Ileal Samples Research Articles

Reduced lesions in chickens with Clostridium perfringens-induced necrotic enteritis by Lactobacillus fermentum 1.2029 ,

Previous studies have demonstrated that Lactobacillus has anti-inflammatory properties, but the protective functions of Lactobacillus and mechanisms of inhibition of necrotic enteritis (NE) in the intestines of chickens have not been fully clarified. In the present study, we selected a probiotic strain, Lactobacillus fermentum 1.2029, which has good adhesive ability and a high survival rate in low pH and bile salts. The objective of this study was to examine the anti-inflammatory properties of L. fermentum 1.2029 against NE in chickens. Two hundred forty 1-d-old male Arbor Acres broilers were blocked into 3 experimental groups as follows: (I) nonchallenge control group, (II) Clostridium perfringens challenge group, and (III) C. perfringens challenge + L. fermentum 1.2029 group. Lactobacillus fermentum 1.2029 (1.0 mL/d, 108 cfu/mL) was orally administered daily to group III during the course of the experiment, and all uninfected control chickens were inoculated accordingly with the same volume of PBS. Clostridium perfringens (0.5 mL on d 1 and 1.0 mL on d 14 to 21, 108 cfu/mL) was administered to chickens in group II. At 28 d, scoring of gross NE lesions was performed. Ileal segments of approximately 2 cm from 24 chickens in each experimental group were collected and fixed in 4% (wt/vol) neutral-buffered formalin solution for histological scoring. Ileal mucosa samples were also collected for mRNA analysis by real-time PCR. The results showed that L. fermentum 1.2029 reduced the severity of NE lesions in chickens. Histological scores revealed that L. fermentum 1.2029 also reduced the inflammation damage of NE in chickens. Changes in cytokines and Toll-like receptors (TLR) were determined, and L. fermentum 1.2029 was found to increase interleukin-10 levels and reduce interferon-γ and TLR2 levels in NE-infected chickens. The results showed that L. fermentum 1.2029 was able to regulate the intestinal mucosal immune response and ameliorate inflammation by changing expression levels of cytokines and TLR.

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.

Evaluation of two external markers for measurement of ileal and total tract digestibility of pigs fed human-type diets1

External markers for determination of nutrient digestibility have often been evaluated in conventional dry feeds but less often in conventional feeds such as human-type diets used in animal model studies. In the present study, 5 ileal-cannulated pigs were fed 5 types of soft bread-based diets supplemented with Cr(2)O(3) and AIA as digestibility markers for 1 wk in a Latin square design. Ileal contents were collected twice for 5 h and a fecal grab sample was obtained once per week. Ileal and total tract digestibility of OM and nonstarch polysaccharides (NSP) based on the 2 markers were compared by linear regression. Across dietary treatments and site of collection, high correlation existed between digestibility values obtained with Cr(2)O(3) and AIA, resulting in a R(2) > 0.77 (P < 0.001) and a linear relation close to unity. For ileal samples, the correlation was poor, particularly for NSP, which had R(2) = 0.09 (P = 0.14) whereas OM had an R(2) = 0.52 (P <0.001). On the other hand, fecal grab samples led to R(2) > 0.92 (P < 0.001) for both OM and NSP. However, AIA gave higher values than Cr(2)O(3), particularly in samples with lower digestibility. The discrepancy is presumably caused by analytical difficulties due to a high fecal ash contents or interference with other components in the human-type diets.

Microbial metabolites, but not other phenolics derived from grape seed phenolic extract, are transported through differentiated Caco-2 cell monolayers

Grape seed phenolic extract (GSE) is predicted to have health benefits, even though its bioavailability, including digestibility, permeability and ultimate metabolism, are still poorly understood. In vitro gastric and pancreatic digestion and in vitro ileal and faecal fermentation were combined with Caco-2 cell permeability studies for GSE samples. Qualitatively, there was no change in type/number of GSE compounds following gastric and pancreatic digestion and LC–MS analysis. However, the monomers were significantly (P<0.05) increased after gastric digestion, along with a significant (P<0.05) decrease in polymers. In addition, all forms of phenolic compounds decreased following pancreatic digestion. However, none of the original GSE phenolic compounds passed the Caco-2 cell monolayer, since all were recovered in the apical compartment. In contrast, the two intestinal microbiota metabolites with deprotonated molecular weights of [M–H]-165/121 and 193/175, that were found both in the ileal and faecal fermented samples, passed the Caco-2 cell monolayer.

Glycerine kinase gene expression, nutrient digestibility and gut microbiota composition in post-weaned pigs fed diets with increasing crude glycerine levels

The effects of substituting maize with crude glycerine (CG) as an energy source, on nutrient digestibility, liver glycerine kinase (GK) gene expression, and gut microbiota composition were investigated in 18 weaned pigs [30days old, 7.8kg initial body weight (BW)]. Pigs were fed one of three diets containing 0, 75 and 150gCG/kg, formulated on equal digestible energy (DE) basis, with similar crude protein (210gCP/kg dry matter; DM) and amino acid (12.9g lysine and 8.2gmethionine+cystine/kg DM) content. The GK relative mRNA expression in liver and microbiota composition in ileal and caecal samples were assessed at 72days of age. The digestibility of GE decreased (P<0.05) at 37days, but increased linearly (P<0.05) at 65days of age with CG increment, due to CG level by age (P<0.01) interactions. Caecal Clostridium spp. and Lactobacillus spp. were linearly reduced (P<0.05) by CG inclusion level. The GK relative mRNA expression in liver increased linearly (P<0.001) with increasing CG. In conclusion, post-weaned pigs after an adaptation to increasing levels of CG may efficiently absorb and activate prior to metabolism, up to 150gCG/kg diet as dietary energy source.

Potential mechanisms explaining why hydrolyzed casein‐based diets outclass single amino acid‐based diets in the prevention of autoimmune diabetes in diabetes‐prone BB rats

It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen®-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen®-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis.

Effect of chito-oligosaccharide on growth performance, intestinal barrier function, intestinal morphology and cecal microflora in weaned pigs1

A total of 180 weanling pigs (21 ± 3 d of age; 5.98 ± 0.04 kg) were used to investigate the effect of chito-oligosaccharide (COS) on growth performance, intestinal barrier function, intestinal morphology, and cecal microflora. Based on initial BW, gender and litter, the pigs were given 5 treatments during a 14-d feeding experiment, including a basal diet (control), 3 diets with COS supplementation (200, 400, or 600 mg/kg), and a diet with colistin sulfate (CSE) supplementation (20 mg/kg). Six randomly selected pigs from each treatment were used to collect serum, duodenal, jejunal, ileal, and cecal samples on d 7 and 14 postweaning. From d 1 to 7 postweaning, pigs fed COS or CSE had greater ADG and ADFI compared with the control pigs. From d 1 to 14, diets with either 400 or 600 mg/kg COS, or 20 mg/kg CSE increased (P < 0.05) ADG and G:F compared with the control diet. No significant differences were observed in ADG, ADFI, and G:F between the pigs fed COS and CSE. Pigs fed either 400 or 600 mg/kg COS, or 20 mg/kg CSE had less (P < 0.05) diamine oxidase (DAO) in the serum, but greater concentration of (P < 0.05) DAO in jejunal mucosa, than the control pigs on d 7 postweaning. Treatments did not affect villous height and crypt depth of the duodenum, jejunum, or ileum. Pigs fed COS at 400 mg/kg had greater (P < 0.05) concentration of Bifidobacteria and Lactobacilli in the cecum than pigs fed the control diet and CSE diet on d 7 postweaning. Supplementation of COS or CSE decreased (P < 0.05) the population of cecal Staphylococcus aureus compared with the control diet on d 7 postweaning. The number of cecal Bifidobacteria in pigs fed 600 mg/kg COS was greater (P < 0.05) than that of pigs fed the control diet or CSE diet on d 14 postweaning. No significant differences were observed in Escherichia coli counts in the cecum among treatments. The present results indicate that dietary supplementation of COS at 400 or 600 mg/kg promotes growth performance and improves gut barrier function, increases the population of Bifidobacteria and Lactobacilli, and decreases S. aureus in the cecum of weanling pigs.

Monomer and Linkage Type of Galacto-Oligosaccharides Affect Their Resistance to Ileal Digestion and Prebiotic Properties in Rats1–3

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) ≥2, 14.0% trisaccharides] and commercial GOS derived from lactose (GOS-La; DP ≥3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 ± 2.6% and 52.9 ± 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1→6) and (1→2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1→4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS.

Inflammatory Bowel Diseases Phenotype, C. difficile and NOD2 Genotype Are Associated with Shifts in Human Ileum Associated Microbial Composition

We tested the hypothesis that Crohn’s disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum–associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1–V3 and V3–V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides – Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides – E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.

Interleukin‐22 and interleukin‐22–producing NKp44+ natural killer cells in subclinical gut inflammation in ankylosing spondylitis

The intestinal inflammation observed in patients with ankylosing spondylitis (AS) is characterized by an overexpression of interleukin-23 (IL-23). IL-23 is known to regulate IL-22 production through lamina propria NKp44+ natural killer (NK) cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44+ NK cells and the expression of IL-22 in the ileum of AS patients. Tissue NKp44+ NK cells, NKp46+ NK cells, and IL-22-producing cells were analyzed by flow cytometry. Quantitative gene expression analysis of IL-22, IL-23, IL-17, STAT-3, and mucin 1 (MUC-1) was performed by reverse transcriptase-polymerase chain reaction on ileal samples from 15 patients with AS, 15 patients with Crohn's disease (CD), and 15 healthy controls. NKp44, pSTAT-3, and IL-22 expression was analyzed by immunohistochemistry. The frequency of NKp44+ but not NKp46+ NK cells was increased in the inflamed ileum of AS patients compared to CD patients and controls. The frequency of NKp46+ NK cells was significantly increased only in CD patients. Among CD4+ lymphocytes and NKp44+ NK cell subsets, the latter were the major source of IL-22 on lamina propria mononuclear cells from AS patients. Significant up-regulation of IL-22, IL-23p19, MUC-1, and STAT-3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and pSTAT-3 expression in inflamed mucosa from AS and CD patients. Our findings indicate that overexpression of IL-22, together with an increased number of IL-22-producing NKp44+ NK cells, occurs in the gut of AS patients, where it appears to play a tissue-protective role.

An Ileal Crohn's Disease Gene Signature Based on Whole Human Genome Expression Profiles of Disease Unaffected Ileal Mucosal Biopsies

Previous genome-wide expression studies have highlighted distinct gene expression patterns in inflammatory bowel disease (IBD) compared to control samples, but the interpretation of these studies has been limited by sample heterogeneity with respect to disease phenotype, disease activity, and anatomic sites. To further improve molecular classification of inflammatory bowel disease phenotypes we focused on a single anatomic site, the disease unaffected proximal ileal margin of resected ileum, and three phenotypes that were unlikely to overlap: ileal Crohn's disease (ileal CD), ulcerative colitis (UC), and control patients without IBD. Whole human genome (Agilent) expression profiling was conducted on two independent sets of disease-unaffected ileal samples collected from the proximal margin of resected ileum. Set 1 (47 ileal CD, 27 UC, and 25 Control non-IBD patients) was used as the training set and Set 2 was subsequently collected as an independent test set (10 ileal CD, 10 UC, and 10 control non-IBD patients). We compared the 17 gene signatures selected by four different feature-selection methods to distinguish ileal CD phenotype with non-CD phenotype. The four methods yielded different but overlapping solutions that were highly discriminating. All four of these methods selected FOLH1 as a common feature. This gene is an established biomarker for prostate cancer, but has not previously been associated with Crohn's disease. Immunohistochemical staining confirmed increased expression of FOLH1 in the ileal epithelium. These results provide evidence for convergent molecular abnormalities in the macroscopically disease unaffected proximal margin of resected ileum from ileal CD subjects.

Tu1850 Expression of Toll-Like Receptor (TLR)-2 and −4 in the Intestinal Crypt Epithelial Cells in Inflammatory Bowel Disease (IBD)

Introduction TLRs are pattern recognition receptors which detect conserved molecular patterns on commensal and pathogenic bacteria. Studies in mice have shown that TLR signalling protects against the development of colitis but may exacerbate established colitis. Epithelial stem cells and their progeny are located in crypts of the small and large intestine. We have investigated TLR2 and TLR4 expression in ulcerative colitis (UC) and Crohn9s disease (CD) crypt intestinal epithelial cells (IEC). Methods Ileal and colonic mucosal samples were obtained from operation resection specimens and were histologically normal (>5 cm from cancer; normal controls) or inflamed (CD or UC). Additionally, paired samples from histologically normal (proximal colon) and inflamed (distal colon) mucosa were obtained from five colectomy specimens with isolated left-sided UC. Crypt IECs were isolated using EDTA and pancreatin. Expression of mRNA transcripts was studied by conventional and real-time RT-PCR. Cell surface TLR protein was studied by flow cytometry (expressed as median fluorescent intensity, MFI). TLR expression was also studied by immunofluorescent (IF) staining of tissue sections. Data are expressed as median (IQR). Results Relative to normal controls, TLR2 mRNA expression in IEC was significantly elevated in inflamed colonic UC [3.18-fold (1.03–10.40), p=0.003] and inflamed colonic CD [3.45 (0.80–5.40), p=0.012]. TLR4 mRNA was significantly elevated in normal colonic UC [1.90 (1.69–4.31], p=0.017), inflamed colonic UC [2.33 (1.15–4.45), p=0.024], inflamed colonic CD [1.71 (1.00–4.32), p=0.042] and inflamed ileal CD [1.84 (1.43–4.66), p=0.03]. In paired analyses, IEC TLR2 and TLR4 mRNA expression was not significantly different in normal UC mucosa compared to inflamed UC mucosa. Compared to IEC from normal control colon, IEC from colonic CD showed significantly greater expression of cell surface TLR2 protein [MFI 10.1 (2.4–29.0) vs 33.1 (17.7–107.5), p=0.05] and TLR4 protein [MFI 12.1 (4.9–25.3) vs 40.9 (23.2–103.5), p=0.04]. IF staining confirmed TLR2 and TLR4 protein expression on the luminal surface of crypt IEC. Conclusion IEC expression of TLR4 mRNA was up-regulated in inflamed and un-inflamed colonic and ileal IBD. TLR2 mRNA was up-regulated in inflamed colonic IBD. Interestingly, TLR2 and TLR4 mRNA expression was similar in IECs isolated from un-inflamed, histologically normal mucosa as it was in IECs isolated from inflamed mucosa in UC. This may suggest TLR2 and TLR4 up-regulation is a primary rather than secondary event in UC. Moreover, TLR2 and TLR4 protein was expressed in greater amounts on the surface of IECs in CD. Competing interests None declared.

Inclusion of chicory (Cichorium intybus L.) in pigs' diets affects the intestinal microenvironment and the gut microbiota.

The content and composition of prebiotic plant fiber in the diet is important in promoting gut-related health. This study investigated the effects of the dietary inclusion of chicory forage and roots on the intestinal microenvironment of pigs. Thirty-seven-week-old pigs were fed 1 of 5 diets for 18 days, including a cereal-based control diet and 4 diets with the inclusion of 80 and 160 g kg(-1) of body weight chicory forage (CF80 and CF160), 80 g kg(-1) chicory root (CR80), and a mix of 80 g kg(-1) forage and 80 g kg(-1) chicory root (CFR). The animals maintained good performance and health irrespective of diet. Bacterial community structure and diversity in ileal and colonic samples was assessed using terminal restriction fragment length polymorphism (T-RFLP), combined with cloning and sequencing. Samples clustered perfectly according to gut segment with a higher bacterial diversity in colon than ileum. Distal ileum was dominated by lactic acid bacteria (LAB), and the relative amount of this group was increased by the CF160 and CFR diets. The colonic bacterial community was dominated by butyrate-producing bacteria and Prevotella. The increased relative abundance of butyrate-producing bacteria in the colon was positively correlated with the molar proportion of acetic acid and furthermore linked to the chicory forage diets (CF80 and CF160). Diets including chicory roots (CR80 and CFR) were correlated with a higher colonic abundance of Megasphaera elsdenii. The fermentation products and pH in digesta responded to diet type and were correlated with shifts in the microbiota, showing that chicory influences the intestinal microenvironment of pigs.

Interactive effects of age, sex, and strain on apparent ileal amino acid digestibility of soybean meal and an animal by-product blend in broilers

The objectives of this study were to determine if age, sex, and strain of broilers affect the apparent ileal amino acid digestibility (AID) of soybean meal and an animal by-product blend. Chicks from 2 broiler strains, 1 commercially available and the other in the test phase, were obtained from a common hatchery, feather-sexed, and placed into floor pens. The birds received common diets while on floor pens. Birds were transferred to metabolism crates for 2 ileal digestibility trials conducted at 3 and 6 wk of age. Two semipurified diets were formulated with each of the feed ingredients providing all of the amino acids (AA) in the diets. Diets were formulated to contain 20% CP, with chromic oxide added to the diet at 0.30% as the indigestible marker. The experimental diets were fed for a 72-h period after an overnight fast to 8 replicate cages of 5 birds per cage. Upon completion of the experimental periods (i.e., d 22 and 43), birds were euthanized and ileal digesta samples were collected and pooled by pen, frozen, lyophilized, and analyzed for AA and chromium concentrations. For soybean meal, no main effects were observed for age, sex, or strain; however, significant (P ≤ 0.05) 3-way interactions were observed for several dispensable and indispensable AA. Statistical analyses of these interactions indicate the AID of soybean meal may be different for 42-d males of the test strain. For the animal by-product blend, only main effects were significant for each AA, with the exception of Ile and Tyr. Main effect of strain was observed for most of the AA, showing that the commercially available strain had higher AID than the test strain. Also, significant main effect of age indicated that the AID for animal by-product blend was higher at 42 d than at 21 d. Evaluation of these 2 ingredients showed that age, sex, and strain may affect digestibility of feedstuffs and should be considered independently or interactively, for some cases, in future investigations as sources of variation in AA digestibility.

Application of Novel PCR-Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances

ABSTRACTGastrointestinal disturbances are commonly reported in children with autism and may be associated with compositional changes in intestinal bacteria. In a previous report, we surveyed intestinal microbiota in ileal and cecal biopsy samples from children with autism and gastrointestinal dysfunction (AUT-GI) and children with only gastrointestinal dysfunction (Control-GI). Our results demonstrated the presence of members of the family Alcaligenaceae in some AUT-GI children, while no Control-GI children had Alcaligenaceae sequences. Here we demonstrate that increased levels of Alcaligenaceae in intestinal biopsy samples from AUT-GI children result from the presence of high levels of members of the genus Sutterella. We also report the first Sutterella-specific PCR assays for detecting, quantitating, and genotyping Sutterella species in biological and environmental samples. Sutterella 16S rRNA gene sequences were found in 12 of 23 AUT-GI children but in none of 9 Control-GI children. Phylogenetic analysis revealed a predominance of either Sutterella wadsworthensis or Sutterella stercoricanis in 11 of the individual Sutterella-positive AUT-GI patients; in one AUT-GI patient, Sutterella sequences were obtained that could not be given a species-level classification based on the 16S rRNA gene sequences of known Sutterella isolates. Western immunoblots revealed plasma IgG or IgM antibody reactivity to Sutterella wadsworthensis antigens in 11 AUT-GI patients, 8 of whom were also PCR positive, indicating the presence of an immune response to Sutterella in some children.

Upregulation of Polymeric Immunoglobulin Receptor Expression by the Heat‐Inactivated Potential Probiotic Bifidobacterium bifidum OLB6378 in a Mouse Intestinal Explant Model

We determined whether a potential probiotic bacterium, Bifidobacterium bifidum OLB6378 (BB6378), exerts beneficial effects on the mucosal immune system in a mouse intestinal explant model. The addition of heat-inactivated BB6378 to intestinal explants prepared from embryonic day 18 BALB/c mice increased the expression of polymeric immunoglobulin receptor (pIgR) mRNA by two- to fivefold. These effects were observed on ileal and colonic explants but not on jejunal explants, suggesting that the BB6378-induced pIgR upregulation is site-specific within the mouse intestine. The upregulation of pIgR protein expression in colonic explants was also detected after 24 h of culture. The results of DNA microarray analysis of ileal and colonic samples indicated that BB6378 increased the gene expression of interleukin (IL)-1α and IL-1β, and IL-1α content in colonic explants was significantly increased after 20 h of culture with BB6378. We then examined the involvement of endogenously induced IL-1α in pIgR mRNA upregulation by using IL-1α knockout (KO) mice. Contrary to our expectations, pIgR mRNA expression was equally upregulated by BB6378 in colonic explants from BALB/c and IL-1α KO mice. Conversely, we examined the involvement of Toll-like receptors in pIgR mRNA upregulation by using MyD88 KO mice. The upregulation of pIgR was completely suppressed in the explants derived from MyD88 KO mice. Taken together, we conclude that in a mouse intestinal explant model, the heat-inactivated potential probiotic BB6378 increases intestinal pIgR expression in a site-specific manner and that the upregulation of pIgR could be explained by a direct microbial effect on the epithelium via Toll-like receptors.

Effects ofLactobacillus plantarumon gut barrier function in experimental obstructive jaundice

To investigate the mechanisms of Lactobacillus plantarum (L. plantarum) action on gut barrier in preoperative and postoperative experimental obstructive jaundice in rats. Forty rats were randomly divided into groups of sham-operation, bile duct ligation (BDL), BDL + L. plantarum, BDL + internal biliary drainage (IBD), and BDL + IBD + L. plantarum. Ten days after L. plantarum administration, blood and ileal samples were collected from the rats for morphological examination, and intestinal barrier function, liver function, intestinal oxidative stress and protein kinase C (PKC) activity measurement. The distribution and expression of the PKC and tight junction (TJ) proteins, such as occludin, zonula occludens-1, claudin-1, claudin-4, junction adhesion molecule-A and F-actin, were examined by confocal laser scanning microscopy, immunohistochemistry, Western blotting, real-time fluorescent quantitative polymerase chain reaction assay. L. plantarum administration substantially restored gut barrier, decreased enterocyte apoptosis, improved intestinal oxidative stress, promoted the activity and expression of protein kinase (BDL vs BDL + L. plantarum, 0.295 ± 0.007 vs 0.349 ± 0.003, P < 0.05; BDL + IBD vs BDL + IBD + L. plantarum, 0.407 ± 0.046 vs 0.465 ± 0.135, P < 0.05), and particularly enhanced the expression and phosphorylation of TJ proteins in the experimental obstructive jaundice (BDL vs BDL + L. plantarum, 0.266 ± 0.118 vs 0.326 ± 0.009, P < 0.05). The protective effect of L. plantarum was more prominent after internal biliary drainage ( BDL + IBD vs BDL + IBD + L. plantarum, 0.415 ± 0.105 vs 0.494 ± 0.145, P < 0.05). L. plantarum can decrease intestinal epithelial cell apoptosis, reduce oxidative stress, and prevent TJ disruption in biliary obstruction by activating the PKC pathway.

Protective Effects of a Phosphatidylcholine-Enriched Diet in Lipopolysaccharide-Induced Experimental Neuroinflammation in the Rat

Our goal was to characterize the neuroprotective properties of orally administered phosphatidylcholine (PC) in a rodent model of systemic inflammation. Sprague-Dawley rats were killed at 3 h, 1 day, 3 days, or 7 days after i.p. administration of lipopolysaccharide (LPS) to determine the plasma levels of tumor necrosis factor α (TNF-α) and interleukin 6 cytokines. The control group and one group of LPS-treated animals were nourished with standard laboratory chow, whereas another LPS-treated group received a special diet enriched with 1% PC for 5 days before the administration of LPS and thereafter during the 7-day observation period. Immunohistochemistry was performed to visualize the bromodeoxyuridine and doublecortin-positive neuroprogenitor cells and Iba1-positive microglia in the hippocampus, whereas the degree of mucosal damage was evaluated on ileal and colon biopsy samples after hematoxylin-eosin staining. The activities of proinflammatory myeloperoxidase and xanthine-oxidoreductase and the tissue nitrite/nitrate (NOx) level were additionally determined, and the cognitive functions were monitored via Morris water maze testing. The inflammatory challenge transiently increased the hippocampal NOx level and led to microglia accumulation and decreased neurogenesis. The intestinal damage, mucosal myeloperoxidase, xanthine-oxidoreductase, and NOx changes were less pronounced, and long-lasting behavioral alterations were not observed. Phosphatidylcholine pretreatment reduced the plasma TNF-α and hippocampal NOx changes and prevented the decreased neurogenesis. These data demonstrated the relative susceptibility of the brain to the consequences of transient peripheral inflammatory stimuli. Phosphatidylcholine supplementation did not reduce the overall extent of peripheral inflammatory activation, but efficiently counteracted the disturbed hippocampal neurogenesis by lowering circulating TNF-α concentrations.

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

BackgroundEndoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.Methodology/Principal FindingsExpression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.Conclusions/SignificanceActivation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.

Comparison of sample source (excreta or ileal digesta) and age of broiler chick on measurement of apparent metabolisable energy of local feed ingredients

The present study was designed to measure the metabolisable energy (ME) values of local feed ingredients, namely corn, rice tips, rice polish, soybean meal, canola meal and sunflower meal for commercial broilers at 18, 26 and 34 days of age using total fecal collection and external marker in ileal and fecal samples. For this purpose, 240 birds of same weight (at 11 days of age) were selected and randomly divided into 24 test groups having 10 chicks each. Six iso-caloric and iso-nitrogenous rations namely corn-soybean meal (basal diet), basal diet + rice tips, basal diet + rice polish, basal diet + sunflower meal, basal diet + canola meal and corn + canola meal were prepared and each diet was fed to four test groups. The excreta were collected over a period of 48 hours on day 18, 26 and 34 days. On 34th day of age, two birds from each test group (eight birds per treatment) were randomly selected and slaughtered for collecting ileal digesta samples. Celite was used as external marker at the rate of 1% of the diet. Feed, fecal and ileal samples were analyzed for gross energy and nitrogen contents. Apparent metabolisable energy corrected to a state of nitrogen equilibrium (AMEn) of the feed was measured by difference of gross energy of feed and fecal or ileal samples. The results showed that AMEn values of corn, rice tips, rice polish, soybean meal, canola meal and sunflower meal were 3.27–3.31, 2.48–2.78, 2.62–3.0, 2.38–2.41, 1.79–1.97 and 2.18–2.44 kcal/g, respectively. The AMEn values of all ingredients did not increase with increase in age and showed no specific trend. The ME values determined using marker or total collection or ileal methods were not different. However, the ME values tended to be lower using marker than total collection method in most of the situations.