occur in childhood.

Anti–Tumor Necrosis Factor α Prevents Bowel Fibrosis Assessed by Messenger RNA, Histology, and Magnetization Transfer MRI in Rats With Crohnʼs Disease

Treatment of Crohn's disease (CD) with anti-tumor necrosis factor α (TNFα) decreases intestinal inflammation, but the effect on fibrosis remains unclear. We hypothesized that treatment with rat-specific anti-TNFα will decrease the development of intestinal fibrosis in a rat model of CD. We further hypothesized that magnetization transfer magnetic resonance imaging (MT-MRI) will be sensitive in detecting these differences in collagen content. Rats were injected in the distal ileum and cecum with peptidoglycan-polysaccharide (PG-PS) or human serum albumin (control) at laparotomy and then received intraperitoneal injections of rat-specific anti-TNFα or vehicle daily for 21 days after laparotomy. Rats underwent MT-MRI abdominal imaging on day 19 or 20. MT ratio was calculated in the cecal wall. Cecal tissue histologic inflammation was scored. Cecal tissue procollagen, cytokine, and growth factor messenger RNAs were measured by quantitative real-time PCR. PG-PS-injected rats treated with anti-TNFα had less histologic inflammation, and cecal tissue expressed lower levels of proinflammatory cytokine messenger RNAs than vehicle-treated PG-PS-injected rats (IL-1β: 5.59 ± 1.53 versus 10.41 ± 1.78, P = 0.02; IL-6: 23.23 ± 9.33 versus 45.89 ± 11.79, P = 0.07). PG-PS-injected rats treated with anti-TNFα developed less intestinal fibrosis than vehicle-treated PG-PS-injected rats by tissue procollagen I (2.87 ± 0.66 versus 9.28 ± 1.11; P = 0.00002), procollagen III (2.25 ± 0.35 versus 7.28 ± 0.76; P = 0.0000009), and MT-MRI (MT ratio: 17.79 ± 1.61 versus 27.95 ± 1.75; P = 0.0001). Insulin-like growth factor I (2.52 ± 0.44 versus 5.14 ± 0.60; P = 0.0007) and transforming growth factor β1 (2.34 ± 0.29 versus 3.45 ± 0.29; P = 0.006) were also decreased in anti-TNFα-treated PG-PS-injected rats. Anti-TNFα prevents the development of bowel wall inflammation and fibrosis in the PG-PS rat model of CD. MT-MRI measurably demonstrates this decrease in intestinal fibrosis.

Effect of the Leptin Receptor Q223R Polymorphism on the Host Transcriptome following Infection with Entamoeba histolytica

Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with the ancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying the mutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genes required for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infected with E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues were analyzed for changes in gene expression. By 72 h postchallenge, all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. Thirty-seven genes were differentially expressed in response to infection at 72 h, including proinflammatory genes (CXCL2, S100A8/9, PLA2G7, ITBG2, and MMP9) and functions pertaining to the movement and activity of immune cells. A comparison at 12 h postchallenge of infected Q223 versus R223 mice identified a subset of differentially expressed genes, many of which were closely linked to leptin signaling. Further analyses indicated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed increased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor polymorphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide into this previously uncharacterized mechanism of mucosal immunity.

Post-hatching ontogeny of intestinal proton-coupled folate transporter and reduced folate carrier in broiler chickens

Folate transporters, including the reduced folate carrier and the proton-coupled folate transporter, encoded by Slc19a1 and Slc46a1 genes respectively, play important roles in the transport of folate across biological membranes given the hydrophilic nature of folates. Although a number of studies have demonstrated that these two transporters are regulated ontogenetically in mammals, little data are available on their developmental patterns of expression in poultry. The objective of this study was to investigate the expression patterns of Slc19a1 and Slc46a1 in jejunal and cecal tissue of broiler chickens during post-hatching development. Post-hatch male chicks (Ross × Ross) had free access to water and a soybean/wheat-based diet. Jejunal, cecal and blood samples were collected on day-of-hatch but before feeding (D0), and on D2, D7, D14, D21 and D35 post-hatch (n = 8 at each time point), respectively. Plasma folate concentrations were low on the day of hatch and increased with maturation; by contrast, plasma homocysteine, a marker of folate status, was highest (P < 0.05) in the day-of-hatch birds and decreased thereafter. Increasing age reduced mRNA abundance of Slc19a1 (P < 0.05) in the jejunum and cecum. Abundance of Slc46a1 mRNA (P < 0.05) gradually decreased in the cecum with increasing age and that of Slc46a1 in the jejunum initially decreased and then increased to level similar to that of day-of-hatch. The study provides some initial data on ontogenetic regulation of Slc19a1 and Slc46a1 in the jejunum and cecum of the chicken and lays the ground work for future nutritional studies. Moreover, the expression of Slc19a1 and Slc46a1 transcripts in the cecum provides evidence of the potential for cecally derived folate to contribute to the folate status of the host.

Muc2 Limits Pathogen Burdens and Epithelial Barrier Dysfunction During Salmonella Typhimurium Colitis

Commensal as well as pathogenic bacteria have been implicated as possible triggers of Inflammatory Bowel Disease (IBD). The ability of pathogens to cause episodes of infectious gastroenteritis could play a role in the initiation, and/or exacerbation of IBD. In fact, an increased risk of IBD has been reported in individuals who suffer an acute episode of Salmonella gastroenteritis. For Salmonella to cause gastroenteritis, it has to directly infect the epithelial cells lining the mammalian intestine. Despite many studies exploring Salmonella interactions with epithelial cells, it is unclear how this pathogen evades and survives the array of host defenses found in the mammalian intestine. In particular it is unclear how Salmonella interacts with as well as crosses the mucus layer that protects the underlying intestinal epithelial cells from such noxious agents. Salmonella enterica serovar Typhimurium is a model organism used to test the virulence factors involved in Salmonella pathogenesis. To study Salmonella’s ability to cause intestinal inflammation, most groups use a colitis model relying on streptomycin to displace intestinal commensal microbes, resulting in heavy pathogen colonization of cecal tissues and severe inflammation. Although intestinal mucus is the first line of defense in the mouse GI tract, its role in providing host defense against Salmonella is still unclear. The mucus barrier is made up of the highly glycosylated mucin Muc2, which is secreted by goblet cells. Muc2 glycosylation occurs within the goblet cell and likely has significant implications for the function and effectiveness of the mucus barrier. Glycosylation involves the actions of several enzymes, for example, Core 3- O derived glycans are synthesized by Core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT). Mice lacking these glycans still produce the Muc2 protein, but display a thinner mucus barrier, and show increased susceptibility to chemical induced colitis. We began our investigations by comparing Salmonella induced colitis and mucus dynamics in Muc2 deficient (-/-) mice, C3GnT-/- mice and wildtype C57BL/6 mice. We observed that mucus secretion increased in response to Salmonella infection in C3GnT-/- and C57BL/6, with Salmonella found within the mucus layer. In contrast, Muc2-/- mice showed dramatic susceptibility to Salmonella infection, carrying 100 fold heavier cecal pathogen burdens and developing significantly increased barrier disruption compared with C57BL/6 mice. As a result, Muc2 -/- mice displayed high rates of morbidity and mortality. We also tested the susceptibility of C3GnT -/- mice, finding they carry WT pathogen burdens but developed exaggerated barrier disruption like Muc2 -/- mice. These data suggest that the intestinal mucus layer plays a critical role in controlling Salmonella intestinal burdens, whereas core-3 glycosylation plays an important role in controlling intestinal epithelial barrier function.

An Oxygen Equilibrium at the Host-Microbial Interface Determined by Phosphorescent Nanoprobe Technology

Although the colonic lumen is known to be largely devoid of oxygen, the mechanism by which this anaerobic environment is maintained remains unclear due to the inability of available technology to quantify oxygen in the intestinal tract. Indeed, most microbes in the gut are obligate anaerobes. Accurate quantification of oxygen in the gut is essential to elucidate the host’s relationship with its densely populated intestinal microbial community and may be relevant to various disease states in humans, including inflammatory bowel disease (IBD). For instance, multiple studies have demonstrated significant alterations in the gut microbiota in IBD, notably phylum level increases in Proteobacteria and Actinobacteria, which contain predominantly oxygen tolerant organisms, and a decrease in oxygen-sensitive Firmicutes. Phosphorescent nanoprobe (Oxyphor G41), whose phosphorescence decay time is exquisitely sensitive to environmental oxygen, was administered to mice either via the drinking water to measure intestinal luminal oxygen or intravascularly via the tail vein to quantify tissue oxygen. A fiber-optic time domain phosphorometer2 was used to excite G4 and detect phosphorescence at λmax = 635 nm and λmax = 810 nm, respectively. A laparotomy was performed under isoflurane anesthesia and the decay pattern of G4 phosphorescence was measured in various organs. 100% oxygen was delivered via the anesthesia apparatus for several minutes to determine the effect of host oxygenation on luminal oxygen content. Intravenous injection of G4 led to signal detection in all tissues, but ingested G4 was only detected at intestinal segments containing fecal material. The partial pressure of oxygen (pO2) was remarkably lower in the cecal feces than in the adjacent tissue. Inspiration of pure O2 led to a rapid, dramatic increase in cecal tissue pO2 and a delayed, more gradual increase in luminal pO2 (Figure 1). Both effects were reversible once animals were returned to room air. This technical advance has revealed a novel complexity in hostmicrobial interactions - namely that oxygenation of host colonic tissue affects the level of oxygen in the feces. Thus, the anaerobic nature of the intestinal lumen is not static but rather a dynamic equilibrium. We hypothesize that oxygen released by colonic tissue is consumed by the gut microbiota, thus creating an anaerobic luminal environment. This concept is supported by our data in human subjects indicating enrichment of aerobic and facultative anaerobic bacteria adherent to the rectal mucosa compared to the feces where obligate anaerobic bacteria predominated (Figure 2). We believe that the colonic oxygen equilibrium may have great relevance to IBD where the dysbiotic microbiota composition may, in part, result from an alteration in the redox potential of the colonic environment due to hyperemia, bleeding, and/or the oxidative nature of the inflammatory response. Studies are underway to further characterize the relationship between oxygen levels in the gut and the “dysbiotic” microbiome in IBD.

Effect of grinding intensity and feed physical form on in vitro adhesion of Salmonella Typhimurium and mannose residues in intestinal mucus receptors for salmonellae

The hypothesis of this study was that feeding a fine, pelleted diet (FP) compared to a coarse meal diet (CM) results in a higher mannose content in the intestinal mucus of pigs and therefore an increased in vitro adhesion of Salmonella Typhimurium DT104 L to the mucus. The 2 diets were fed to a total of 24 weaned pigs for 6 wk after which mannose content in the mucus was evaluated histochemically using the α1-3-d-mannose-specific lectin Galanthus nivalis agglutinin. The crypt width was determined as an indirect measure for the amount of secreted mucus. Ileal and cecal tissue samples were incubated with approximately 7.77 × 10(7) cfu Salmonella Typhimurium and numbers of salmonellae adhering to the mucus and/or mucosa were determined by culture techniques. There was no effect of feed physical form on the in vitro adhesion of S. Typhimurium either in the ileum (7.1 ± 0.19 log(10) cfu/g tissue) or in the cecum (6.8 ± 0.26 log(10) cfu/g). The mannose content of the mucus also did not differ between the treatment groups. The crypts of the duodenum, jejunum, and cecum were wider (P < 0.05) after feeding the CM diet. This might be an indication for a higher mucus production in these pigs.

Detection of 793/B serotype of infectious bronchitis virus in tissue sample by indirect immunoperoxidase assay.

The indirect immunoperoxidase (IIP) assay was compared with the reverse transcription–polymerase chain reaction (RT-PCR) for detection of 793/B serotype of infectious bronchitis virus in tissues samples collected from experimentally infected chickens. This technique was optimized in specific pathogen-free (SPF)-embryonated chicken eggs and broiler chickens inoculated with the Iranian IR/773/2001 strain of 793/B serotype The trachea, lung, kidney, and cecal tonsil tissue samples from experimentally infected chicken embryos and chickens were collected in order to prepare tissue sections in IIP assay and to detect in RT-PCR. The sensitivity and specificity values of IIP assay were, respectively, 83 and 84 %, and the positive and negative prediction values were 71 and 91 % when compared with RT-PCR.

Immunization with outer membrane protein A from Salmonella enterica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens

Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. We evaluated the efficacy of outer membrane protein A (OmpA) as a novel vaccine candidate against Salmonella in poultry. Native OmpA purified from Salmonella enterica serovar Enteritidis was mixed with adjuvant and administered intramuscularly to 41-d-old chicks. The vaccinated birds showed no decrease in cecal excretion and tissue colonization compared with the unvaccinated birds after oral challenge with 10(9) cfu of the homologous strain at 28 d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgG. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efficacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal IgA.