Ileal Samples Research Articles

Evaluation of optimal biopsy location for assessment of histological activity, transcriptomic and immunohistochemical analyses in patients with active Crohn's disease.

The appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown. To explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells. Fifty-one patients with Crohn's disease and ulcers >0.5cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0mm, 7 to 8mm and 21 to 24mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins-6, -8 and -23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase-positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient. Histological disease activity scores (P<0.0001), proinflammatory gene expression levels (P<0.005) and numbers of myeloperoxidase-positive cells (P<0.0001) were highest in biopsies from the ulcer edge in the colon and ileum, with decreasing gradients observed with distance from the edge (P<0.05). No differences between colonic and ileal samples were detected for the parameters measured at any location. Biopsies from the ulcer edge in patients with Crohn's disease yielded the greatest histological disease activity and mRNA levels and had similar readouts in the colon and ileum. Research is needed to confirm this conclusion for other measures.

Derivation of adult canine intestinal organoids for translational research in gastroenterology

BackgroundLarge animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures.ResultsOrganoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research.ConclusionsWe establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans

Introduction:A major component of the digesta reaching the colon from the distal ileum is carbohydrate. This carbohydrate is subject to microbial fermentation and can radically change bacterial populations in the colon and the metabolites they produce, particularly short-chain fatty acids (SCFA). However, very little is currently known about the forms and levels of carbohydrate in the ileum and the composition of the ileal microbiota in humans. Most of our current understanding of carbohydrate that is not absorbed by the small intestine comes from ileostomy models, which may not reflect the physiology of an intact gastrointestinal tract.Methods:We will investigate how ileal content changes depending on diet using a randomised crossover study in healthy humans. Participants will be inpatients at the research facility for three separate 4-day visits. During each visit, participants will consume one of three diets, which differ in carbohydrate quality: 1) low-fibre refined diet; 2) high-fibre diet with intact cellular structures; 3) high-fibre diet where the cellular structures have been disrupted (e.g. milling, blending). On day 1, a nasoenteric tube will be placed into the distal ileum and its position confirmed under fluoroscopy. Ileal samples will be collected via the nasoenteric tube and metabolically profiled, which will determine the amount and type of carbohydrate present, and the composition of the ileal microbiota will be measured. Blood samples will be collected to assess circulating hormones and metabolites. Stool samples will be collected to assess faecal microbiota composition. Subjective appetite measures will be collected using visual analogue scales. Breath hydrogen will be measured in real-time as a marker of intestinal fermentation. Finally, anin vitrocontinuous fermentation model will be inoculated with ileal fluid in order to understand the shift in microbial composition and SCFA produced in the colon following the different diets.Registration:ISRCTN11327221.

Metformin Triggers PYY Secretion in Human Gut Mucosa.

The antidiabetic drug metformin causes weight loss, but the underlying mechanisms are unclear. Recent clinical studies show that metformin increases plasma levels of the anorectic gut hormone, peptide YY (PYY), but whether this is through a direct effect on the gut is unknown. We hypothesized that exposure of human gut mucosal tissue to metformin would acutely trigger PYY secretion. Mucosal tissue was prepared from 46 human colonic and 9 ileal samples obtained after surgical resection and ex vivo secretion assays were performed. Tissue was exposed to metformin, as well as a series of other compounds as part of our mechanistic studies, in static incubations. Supernatant was sampled after 15 minutes. PYY levels in supernatant, measured using ELISA. Metformin increased PYY secretion from both ileal (P < 0.05) and colonic (P < 0.001) epithelia. Both basal and metformin-induced PYY secretion were unchanged across body mass index or in tissues obtained from individuals with type 2 diabetes. Metformin-dependent PYY secretion was blocked by inhibitors of the plasma membrane monoamine transporter (PMAT) and the serotonin reuptake transporter (SERT), as well as by an inhibitor of AMP kinase (AMPK). This is a report of a direct action of metformin on the gut epithelium to trigger PYY secretion in humans, occurring via cell internalization through PMAT and SERT and intracellular activation of AMPK. Our results provide further support that the role of metformin in the treatment of metabolic syndrome has a gut-based component.

Protective Effects of Live Combined B. subtilis and E. faecium in Polymicrobial Sepsis Through Modulating Activation and Transformation of Macrophages and Mast Cells.

Aims: Clinical studies showed that the use of probiotics during critical illness reduced nosocomial infection and improved clinical outcome. However, the functional mechanisms of probiotics is remains unclear. Therefore the aim of current study is to explore the protective effects and understand the underlying mechanisms for the beneficial effects of live combined Bacillus subtilis and Enterococcus faecium (LCBE) in cecal ligation puncture (CLP)-induced sepsis.Methods and Results: Seven-week-old C57BL/6J mice were divided into three groups: sham group (6 mice), CLP-control group (20 mice, pretreatment with saline for 7 days before CLP surgery) and CLP-probiotics group (14 mice, pretreatment with LCBE enteric-coated capsules for 7 days before CLP surgery). In survival experiment, mice were monitored for 7 days after CLP. After the procedure, mice were sacrificed, and, serum, and peritoneal lavage fluid were collected and intestinal ileal samples were harvested.Results: Our results showed that the mortality was significantly reduced in mice CLP-probiotics group vs. CLP-control group (P < 0.05). Also, treatment CLP-probiotics group decreased the injury scores CLP-probiotics group when compared to CLP-control group. Additionally, levels of pro-inflammatory cytokines IL-6 and TNF-α levels in the serum and intestinal ileal tissues of CLP-probiotics group were reduced when compared to CLP-control group (P < 0.05). However, no significant differences in anti-inflammatory levels of IL-10 and TGF-β1 were observed between CLP-control and CLP-probiotic groups. Furthermore, our experiments showed that that probiotic treatment suppressed the macrophage activation and transformation from M-type to M1-type, inhibited the mast cells (MCs) degranulation, and activation of AKT (kinase B) pathway.Conclusion: In conclusion, our data shows that probiotics have a protective role in CLP septic mice through reducing intestinal inflammation, altering macrophage polarization and MCs degranulation, and regulating AKT signaling.Significance and Impact of Study: This study demonstrated the protective effects and mechanisms involved in the protective role of live combined Bacillus subtilis and Enterococcus faecium (LCBE) in CLP-induced septic mice model.

In vitro synergy of sertraline and tetracycline cannot be reproduced in pigs orally challenged with a tetracycline resistant Escherichia coli

BackgroundAntimicrobial helper-compounds may reverse antimicrobial resistance. Sertraline, a antidepressant drug, has been suggested as a tetracycline helper-compound. Tetracycline is the preferred antimicrobial for treatment of enteric diseases in pigs. This study is the first to evaluate the potency of sertraline as a tetracycline adjuvant in pigs.MethodsForty-eight nursery pigs were divided into four treatment groups: Tetracycline, sertraline, tetracycline/sertraline or un-medicated control. Fecal and ileal samples were obtained before treatment, 48 h and nine days after five days of treatment, respectively. Colony forming units (CFU) of tetracycline resistant coliforms in each sample (ileal or fecal) and CFU of an orally inoculated tetracycline-resistant strain of Escherichia coli were determined at each sampling point. The microbiome of fecal and ileal and samples was analyzed by sequencing of the 16S V3-V4 region.ResultsThe results did not provide evidence that sertraline in combination with tetracycline has any impact on tetracycline resistant bacteria in either fecal or ileum samples, while in the tetracycline treated group of pigs, an increase in the prevalence of a tetracycline resistant indicator strain of Escherichia coli shortly after ended five-day treatment was observed. The ileal samples obtained shortly after ended treatment showed treatment-associated changes in the composition of the microbiota in the groups of pigs treated with tetracycline (+/−) sertraline. While tetracycline treatment increased the abundance in the reads of E. coli, sertraline/tetracycline treatment led to increased abundances of Streptococcus spp. and decreased abundances of Lactobacillus spp. However, all observed differences (on CFU counts and microbiota composition) between groups shortly after treatment had diminished in less than two weeks after last treatment day.ConclusionsSertraline (+/−) tetracycline treatment did not reduce the long-term level of tetracycline-resistant bacteria in the feces or small intestine contents of piglets compared to the un-medicated control group of pigs. The result of this study reflects the importance of in vivo studies for confirmation of the antimicrobial helper-compound potential of an in vitro active compound.

Effect of olive leaf powder on the performance and ileal bacterial count of broilers.

This experiment was conducted to investigate the effects of olive leaf (OL) on the performance, abdominal fat pad and some ileal bacterial population of Cobb broiler chickens. A total number of 400 day-old chicks were randomly distributed into floor pens and reared under the same condition until 14 days of age. On day 14, each pen was randomly assigned to one of the five experimental treatments with four replicates of 20 male and female chicks. The dietary treatments were consisted of a control group which fed basal diet without OL entire period of the study and groups 2 to 5 that fed diets supplemented with 0.25, 0.50, 0.75 and 1.00% OL powder, respectively. On days 21 and 42 of the experiment, ileal digesta samples were collected under the sterile condition to evaluate ileal bacterial population. The results indicated that birds fed diets containing various levels of OL, had higher body weight gain (except for 1.00% OL) and lower feed conversion ratio compared to that of the control group. Dietary inclusion of OL resulted in a higher count of Lactobacillus sp. compared to the control group on 42 days of age, while Escherichia coli count significantly was not influenced. The abdominal fat pad was lower in birds fed OL supplemented diets. In conclusion, findings of the current experiment showed that the OL had positive effects on feed conversion ratio, abdominal fat pad deposition and ileal bacterial count of broiler chickens.

Exogenous xylanase or protease for pigs fed barley cultivars with high or low enzyme inhibitors

Xylanase and protease inhibitors in cereals may be a reason for the varying efficacy of exogenous xylanase and protease in pig diets. The present study was conducted to evaluate the effect of xylanase and protease on the digestibility in pigs fed three barley varieties selected to be high and low in inhibitors. The cv. Invictus (spring barley) was selected to be low in xylanase inhibitors and cv. Hejmdal (winter barley) to be low in protease inhibitors, while SJ115158 (winter barley) was high in both xylanase and protease inhibitors. Growing pigs (34 ± 3 kg) were surgically fitted with a T-cannula in the terminal ileum and allotted to a 7 × 6 Youden square design, where 7 animals were fed with 7 diets for 6 weeks. Ileal samples were collected for 8 h on day 5 and 7 of each experimental week and fecal samples were collected from the first feces excreted on the same collection days. Titanium dioxide was added to all diets as an indigestible solid phase marker for the calculation of apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD). There was no consistent effect of variety or enzymes on the viscosity of ileal digesta. The ATTD of dry matter and organic matter was higher (P < 0.05) for Invictus than SJ115158, which was higher (P < 0.05) than Hejmdal. For Hejmdal, the AID of dry matter and organic matter was also lower (P < 0.05) compared to SJ115158. Supplementation of exogenous xylanase increased AID of dry matter, whereas the other evaluated components were unaffected. The ATTD of crude protein was lower (P = 0.027) for Hejmdal than for SJ115158 and exogenous protease increased (P = 0.004) ATTD of crude protein in both SJ115158 and Hejmdal. In conclusion, this study shows differences in nutrient digestibility among barley varieties and low efficacies of the selected exogenous enzymes independent of barley variety.

Digestibility of amino acid in full-fat canola seeds, canola meal, and canola expellers fed to broiler chickens and pigs1

Canola products including full-fat canola seeds (FFCS), canola meal (CM), and canola expellers (CE) have been used in diets for both broiler chickens and pigs. However, their ability to utilize the AA in canola products might be different from each other. Therefore, this study was conducted to compare the apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of CP and AA in broiler chickens and growing pigs fed FFCS, CM, and CE. Three diets were prepared to contain FFCS, CM, or CE as a sole source of nitrogen. A nitrogen-free diet was prepared. In Exp. 1, a total of 272 twenty-one-day-old male broiler chickens with an initial BW of 932 ± 80.6 g were assigned to diets in a randomized complete block design with BW as a blocking factor. After 5 d of feeding, birds were euthanized by sodium pentobarbital, and ileal digesta samples were collected from distal two-third of the ileum. In Exp. 2, 16 barrows were surgically fitted with T-cannulas at the distal ileum. After 8-d recovery period, pigs (initial BW = 18.9 ± 1.17 kg) were divided into 4 blocks based on BW and assigned to a quadruplicate 4 × 2 incomplete Latin Square design with 4 diets and 2 periods. Each period consisted of 5-d adaptation and 2-d ileal digesta collection periods. Data were analyzed as a 2 × 3 factorial treatment arrangement with effects of species (broiler chickens or pigs) and diets (FFCS, CM, or CE). There were interactions (P < 0.05) between species and experimental diets in the AID of all indispensable AA except for Lys. The AID of indispensable AA in FFCS for broiler chickens was greater (P < 0.05) than for pigs. Broiler chickens also had greater (P < 0.05) AID of Arg, His, Leu, Phe, and Val in CM compared with pigs; however, there were no differences in the AID of indispensable AA in CE between broiler chickens and pigs. The basal ileal endogenous losses of CP and AA, except Trp, in pigs were greater (P < 0.05) than in broiler chickens. There were also interactions (P < 0.05) between species and experimental diets in the SID of all indispensable AA except for Lys. Broiler chickens fed the diet containing FFCS had greater (P < 0.05) SID of indispensable AA compared with pigs fed the same diet; however, the SID of indispensable AA in CM or CE were not different between broiler chickens and pigs. In conclusion, differences in digestibility of AA in canola products were affected by nonruminant animal species.

Autolyzed Yeast (Saccharomyces cerevisiae) Supplementation Improves Performance While Modulating the Intestinal Immune-System and Microbiology of Broiler Chickens

The objective of this study was to evaluate the effects of dietary autolyzed yeast (AY; Saccharomyces cerevisiae) supplementation on growth performance, immune system, and intestinal bacterial count in broiler chickens. A total of 1,260 one-day-old male Ross AP95 chicks were placed in a completely randomized design (4 treatments, 7 replicates each, and 45 birds/replicate). The treatments were: basal diet – negative control (NC); basal diet supplemented with 55 ppm of zinc bacitracin – positive control (PC); NC + 0.2% of AY; NC + 0.4% of AY. The diets were formulated based on corn-soybean meal with 5% inclusion of wheat bran and 5% of poultry by-product meal. At 7 days of age, all birds were eye drop-vaccinated with live vaccine against coccidiosis. At 8 and 21 d of age, one chicken per pen was then euthanized by cervical dislocation to collect ileal and cecal contents for enumeration of Enterococcus sp., Escherichia coli and Lactobacillus sp. Yet, ileal samples were collected to analyze the gene expression of Claudin-1, IL-1β, IL-4, TLR-4 and MUC-2 through real time PCR. On d 21, it was observed that the inclusion of 0.2% of AY improved FCR (P < 0.05) when compared to the NC treatment. In the overall experimental period, the inclusion of zinc bacitracin and 0.4% of AY improved FCR (P < 0.05) compared to the NC group. On d 8, supplementation of 0.2 % of AY increased Enterococcus and both concentrations of AY reduced Lactobacillus in the ileal digesta compared to birds supplemented with zinc bacitracin. On d 21, 0.2% of AY reduced E. coli in the cecal digesta. On d 8, AY supplementation downregulated the expression of TLR-4 vs. the PC group (P = 0.04). On d 21, supplementation of AY upregulated the expression of IL-1β (P < 0.05) vs. the NC group. Supplementation of AY improved the growth performance of broiler chickens vaccinated against coccidiosis, partially explained by the modulation of the intestinal microbiota and immune-system.

Oven drying of ileal digesta from growing pigs reduces the concentration of AA compared with freeze drying and results in reduced calculated values for endogenous losses and elevated estimates for ileal digestibility of AA.

Two experiments were conducted to evaluate the influence of drying method on the composition of ileal digesta and the standardized ileal digestibility (SID) of CP and AA in feed ingredients fed to pigs. The hypothesis was that oven drying of ileal digesta samples results in loss of N and AA, and therefore, in greater calculated values for SID of CP and AA compared with lyophilized samples. In Exp. 1, eight barrows (13.8 ± 0.5 kg BW) were equipped with a T-cannula in the distal ileum and randomly allotted to a replicated 4 × 4 Latin square design with four diets and four periods, for a total of eight replicate pigs per diet. Three diets containing three different sources of soybean meal as the sole source of AA and an N-free diet were used. In Exp. 2, 18 cannulated growing barrows (72.5 ± 9.2 kg BW) were allotted to a completely randomized design with three diets and six replicate pigs per diet. The three diets included two diets based on two sources of distillers dried grains with solubles and an N-free diet. In both experiments, ileal digesta samples were collected for 8 h on days 6 and 7 of the 7-d feeding period. At the conclusion, two representative sub-samples were collected from each ileal digesta sample. One sub-sample was lyophilized and the other was oven dried at 60 °C using a forced air oven. Results indicated that in both experiments, DM was greater (P < 0.05) in lyophilized samples than in oven dried samples. There was no difference in the SID of CP between the two drying methods. However, except for Ala and Val in Exp. 1, the concentration of AA (88% DM-basis) was greater (P < 0.05) in lyophilized samples than in oven dried samples, which resulted in reduced calculated values for basal endogenous losses of AA. Therefore, values for the SID of AA (except for Ala in Exp. 1 and Trp and Gly in Exp. 2) were greater (P < 0.05) when calculated from oven dried samples than from lyophilized samples. In conclusion, regardless of the diet, oven drying of ileal digesta samples does not result in loss of N, but appears to damage or convert AA to other N-containing compounds, which results in reduced estimates for basal endogenous losses and greater calculated values for SID of AA. Thus, freeze drying of ileal digesta samples is recommended when data for the SID of AA are calculated for feed ingredients fed to pigs.

IL23R-Protective Coding Variant Promotes Beneficial Bacteria and Diversity in the Ileal Microbiome in Healthy Individuals Without Inflammatory Bowel Disease.

This study aimed to characterize the mucosa-associated microbiota in ileal Crohn's disease [CD] patients and in healthy controls in terms of host genotype and inflammation status. The mucosa-associated microbiotas of intestinal pinch biopsies from 15 ileal CD patients with mild and moderate disease and from 58 healthy controls were analysed based on 16S ribosomal sequencing to determine microbial profile differences between [1] IL23R, NOD2 and ATG16L1 genotypes in healthy subjects, [2] ileal CD patients and control subjects, and [3] inflamed and non-inflamed mucosal tissue in CD patients. The protective variant of the IL23R gene [rs11209026] significantly impacted the microbial composition in the ileum of healthy subjects and was associated with an increased abundance of phylotypes within the family Christensenellaceae as well as increases in diversity and richness. Comparative analysis of healthy and non-inflamed CD microbiome samples indicated a notable decrease in the abundance of Faecalibacterium prausnitzii as well as Shannon diversity and richness. Inflamed and non-inflamed ileal samples of CD subjects had high intra-individual stability and inter-individual variability, but no significant alterations in diversity, richness or taxa were identified. Calprotectin correlated positively with the abundance of Proteobacteria and negatively with diversity in the samples from healthy subjects. The observation of low diversity and low abundance of beneficial bacteria in healthy control subjects carrying the IL23R [rs11209026] wild-type GG genotype indicates that the gut microbiome is influenced by host genetics and is altered prior to disease diagnosis. Faecal calprotectin may be a potential non-invasive screening tool for dysbiosis in subjects without disorders of intestinal inflammation.

Pre-cecal phosphorus digestibility for corn, wheat, soybean meal, and corn gluten meal in growing Japanese quails from 28 to 32 d of age.

The optimization of dietary phosphorus (P) depends on precise details of the P availability in feed ingredients to avoid excess or deficient P in a mixed diet. This study was carried out to measure the apparent ileal digestibility of P for corn, wheat, soybean meal, and corn gluten meal in growing Japanese quails from 28 to 32 d posthatch. A total of 400 quail chicks were randomly distributed across 5 treatments with 4 replicates and 20 birds in each floor pen. The P-free diet (PFD) was formulated based on cornstarch to measure the basal endogenous P losses (EPL). Digestibility coefficients were determined by ileal digesta sampling using TiO2 as an indigestible marker. The EPL was estimated at 384 mg/kg DMI. The apparent ileal P digestibility (AIPD) for corn, soybean meal, wheat, and corn gluten meal were determined to be 0.38, 0.53, 0.38, and 0.78, respectively. The corresponding values for true ileal P digestibility (TIPD) were 0.48, 0.61, 0.50, and 0.83, respectively. The t-test analysis showed that the difference of AIPD and TIPD values for corn (P = 0.031) and wheat (P = 0.015) were statistically significant, however, no significant differences were observed for corn gluten meal (P = 0.318) and soybean meal (P = 0.104). In conclusion, the correction of AIPD coefficients for EPL in low-P ingredients such as corn and wheat may be much more important than that in high-P feedstuffs such as corn gluten meal and soybean meal in growing quails.

Prevalence of Yersinia Species in the Ileum of Crohn's Disease Patients and Controls.

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored.

Importance of TLR9-IL23-IL17 axis in inflammatory bowel disease development: Gene expression profiling study

Background and aimsMucosal gene expression have not been fully enlightened in inflammatory bowel disease (IBD). Aim of this study was to define IL23A, IL17A, IL17F and TLR9 expression in different IBD phenotypes. MethodsEvaluation of mRNA levels was performed in paired non-inflamed and inflamed mucosal biopsies of newly diagnosed 50 Crohn's disease (CD) and 54 ulcerative colitis (UC) patients by quantitative real-time PCR analysis. ResultsIL17A and IL17F expression levels were significantly increased in inflamed IBD mucosa. Inflamed CD ileal and UC mucosa showed increased IL23A, while only inflamed CD ileal samples showed increased TLR9 mRNA level. Correlation between analysed mRNAs levels and endoscopic and clinical disease activity were found in UC, but only with clinical activity in CD. ConclusionBoth CD and UC presented expression of Th17-associated genes. Nevertheless, expression profiles between different disease forms varies which should be taken into account for future research and therapeutics strategies.

Determination of Growth Performance, Viscera Organ Weights and Ileal Intestinal Architecture of Broilers in Response to Drinking Water Added Extractions from Wooden Chips for the Starter Period

A total of 90 one-day-old male broilers (Ross 308) were randomly assigned to one of the three dietary treatments, each consisting of six replicates (5 broilers/cage). The dietary treatments were 1) control (CON: fresh clean water with no supplement); 2) low dose [LD: CON + 1.56% extractions from the wooden chips (EWC)] and 3) high dose (HD: CON + 12.5% EWC). Drinking water supplemented with EWC was provided using specifically designed individual nipple drinker units. Average daily water intake (ADWI), average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were measured weekly for 21 days. One broiler from each cage was euthanized for measuring the visceral organ weights and collecting ileal tissue samples for ileal architecture analysis on day 21. Broilers assigned to the LD and HD watery groups showed higher ADWI than that in broilers consumed CON on day 7 (<italic>P</italic>&lt;0.05). The broilers subjected to HD treatment showed a deeper crypt depth (<italic>P</italic>&lt;0.05) than that in broilers subjected to LD and CON on day 21. Therefore, broilers consumed HD showed a lower (<italic>P</italic>&lt;0.05) villus height:crypt depth ratio than that broilers consumed CON on day 21. Broilers provided drinking water containing any of the tested concentrations of EWC showed no effect (P &gt; 0.05) on growth performance, ileal villus height, and visceral organ weights as compared with those in the CON from hatch to 21 days. In conclusion, broilers fed HD showed reduction in villus height:crypt depth ratio without impairing growth performance and visceral organ weights for the experimental period.

Moderated milk replacer restriction of ewe lambs alters gut immunity parameters during the pre-weaning period and impairs liver function and animal performance during the replacement phase

Undernutrition during the first stages of life may impair maturation of the immune system, harming the health status permanently. Thus, the aim of the present study was to define the impact of moderated milk replacer (MR) restriction during the artificial rearing period on the gut-associated lymphoid tissues of pre-weaned ewe lambs and long-term effects on biochemical profile and animal performance during the replacement period. Forty female Assaf lambs [average live body weight (LBW) 4.73 ± 0.116 kg] were penned individually. The first group of lambs (n = 20) was fed MR ad libitum (ADL) whereas the second one (restricted, RES; n = 20) only received approximately 62.5% of the MR intake measured in the ADL group. All the lambs were weighed twice a week until they were 35-days-old. Then, 8 lambs from each group (pre-weaned) were harvested and ileal samples collected. The rest of the lambs (12 ADL vs. 12 RES) were weaned progressively, and then offered a total mixed ration (TMR) ad libitum until the end of the experiment (7.5-months-old). The lower nutrient intake of RES pre-weaned lambs caused greater percentages for T lymphocytes (CD4+, CD8+, WC1+) and an increased secretory immunoglobulin A (sIgA) production in the ileum compared to the ADL group (P < 0.05). These results, together with the significant differences in the expression of toll-like receptors and cytokines at this level (e.g., interferon gamma was increased in the RES group; P<0.05) suggest the existence of pro-inflammatory conditions in the ileum of pre-weaned lambs promoted by early feed restriction. Moreover, the biochemical profile revealed that liver function (e.g., albumin, gamma glutamyl transpeptidase) was impaired during several months once the feed restriction period was finished, whereas the average daily gain during the replacement phase was reduced for the early feed restricted lambs (182 vs. 202 g/day for RES and ADL groups, respectively; P < 0.05). Therefore it can be concluded that moderated MR restriction promoted both, pro-inflammatory conditions at ileal level in the pre-weaned lambs and an altered liver function which lasted several months after the end of feed restriction. Both circumstances might have impaired animal performance during the replacement phase of the ewe lambs.

Effect of Early Weaning on the Intestinal Microbiota and Expression of Genes Related to Barrier Function in Lambs.

Weaning stress has been reported to impair intestinal health. The gut microbiota plays a vital role in the long-term health of the host. However, our understanding of weaning stress on gut microbiota and barrier function is very limited in livestock species, especially lambs. We investigated the effects of early weaning stress on intestinal bacterial communities and intestinal barrier function in lambs. A total of 24 neonatal male Hu lambs were randomly allocated into two groups, one weaned on day 28 and the other weaned on day 56. At 42 and 84 days, six lambs from each group were randomly selected and sacrificed. Ileal tissue and ileal digesta were collected to compare the differences in ileal microbiota and the mRNA levels of Toll-like receptors (TLRs) and tight junction proteins between the early weaning group and the control group at day 42 when the early weaning group have been weaned for 14 days, and at day 84 when the 28 and 56 days weaning groups had been weaned for 56 and 28 days, respectively. 16S rRNA gene sequencing of ileal samples revealed that the ileal microbiota was very different between the two groups, even at 84 days of age. Early weaning significantly increased alpha diversity and altered the relative abundance of several bacterial taxa. The expression of genes related to intestinal barrier function was affected by early weaning. Early weaning significantly increased ileal mRNA levels of TLR1 on days 42 and 84; TLR2, TLR4, and TLR5 on day 84; claudin1 and claudin4 on day 42; and occludin on day 84. We demonstrate that early weaning not only altered the ileal microbiota on day 42 (compared with lambs that were not weaned), but also had lasting effects on the ileal microbiota at day 84; furthermore, early weaning impacts expression levels of genes related to intestinal barrier function.

Assessing effects of yeast cell wall supplementation on threonine requirements in broilers as measured by performance and intestinal morphology

The objective of this study was to evaluate whether or not the use of a prebiotic yeast cell wall (YCW) affects threonine requirements in starter broilers. Two hundred and forty 1-d-old Ross 308 broiler chickens were distributed among 2 Petersime battery brooder units (48 pens; 5 birds per pen). Different threonine to lysine ratios (0.60, 0.65, and 0.70 to 1.0) with 1.22% available lysine with and without YCW at 250 ppm was fed to the chickens in order to evaluate performance and intestinal morphology over a 21-d trial. A basal diet with 22% protein and 2,980 kcal/kg ME was prepared to create the 6 dietary treatments. The calculated lysine concentration was 1.34%, whereas threonine was 0.81, 0.88, and 0.94%, respectively. Pen weights and feed consumption were recorded at d 1, 7, 14, and 21 of the experiment. Jejunal and ileal samples were collected on d 21 for histology analysis to evaluate villi height (VH), crypt depth, villi width (VW), and muscularis thickness. A threonine to lysine ratio of 0.7:1.0 resulted in significant improvement of performance variables with increased jejunum VW and ileum VH compared with the other threonine to lysine ratios. Adding YCW to starter broiler did not affect the requirement for threonine.

The Effect of Hepatocyte Growth Factor on Intestinal Adaption in an Experimental Model of Short Bowel Syndrome

Introduction Short bowel syndrome (SBS) is a relatively uncommon syndrome which is defined as the functional loss of the 75% of normal short bowel length at least. To date, the standard therapy is the administration of total parenteral nutrition (TPN). However, long-term complications in patients with protracted PN are occurred. Intestinal transplantation is a potentially life-saving treatment for patients with severe SBS; its increased morbidity and mortality rates imply a careful preoperative consideration though. Moreover, the lack of compatible donor organs may delay significantly the transplantation. As a result, alternative therapies, like the administration of growth factors, in order to enhance intestinal adaptation acting as a bridge to transplantation have been emerged. The purpose of the present study is to evaluate the effect of hepatocyte growth factor (HGF) on structural intestinal adaptation, cell proliferation and apoptosis in a rat model of short bowel syndrome (SBS). Materials and Methods Thirty Sprague-Dawley female rats were divided into three groups, sham rats underwent bowel transection (group A); SBS rats underwent a 75% bowel resection (group B); and SBS/HGF rats underwent bowel resection and were treated with HGF (75 μg/kg/d) (group C) starting at fourth postoperative day. All rats were sacrificed at 15th postoperative day. Parameters of intestinal adaptation (body weight, villus height and crypt depth in jejunum and ileum), enterocyte proliferation and enterocyte apoptosis were determined. A microarray analysis of RNA extracted from ileal samples was also carried out. Statistical analysis was performed using ANOVA test, with P<0.05 considered statistically significant. Results Treatment with HGF (group C rats) resulted in significant increase in body weight compared to SBS rats (group B). The histopathologic analysis showed that jejunal and ileal villus height and crypt depth were significantly increased in HGF/SBS rats compared to SBS rats (36%, P<0.05 and 27%, P<0.05 respectively), showing that treatment with HGF enhanced the intestinal adaptation making intestinal absorption more efficient. Enterocyte proliferation was also significantly increased in HGF rats (21% P<0.05) compared to SBS rats; however we did not found any significant differences in enterocyte apoptosis rate between HGF/SBS and SBS rats. These findings indicate the augmentation of intestinal hyperplasia through HGF therapy. Microarray analysis of 22 genes showed that 12 genes were significantly up-regulated, and four genes were down-regulated in HGF/SBS rats compared to SBS rats; of note, some of these genes are involved in enterocyte proliferation/apoptosis pathway, indicating also that HGF therapy induces cell proliferation. Conclusion HGF emerges as a growth factor that enhances intestinal adaptation in patients with short bowel syndrome. A future use of HGF may facilitate the prolonged TPN therapy while patients wait for intestinal transplantation.