Heat stress directly impairs gut integrity and recruits distinct immune cell populations into the bovine intestine.

High ambient temperature has multiple potential effects on the organism such as hyperthermia, endotoxemia, and/or systemic inflammation. However, it is often difficult to discriminate between cause and consequence of phenotypic effects, such as the indirect influence of heat stress via reduced food intake. Lactating dairy cows are a particularly sensitive model to examine the effects of heat stress due to their intensive metabolic heat production and small surface:volume ratio. Results from this model show heat stress directly induced a so-far unknown infiltration of yet uncategorized cells into the mucosa and submucosa of the jejunum. Due to a pair-feeding design, we can exclude this effect being a consequence of the concurrent heat-induced reduction in feed intake. Isolation and characterization of the infiltrating cells using laser capture microdissection and RNA sequencing indicated a myeloic origin and macrophage-like phenotype. Furthermore, targeted transcriptome analyses provided evidence of activated immune- and phagocytosis-related pathways with LPS and cytokines as upstream regulators directly associated with heat stress. Finally, we obtained indication that heat stress may directly alter jejunal tight junction proteins suggesting an impaired intestinal barrier. The penetration of toxic and bacterial compounds during heat stress may have triggered a modulated immune repertoire and induced an antioxidative defense mechanism to maintain homeostasis between commensal bacteria and the jejunal immune system. Our bovine model indicates direct effects of heat stress on the jejunum of mammals already at moderately elevated ambient temperature. These results need to be considered when developing concepts to combat the negative consequences of heat stress.

Water intake releases serotonin from enterochromaffin cells in rat jejunal villi.

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.

A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release.

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology.

The Potential Protective Role of RUNX1 in Nonalcoholic Fatty Liver Disease.

The pathogenic mechanisms underlying nonalcoholic fatty liver disease (NAFLD) are beginning to be understood. RUNX1 is involved in angiogenesis, which is crucial in inflammation, but its role in nonalcoholic steatohepatitis (NASH) remains unclear. The aim of this study was to analyze RUNX1 mRNA hepatic and jejunal abundance in women with morbid obesity (MO) and NAFLD. RUNX1, lipid metabolism-related genes, and TLRs in women with MO and normal liver (NL, n = 28), NAFLD (n = 41) (simple steatosis (SS, n = 24), or NASH (n = 17)) were analyzed by RT-qPCR. The RUNX1 hepatic expression was higher in SS than in NL or NASH, as likewise confirmed by immunohistochemistry. An increased expression of hepatic FAS was found in NAFLD. Hepatic RUNX1 correlated positively with FAS. There were no significant differences in the jejunum RUNX1 expressions in the different groups. Jejunal FXR expression was lower in NASH than in NL, while the TLR9 expression increased as NAFLD progressed. Jejunal RUNX1 correlated positively with jejunal PPARγ, TLR4, and TLR5. In summary, the hepatic expression of RUNX1 seems to be involved in the first steps of the NAFLD process; however, in NASH, it seems to be downregulated. Our findings provide important insights into the role of RUNX1 in the context of NAFLD/NASH, suggesting a protective role.

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1.

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

Dietary Supplementation of dl-Methionine Potently Induces Sodium-Dependent l-Methionine Absorption in Porcine Jejunum Ex Vivo.

BACKGROUND: Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock. OBJECTIVES: The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets. METHODS: A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met + cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 µM and a high concentration of 5 mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA. RESULTS: Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P < 0.05). Pigs supplemented with dl-Met had greater (P < 0.085) absorptive fluxes at 50 µM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 µM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P < 0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P < 0.05). CONCLUSIONS: Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.

Influence of the effective microorganisms (EM) on performance, intestinal morphology and gene expression in the jejunal mucosa of pigs fed different diets.

The aim of the study was to determine the influence of the effective microorganisms (EM) on performance parameters, intestinal morphology and gene expression in the jejunal mucosa in pigs under different feeding regimes. The study group comprised of 150 piglets divided into three feeding groups: C, E1 and E2. Feeding groups included: C-standard fodder, blend with a full share of post-extracted soy meal, E1-in the phase I of fattening: pea and lupin/soybean 50/50%; in the phase II of fattening: pea and lupin/soybean 75/25%, and E2-in the phase I of fattening: pea and lupin/soybean 50/50%; in the phase II of fattening: pea and lupin 100%. The experimental factor was addition of a probiotic EM Carbon Bokashi to the diets (C + EM, E1 + EM and E2 + EM). After slaughter, histological evaluation and gene expression analysis were performed. The highest intestinal villi were reported in E2 + EM. A higher intestinal absorption area was demonstrated in groups C + EM and E2 + EM. An interaction between feeding and EM Bokashi supplementation was found in villus surface area crypt depth, villus height/crypt depth and number of goblet cells. Mucosa thickness and number of goblet cells was the largest in E2 + EM. Gene expression of FABP4 increased in E1, and GLUT2 decreased in E2. Gene expression of IL10 and FABP4 increased in E2 + EM. The results indicate that the E2 diet is more optimal for EM Bokashi supplementation, because in this group, EM positively influenced the morphological characteristics of the porcine jejunum and caused an increase in the expression of genes related to the metabolism and functioning of the gastrointestinal tract.

Effects of dietary grape pomace on the intestinal microbiota and growth performance of weaned piglets.

Grape pomace (GP) is an abundant by-product from wine production and is rich in phenolic compounds, unsaturated fatty acids, dietary fibre and beneficial bacteria. In this study, weaned piglets were fed a basic diet supplemented with 5% GP for 4 weeks. Compared with those in the control (CON) group, it was found that the proportion of Lactobacillus delbrueckii, Olsenella umbonata and Selenomonas bovis in the caecum and the villus height and villus height/crypt depth ratio (VCR) of the jejunum were both significantly increased in the GP group (p < 0.05). Meanwhile, at the mRNA expression level, several proinflammatory cytokines (IL-1ß, IL-8, IL-6 and TNF-α) were significantly downregulated (p < 0.05) in piglet caecal tissue, and the short-chain fatty acid receptors (GPR41 and GPR43) were not significantly upregulated. In contrast, the levels of IgG was significantly increased (p < 0.05) in the sera of weaned piglets in the GP group. However, no difference in growth performance between the two groups of piglets was detected. These results show that GP had no adverse effects on the growth performance of piglets, but GP can promote the content of some beneficial bacteria in the caecum; this effect is conducive to improving the disease resistance potential of piglets.

A flavonoid driven phyto-pharmacological effects of Capparis decidua Edgew. in rodents.

This study elicits the underlying mechanism(s) of Capparis decidua when used for different gut disorders. HPLC chromatogram of C. decidua extract (CD.Cr) and its respective fractions showed a variety of phytochemicals of which, kaempferol being in a high proportion. In mice, CD.Cr at doses of 70 and 150 mg/kg enhanced the wet feces output to 33 and 44% respectively as compared to carbachol (47.6%), while doses of 500 and 700 mg/kg, presented 41 and 70% safety against castor oil-driven diarrhea, respectively. Its flavonoid constituent, kaempferol at doses of (50 and 100 mg/kg) produced 51.7 and 82% safety when compared to nifedipine which provided 95% safety at dose of 40 mg/kg against castor oil-driven diarrhea like loperamide. In isolated jejunum preparations, C. decidua extract and its respective fractions (except pet-ether) produced atropine-sensitive inhibitory effects, whereas kaempferol and nifedipine showed atropine insensitive effects. Against high K+-induced contractions, C. decidua's fractions and kaempferol both exhibited a concentration-related non-specific inhibition while displacing the Ca++ -CRCs to right-ward with suppression in maximal response like nifedipine. In isolated rat ileal preparations, CD.Cr and respective fractions elicited atropine-sensitive gut excitatory responses. In summary, this article reports C. decidua's laxative effect through cholinergic receptor activation as well as its antidiarrheal effects, where its flavonoid constituent kaempferol produces Ca++ antagonist like activity, thus justifying C. decidua folk use in constipation and diarrhea.

Inducible nitric oxide synthase-derived nitric oxide reduces vagal satiety signalling in obese mice.

KEY POINTS: Obesity is associated with disrupted satiety regulation. Mice with diet-induced obesity have reduced vagal afferent neuronal excitability and a decreased afferent response to satiety signals. A low grade inflammation occurs in obesity with increased expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS in diet-induced obese mice restored vagal afferent neuronal excitability, increased the afferent response to satiety mediators and distention of the gut, and reduced short-term energy intake. A prolonged inhibition of iNOS reduced energy intake and body weight gain during the first week, and reduced amounts of epididymal fat after 3 weeks. We identified a novel pathway underlying disrupted satiety regulation in obesity. Blocking of this pathway might be clinically useful for the management of obesity. ABSTRACT: Vagal afferents regulate feeding by transmitting satiety signals to the brain. Mice with diet-induced obesity have reduced vagal afferent sensitivity to satiety signals. We investigated whether inducible nitric oxide synthase (iNOS)-derived NO contributed to this reduction. C57BL/6J mice were fed a high- or low-fat diet for 6-8 weeks. Nodose ganglia and jejunum were analysed by immunoblotting for iNOS expression; NO production was measured using a fluorometric assay. Nodose neuron excitability and intestinal afferent sensitivity were evaluated by whole-cell patch clamp and in vitro afferent recording, respectively. Expression of iNOS and production of NO were increased in nodose ganglia and the small intestine in obese mice. Inhibition of iNOS in obese mice by pre-treatment with an iNOS inhibitor increased nodose neuron excitability via 2-pore-domain K+ channel leak currents, restored afferent sensitivity to satiety signals and reduced short-term energy intake. Obese mice given the iNOS inhibitor daily for 3 weeks had reduced energy intake and decreased body weight gain during the first week, compared to mice given saline, and lower amounts of epididymal fat at the end of 3 weeks. Inhibition of iNOS or blocking the action of iNOS-derived NO on vagal afferent pathways might comprise therapeutic strategies for hyperphagia and obesity.

Metabolic improvement in obese patients after duodenal-jejunal exclusion is associated with intestinal microbiota composition changes.

BACKGROUND: Intestinal microbiota have been suggested to play an important role in the pathogenesis of obesity and type 2 diabetes. Bariatric surgery improves both conditions and has been associated with changes in intestinal microbiota composition. We investigated the effect of a nonsurgical bariatric technique on intestinal microbiota composition in relation to metabolic improvement. METHODS: Seventeen patients with obesity and type 2 diabetes were treated with the nonsurgical duodenal-jejunal bypass liner, which excludes the proximal 60 cm small intestine from food. Fecal samples as well as metabolic parameters reflecting obesity and type 2 diabetes were obtained from the patients at baseline, after 6 months with the device in situ, and 6 months after explantation. RESULTS: After 6 months of treatment, both obesity and type 2 diabetes had improved with a decrease in weight from 106.1 [99.4-123.5] to 97.4 [89.4-114.0] kg and a decrease in HbA1c from 8.5% [7.6-9.2] to 7.2% [6.3-8.1] (both p < 0.05). This was paralleled by an increased abundance of typical small intestinal bacteria such as Proteobacteria, Veillonella, and Lactobacillus spp. in feces. After removal of the duodenal-jejunal bypass liner, fecal microbiota composition was similar to that observed at baseline, despite persistent weight loss. CONCLUSION: Improvement of obesity and type 2 diabetes after exclusion of the proximal 60 cm small intestine by treatment with a nonsurgical duodenal-jejunal bypass liner may be promoted by changes in fecal microbiota composition.

Intestinal Electrical Stimulation to Increase the Rate of Peristalsis.

BACKGROUND: Pediatric gastrointestinal motility disorders are a large and broad group. Some of these disorders have been effectively treated with electrical stimulation. The goal of our present study is to determine whether the rate of intestinal peristalsis can be increased with electrical stimulation. METHODS: Juvenile mini-Yucatan pigs were placed under general anesthesia and a short segment of the jejunum was transected. Ultrasound gel was placed inside the segment. The segment of the jejunum was first monitored for 20 min under no stimulation, followed by direct electrical stimulation using a planar electrode. The gel extruded out of the intestine via peristalsis was collected and weighed for each 20-min time interval. RESULTS: Effective delivery of the current to the intestine was confirmed via direct measurements. When there was no direct intestinal electrical stimulation, an average of 0.40 g of gel was expelled in 20 min, compared to 1.57 g of gel expelled during direct electrical stimulation (P < 0.01). CONCLUSIONS: Direct intestinal electrical stimulation accelerates the transit of gastrointestinal contents. This approach may be useful in the treatment of a range of pediatric motility disorders.

Montmorillonite improved the intestinal mucosal barrier functions of laying hens in late production.

This study was conducted to investigate the effects of dietary supplementation with montmorillonite (MMT) on performance, intestinal endotoxin concentration, gut mucosal oxidation status, intestinal morphology and permeability, and immunological barrier function of laying hens during late production. Four hundred and eighty 75-week-old laying hens (Lohmann Brown) were randomly assigned to five treatments with eight replicates per treatment and 12 hens in each replicate. The hens were fed the basal diet supplemented with 0 (control), 0.3, 0.6, 0.9, or 1.2 g MMT/kg for 70 days. Compared with the control, supplemented with 0.9 g MMT/kg increased egg mass significantly (p < 0.05) during weeks 1-5 of the experiment. Supplemented with 0.6 and 0.9 g MMT/kg also increased the endotoxin concentration in the ileal digesta (p < 0.05), but decreased the MDA concentration in the ileum significantly (p < 0.05). The T-AOC in the jejunum of the group fed 0.3 g MMT/kg was significantly increased (p < 0.05). Compared with the control, the villus height:crypt depth of ileum from the groups fed 0.6, 0.9, and 1.2 g MMT/kg increased significantly (p < 0.05). The sIgA concentration of jejunum in the groups fed 0.6 and 0.9 g MMT/kg was higher (p < 0.05) than the control. The MMT supplementation linearly increased (p < 0.05) the mRNA expression of claudin-1 and claudin-5 in the jejunum. Dietary MMT supplementation down-regulated the mRNA expression of NF-κB P65 and TNF-α in the jejunum in a linear and quadratic manner (p < 0.05). The IL-1ß mRNA expression of jejunum in the group fed 0.6 g MMT/kg was lower (p < 0.05) than the control. In conclusion, dietary supplementation with MMT may improve the gut barrier functions and suggests that 0.9 g/kg of MMT in diets may be the optimal supplemental level for laying hens in late production.

Porcine Small and Large Intestinal Microbiota Rapidly Hydrolyze the Masked Mycotoxin Deoxynivalenol-3-Glucoside and Release Deoxynivalenol in Spiked Batch Cultures In Vitro.

Mycotoxin contamination of cereal grains causes well-recognized toxicities in animals and humans, but the fate of plant-bound masked mycotoxins in the gut is less well understood. Masked mycotoxins have been found to be stable under conditions prevailing in the small intestine but are rapidly hydrolyzed by fecal microbiota. This study aims to assess the hydrolysis of the masked mycotoxin deoxynivalenol-3-glucoside (DON3Glc) by the microbiota of different regions of the porcine intestinal tract. Intestinal digesta samples were collected from the jejunum, ileum, cecum, colon, and feces of 5 pigs and immediately frozen under anaerobic conditions. Sample slurries were prepared in M2 culture medium, spiked with DON3Glc or free deoxynivalenol (DON; 2 nmol/ml), and incubated anaerobically for up to 72 h. Mycotoxin concentrations were determined using liquid chromatography-tandem mass spectrometry, and the microbiota composition was determined using a quantitative PCR methodology. The jejunal microbiota hydrolyzed DON3Glc very slowly, while samples from the ileum, cecum, colon, and feces rapidly and efficiently hydrolyzed DON3Glc. No further metabolism of DON was observed in any sample. The microbial load and microbiota composition in the ileum were significantly different from those in the distal intestinal regions, whereas those in the cecum, colon and feces did not differ.IMPORTANCE Results from this study clearly demonstrate that the masked mycotoxin DON3Glc is hydrolyzed efficiently in the distal small intestine and large intestine of pigs. Once DON is released, toxicity and absorption in the distal intestinal tract likely occur in vivo This study further supports the need to include masked metabolites in mycotoxin risk assessments and regulatory actions for feed and food.

Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum.

NEW FINDINGS: What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl- homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl- homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl- (Cl-o ) with the impermeant anions gluconate and isethionate. On reducing Cl-o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em ). Restoration of Cl- hyperpolarized smooth muscle Em proportional to the change in Cl-o concentration. Replacement of 90% of Cl-o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl- and 0.13 mm free Ca2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl-o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl- homeostasis in ICCs can alter smooth muscle Em .

Segment-specific responses of intestinal epithelium transcriptome to in-feed antibiotics in pigs.

Despite widespread use of antibiotics for treatment of human diseases and promotion of growth of agricultural animals, our understanding of their effects on the host is still very limited. We used a model in which pigs were fed with or without a cocktail of antibiotics and found, based on the denaturing gradient gel electrophoresis (DGGE) patterns, that the fecal bacteria from the treatment and control animals were distinct. Furthermore, the total bacterial population in the feces tended to be decreased by the antibiotic treatment (P = 0.07), and the counts of Lactobacillus and Clostridium XIVa were significantly reduced (P < 0.05). To explore the effects of antibiotics on host intestinal epithelium, we assessed gene expression profiles of the jejunum and ileum and their response to antibiotic administration. The results indicate that in-feed antibiotics increased expression of genes involved in immune functions in both the jejunum and ileum, some of which were clustered in the coexpression network. Gene ontology terms of metabolic processes were altered predominantly in the jejunum but not in the ileum. Notably, antibiotics diminished intestinal segment-specific transcriptional changes, especially for genes associated with metabolic functions. This study reveals segment-specific responses of host intestinal epithelium to in-feed antibiotics, which can be a valuable resource for deciphering antibiotic-microbiota-host interactions.

Birth delivery method affects expression of immune genes in lung and jejunum tissue of neonatal beef calves.

BACKGROUND: Caesarean section is a routine veterinary obstetrical procedure employed to alleviate dystocia in cattle. However, CS, particularly before the onset of labour, is known to negatively affect neonatal respiration and metabolic adaptation in humans, though there is little published information for cattle. The aim of this study was to investigate the effect of elective caesarean section (ECS) or normal trans-vaginal (TV) delivery, on lung and jejunal gene expression profiles of neonatal calves. RESULTS: Paternal half-sib Angus calves (gestation length 278 + 1.8 d) were delivered either transvaginally (TV; n = 8) or by elective caesarean section (ECS; n = 9) and immediately euthanized. Lung and jejunum epithelial tissue was isolated and snap frozen. Total RNA was extracted using Trizol reagent and reverse transcribed to generate cDNA. For lung tissue, primers were designed to target genes involved in immunity, surfactant production, cellular detoxification, membrane transport and mucin production. Primers for jejunum tissue were chosen to target mucin production, immunoglobulin uptake, cortisol reaction and membrane trafficking. Quantitative real-time PCR reactions were performed and data were statistically analysed using mixed models ANOVA. In lung tissue the expression of five genes were affected (p < 0.05) by delivery method. Four of these genes were present at lower (LAP, CYP1A1, SCN11α and SCN11ß) and one (MUC5AC) at higher abundance in ECS compared with TV calves. In jejunal tissue, expression of TNFα, Il-1ß and 1 l-6 was higher in ECS compared with TV calves. CONCLUSIONS: This novel study shows that ECS delivery affects the expression of key genes involved in the efficiency of the pulmonary liquid to air transition at birth, and may lead to an increased inflammatory response in jejunal tissue, which could compromise colostral immunoglobulin absorption. These findings are important to our understanding of the viability and management of neonatal calves born through ECS.

Optimization of the Ussing chamber setup with excised rat intestinal segments for dissolution/permeation experiments of poorly soluble drugs.

CONTEXT: Prediction of the in vivo absorption of poorly soluble drugs may require simultaneous dissolution/permeation experiments. In vivo predictive media have been modified for permeation experiments with Caco-2 cells, but not for excised rat intestinal segments. OBJECTIVE: The present study aimed at improving the setup of dissolution/permeation experiments with excised rat intestinal segments by assessing suitable donor and receiver media. METHODS: The regional compatibility of rat intestine in Ussing chambers with modified Fasted and Fed State Simulated Intestinal Fluids (Fa/FeSSIFmod) as donor media was evaluated via several parameters that reflect the viability of the excised intestinal segments. Receiver media that establish sink conditions were investigated for their foaming potential and toxicity. Dissolution/permeation experiments with the optimized conditions were then tested for two particle sizes of the BCS class II drug aprepitant. RESULTS: Fa/FeSSIFmod were toxic for excised rat ileal sheets but not duodenal sheets, the compatibility with jejunal segments depended on the bile salt concentration. A non-foaming receiver medium containing bovine serum albumin (BSA) and Antifoam B was nontoxic. With these conditions, the permeation of nanosized aprepitant was higher than of the unmilled drug formulations. DISCUSSION: The compatibility of Fa/FeSSIFmod depends on the excised intestinal region. The chosen conditions enable dissolution/permeation experiments with excised rat duodenal segments. The experiments correctly predicted the superior permeation of nanosized over unmilled aprepitant that is observed in vivo. CONCLUSION: The optimized setup uses FaSSIFmod as donor medium, excised rat duodenal sheets as permeation membrane and a receiver medium containing BSA and Antifoam B.

Comparison of electrogenic glucose transport processes and permeability between proximal and distal jejunum of laying hens.

1. The current objective was to assess (1) differences in mucosal transepithelial short-circuit current (Isc) and tissue conductance (GT), (2) the effect of a glucose stimulus and (3) epithelial paracellular permeability in the proximal and distal jejunum of laying hens. 2. Proximal and distal jejunal segments used in the Ussing chambers were collected at 9 ± 0.5 and 73 ± 3.4% (SEM) of jejunal length, respectively. The proximal jejunal mucosa showed a small negative Isc (-1.3 µA/cm2), whereas the distal jejunum had a higher Isc (32.9 µA/cm2). Similarly, GT was 2.5-fold greater in the distal compared to the proximal jejunum. 3. Increased paracellular permeability in the distal jejunum was displayed as demonstrated by a 5-fold higher mucosal to serosal flux of fluorescein isothiocyanate and horseradish peroxidase, representing molecules of low and high molecular weight, respectively. 4. Addition of glucose to the mucosal side (5 mmol/l, final concentration in the chamber) to stimulate an absorptive effect caused 3-fold greater GT in the distal compared to the proximal jejunum. 5. In conclusion, the present results supported site-specific electrogenic transport processes for the jejunal mucosa of laying hens. Therefore, precise description of the jejunal site may contribute to an improved comparability of electrophysiological data.

Gamma-aminobutyric acid (GABA) permeates ovine ruminal and jejunal epithelia, mainly by passive diffusion.

Gamma-aminobutyric acid (GABA) represents the most abundant inhibitory neurotransmitter in the mammalian brain. GABA is also produced in plants and/or by the microbial conversion of amino acids. Thus, ruminants may be forced to take up significant amounts of GABA from their diet. However, it is not known whether exogenously acquired GABA might permeate the gastrointestinal barrier in such quantities as to induce systemic alterations. Thus, this study pursues the question of where within the ruminant's GI tract and by which pathways GABA may be taken up from the ingesta. The jejunal and ruminal epithelia of sheep were mounted in Ussing chambers under short-circuit conditions. The flux rates of radiolabelled GABA from the mucosal to the serosal side (Jms ) and vice versa (Jsm ) were measured. GABA was applied in various concentrations with adjustment of the mucosal pH to 6.1 or 7.4. Furthermore, beta-alanine or glycine was used as a competitive inhibitor for GABA transport. In both the jejunal and ruminal epithelium, the Jms of GABA was linearly correlated to the mucosal GABA concentration. However, Jms across the jejunal epithelium was approximately 10-fold higher than Jms across the ruminal epithelium. When 0.5 mmol/l GABA was applied on both sides of the epithelium, no net flux could be observed in the jejunal epithelia. Additionally, there was no effect of decreased mucosal pH or the application of glycine or beta-alanine under these conditions. The Jms and Jsm of GABA were linearly correlated to the transepithelial conductance. Our results suggest that GABA is taken up from the small intestine rather than from the rumen. Due to the lack of influence of pH and competitive inhibitors, this uptake seems to occur primarily via passive diffusion.