Colonic Paracellular Permeability Research Articles

Infant nutrition affects the microbiota-gut-brain axis: Comparison of human milk vs. infant formula feeding in the piglet model.

Early nutrition plays a dominant role in infant development and health. It is now understood that the infant diet impacts the gut microbiota and its relationship with gut function and brain development. However, its impact on the microbiota-gut-brain axis has not been studied in an integrative way. The objective here was to evaluate the effects of human milk (HM) or cow’s milk based infant formula (IF) on the relationships between gut microbiota and the collective host intestinal-brain axis. Eighteen 10-day-old Yucatan mini-piglets were fed with HM or IF. Intestinal and fecal microbiota composition, intestinal phenotypic parameters, and the expression of genes involved in several gut and brain functions were determined. Unidimensional analyses were performed, followed by multifactorial analyses to evaluate the relationships among all the variables across the microbiota-gut-brain axis. Compared to IF, HM decreased the α-diversity of colonic and fecal microbiota and modified their composition. Piglets fed HM had a significantly higher ileal and colonic paracellular permeability assessed by ex vivo analysis, a lower expression of genes encoding tight junction proteins, and a higher expression of genes encoding pro-inflammatory and anti-inflammatory immune activity. In addition, the expression of genes involved in endocrine function, tryptophan metabolism and nutrient transport was modified mostly in the colon. These diet-induced intestinal modifications were associated with changes in the brain tissue expression of genes encoding the blood-brain barrier, endocrine function and short chain fatty acid receptors, mostly in hypothalamic and striatal areas. The integrative approach underlined specific groups of bacteria (Veillonellaceae, Enterobacteriaceae, Lachnospiraceae, Rikenellaceae, and Prevotellaceae) associated with changes in the gut-brain axis. There is a clear influence of the infant diet, even over a short dietary intervention period, on establishment of the microbiota-gut-brain axis.

Changes in intestinal barrier protein expression and intestinal flora in a rat model of visceral hypersensitivity.

Destruction of the intestinal mucosal barrier and visceral hypersensitivity are main pathogenesis of irritable bowel syndrome (IBS). The study aimed to establish a rat model of visceral hypersensitivity and explore mechanisms involved the changes of the intestinal barrier protein expression and intestinal flora. A rat model of visceral hypersensitivity was established and evaluated using abdominal withdrawal reflex (AWR) scores, colonic paracellular permeability, and gastrointestinal motility. The expression of tight junction proteins, aquaporin proteins (AQPs), phosphorylated ERK, and proteinase-activated receptor-2 (PAR-2) was determined. The intestinal microflora was evaluated by high-throughput sequencing of the 16S rRNA gene. In model rats, AWR score and fecal water content were significantly increased, gastrointestinal motilities were disorder and characterized by an inhibition of gastric motility and an enhancement of small intestinal and colonic movement. The expressions of colonic occludin, ZO-1, AQP3, and AQP8 were decreased but claudin-2 and claudin-4 were markedly increased. Imbalance of intestinal flora appeared and showed an obvious decrease of Lactobacillus and an increase of Clostridiales_bacterium. Additionally, the total serine protease activity in feces, the expressions of PAR2 and phosphorylated ERK in the colon tissues were increased significantly. The model rats of visceral hypersensitivity possess the decreased expression of occludin, ZO-1, AQP3, AQP8, and the increased expression of claudin-2 and claudin-4, meanwhile develop an imbalance of intestinal flora which probably increase serine protease activity, thereby activating the PAR2/ERK signaling and causing the intestinal barrier disorder.

The protective effect of Jangkanghwan (Korean traditional food) on lipopolysaccharide-induced disruption of the colonic epithelial barrier

Jangkanghwan (JKH) is a Korean traditional food that is a mixture of food ingredients and traditional Korean medicine ingredients, and it has been observed to produce satisfactory anti-inflammatory, antioxidant, and antibacterial effects. In the current study, JKH was administered by gavage to BALB/C mice with lipopolysaccharide (LPS)-induced colonic epithelial dysfunction, and mouse body weight and food intake were recorded. Indexes such as colonic paracellular permeability, serum inflammatory cytokines, and bacterial translocation were used to comprehensively evaluate the regulatory effect of JKH on mouse colonic epithelial function, and qPCR and Western blot were also used to analyze the expression of tight junction (TJ)-related genes, such as occludin, claudin, zonula occludens (ZOs) proteins, and junction adhesion molecules (JAM) in the colonic epithelial tissue. The experimental results indicated that JKH relieved the edema of the liver, spleen, and mesenteric lymph node tissues, and reduced the loss of appetite and diarrhea caused by LPS injection in mice. It increased the amount of mice food intake from 3.7 g/day in the LPS group to 4.7 g per day; the water content in the feces of mice in the JKH group was 13.86% less than that in the LPS group. JKH reduced the inflammatory response in mice caused by LPS, protected the integrity of the colon, the permeability of fluorescent macromolecules was one-fourth of the LPS group, and enhanced the mRNA and protein expression of TJ-related proteins in colon tissue. Our findings highlight that JKH has benefits in intestinal health and relieving systemic inflammation, relevant aspects of its use as a functional food.

Colonic paracellular permeability and circulating zonulin-related proteins

Objective Irritable bowel syndrome (IBS) is a gut-brain disorder associated with increased gut permeability. Zonulin has been suggested to regulate the gut barrier and claimed to be pre-haptoglobin 2 (pre-HP2) and circulating zonulin is often used as a proxy for gastrointestinal permeability. This study investigated the correlation between colonic paracellular permeability and levels of circulating zonulin and pre-HP2. Materials and Methods Colonic biopsies from 32 patients with IBS and 15 healthy controls (HC) were used to measure permeability in Ussing chambers and levels of zonulin (Cusabio ELISA). Zonulin was also measured in blood samples from 40 HC, 78 patients with IBS and 20 patients with celiac disease (CeD), before and after a gluten-free diet. In addition, we verified HP genotype and circulating pre-HP2 using a monoclonal pre-HP2 antibody (Bio-Rad) by ELISA. Results Increased colonic paracellular permeability correlated positively with zonulin levels in IBS biopsies, but negatively with plasma zonulin. We found no agreement between circulating zonulin and pre-HP2. Genotyping revealed non-specificity of the zonulin kit, as all pre-HP2 non-producers presented detectable levels. Patients with CeD displayed higher pre-HP2 and zonulin levels compared to HC. A gluten-free diet in patients with CeD led to lower serum zonulin and pre-HP2 concentrations. Conclusions Our study suggests that neither circulating zonulin nor pre-HP2 mirror colonic permeability. Our data corroborate previous reports showing the inability of the Cusabio zonulin kit to target zonulin and highlights that the results of studies using this kit must be re-examined with caution.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity

The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting thatposttranscriptional mechanisms play a key role in gutbarrier dysfunction. To our knowledge,our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.

The role of the gut microbiome in chronic liver disease: the clinical evidence revised.

SummaryRecent research has suggested a role for the intestinal microbiota in the pathogenesis and potential treatment of a wide range of liver diseases. The intestinal microbiota and bacterial products may contribute to the development of liver diseases through multiple mechanisms including increased intestinal permeability, chronic systemic inflammation, production of short-chain fatty acids and changes in metabolism. This suggests a potential role for pre-, pro- and synbiotic products in the prevention or treatment of some liver diseases. In addition, there is emerging evidence on the effects of faecal microbial transplant. Herein, we discuss the relationship between the intestinal microbiota and liver diseases, as well as reviewing intestinal microbiota-based treatment options that are currently being investigated.

Glutamine, but not Branched-Chain Amino Acids, Restores Intestinal Barrier Function during Activity-Based Anorexia.

Background: During activity-based anorexia (ABA) in mice, enhanced paracellular permeability and reduced protein synthesis have been shown in the colon while the gut–brain axis has received increasing attention in the regulation of intestinal and mood disorders that frequently occur during anorexia nervosa, a severe eating disorder for which there is no specific treatment. In the present study, we assessed the effects of oral glutamine (Gln) or branched-chain amino acids (BCAA) supplementation during ABA to target intestinal functions, body composition and feeding behavior. Methods: C57BL/6 male mice were randomized in Control (CTRL) and ABA groups. After ABA induction, mice received, or not, either 1% Gln or 2.5% BCAA (Leu, Ile, Val) for one week in drinking water. Results: Neither Gln nor BCAA supplementation affected body weight and body composition, while only Gln supplementation slightly increased food intake. ABA mice exhibited increased paracellular permeability and reduced protein synthesis in the colonic mucosa. Oral Gln restored colonic paracellular permeability and protein synthesis and increased the mucin-2 mRNA level, whereas BCAA did not affect colonic parameters. Conclusion: In conclusion, oral Gln specifically improves colonic response during ABA. These data should be further confirmed in AN patients.

A Novel Role of SLC26A3 in Maintenance of Intestinal Barrier Function

Inflammatory Bowel Disease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. The pathogenesis of IBD is still unclear due to the multifactorial nature of the disease. Compromised intestinal barrier is one of the most critical early events linked to the onset of intestinal inflammation. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) is an essential transporter for chloride absorption in the mammalian intestine and has been identified as an IBD susceptibility gene in certain cohorts. Further, DRA levels are markedly decreased in colonic mucosa of IBD patients and in mouse models of gut inflammation. Also, DRA KO mice are more susceptible to DSS colitis. However, the mechanisms underlying increased susceptibility to inflammation by loss of DRA are not fully understood.AimsTo examine the impact of loss of DRA (in DRA KO mice) on epithelial barrier function and expression of key tight junction and adherens junction proteins and elucidate underlying mechanisms.MethodsWild type and DRA KO mice (8–10 weeks old, M/F) were used. Protein expression was measured by immunoblotting and immunofluorescence. FITC‐dextran flux was examined utilizing Ussing chamber.ResultsDRA KO mice exhibited an increase in colonic paracellular permeability (~ 5 fold, p<0.05) indicating impaired barrier function. This was associated with a decrease in levels of TJ protein ZO‐1 and occludin (~ 65%, p<0.05) and major AJ protein, E‐cadherin (~ 60%, p<0.05). Also, the expression of pore forming, Claudin 2 was significantly upregulated in DRA KO mouse colon (~ 3 fold, p<0.05). No significant changes were observed in claudin 1 or 3. Since mouse intestine is a complex physiological system, we next examined if DRA silencing in a model epithelial Caco‐2 cell line, affects barrier function and tight junction protein expression. shRNA Knock‐down of DRA in Caco‐2 cells resulted in a marked decrease in TEER (~ 40%, p<0.05) and occludin expression (~ 60%, p<0.05). Similarly, enteroids derived from DRA KO mice exhibited decreased expression of occludin and E‐cadherin proteins (~ 50%, p<0.05). There was no alteration in mRNA levels of TJ/AJ proteins in DRA KO mice indicating role of post‐transcriptional mechanisms. To understand the mechanisms underlying these alterations, we next examined the expression of RNA‐binding proteins that are known to regulate the translation of occludin and E‐cadherin. Our results showed a marked increase in expression of CUGBP1 (a negative regulator of TJ/AJ proteins) and a marked decrease in expression of intact HuR (a positive regulator).ConclusionOur studies suggest a novel role of a membrane chloride transporter DRA in maintenance of intestinal barrier function and thus a down regulation of this protein may play a key role in pathogenesis of IBD.Support or Funding InformationSupported by NIDDK and Department of Veterans AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced gastrointestinal passive paracellular permeability contributes to the obesity-associated hyperoxaluria.

Most kidney stones (KS) are composed of calcium oxalate and small increases in urine oxalate enhance the stone risk. Obesity is a risk factor for KS, and urinary oxalate excretion increases with increased body size. We previously established the obese ob/ob ( ob) mice as a model (3.3-fold higher urine oxalate) to define the pathogenesis of obesity-associated hyperoxaluria (OAH). The purpose of this study was to test the hypothesis that the obesity-associated enhanced small intestinal paracellular permeability contributes to OAH by increasing passive paracellular intestinal oxalate absorption. ob Mice have significantly higher jejunal (1.6-fold) and ileal (1.4-fold) paracellular oxalate absorption ex vivo and significantly higher (5-fold) urine [13C]oxalate following oral gavage with [13C]oxalate, indicating increased intestinal oxalate absorption in vivo. The observation of higher oxalate absorption in vivo compared with ex vivo suggests the possibility of increased paracellular permeability along the entire gut. Indeed, ob mice have significantly higher fractions of the administered sucrose (1.7-fold), lactulose (4.4-fold), and sucralose (3.1-fold) excreted in the urine, reflecting increased gastric, small intestinal, and colonic paracellular permeability, respectively. The ob mice have significantly reduced gastrointestinal occludin, zonula occludens-1, and claudins-1 and -3 mRNA and total protein expression. Proinflammatory cytokines and oxidative stress, which are elevated in obesity, significantly enhanced paracellular intestinal oxalate absorption in vitro and ex vivo. We conclude that obese mice have significantly higher intestinal oxalate absorption and enhanced gastrointestinal paracellular permeability in vivo, which would likely contribute to the pathogenesis of OAH, since there is a transepithelial oxalate concentration gradient to drive paracellular intestinal oxalate absorption. NEW & NOTEWORTHY This study shows that the obese ob/ob mice have significantly increased gastrointestinal paracellular oxalate absorption and remarkably enhanced paracellular permeability along the entire gut in vivo, which are likely mediated by the obesity-associated increased systemic and intestinal inflammation and oxidative stress. A transepithelial oxalate concentration gradient driving gastrointestinal paracellular oxalate absorption exists, and therefore, our novel findings likely contribute to the hyperoxaluria observed in the ob/ob mice and hence to the pathogenesis of obesity-associated hyperoxaluria.

Lipopolysaccharide induces the early enhancement of mice colonic mucosal paracellular permeability mainly mediated by mast cells.

The alteration of intestinal mucosal barrier is considered to be the central pathophysiological process in response to gastrointestinal infections, and mucosal microstructural damage is a major factor for enhancing epithelial permeability in persistent bacterial infections. However, the mechanism involved in hyperpermeability in the early stage of acute bacterial infections is not fully understood. In the present study, fluorescein isothiocyanate-dextran across and transepithelial resistance measured in Ussing chambers were used to assess the intestinal paracellular permeability. Mast cell activation was evaluated by western blotting for the presence of tryptase released from mast cells. Serum levels of interleukin-6 were evaluated using enzyme-linked immunosorbent assay. Our results indicated that mast cells played a pivotal role in colonic mucosal hyperpermeability in wild type mice treated with lipopolysaccharide (LPS) for 2 h. And the effect of LPS was mainly dependent on mast cell degranulation, while no change in permeability was observed in the mast cell-deficient mice (Wads⁻/⁻) after LPS administration. No obvious changes of the mucosal structure including histomorphological architecture and expression of intercellular junction proteins were obtained in either wild type or Wads⁻/⁻ mice after LPS stimulation by hematoxylin and eosin staining, immunofluorescence staining and western blot analysis. Furthermore, the self-renewal of intestinal epithelia, detected by using proliferation marker 5'-bromo-2'-deoxyuridine, was not involved in increased permeability. Collectively, activation of mast cells induced by LPS mediated intestinal hyperpermeability in the initial stage, and played a crucial role in barrier dysfunction rather than mucosal microstructural damage in acute enterogenous bacterial infection.

Glutamine restores the intestinal barrier function in anorectic mice

Rationale: Mood and gastrointestinal disorders are frequently observed in patients with anorexia nervosa, while the role of gut-brain axis has been recently highlighted in food intake and behavior regulation. In the activity-based anorexia (ABA) model, an increase of colonic paracellular permeability has been reported. Thus, we aimed to evaluate the effects of glutamine (Gln), the preferential substrate for rapidly dividing cells, on intestinal barrier function in ABA mice.

Function of colonic mucosal barrier of patients with colonic slow transit constipation

Objective To explore the function of colonic mucosal barrier of patients with colonic slow transit constipation (STC).Methods From June 2008 to June 2012,a total of 136 patients with STC were enrolled.Among them,course of disease of 55 cases was between one and six years,of 43 cases was between six and 10 years,and of 38 cases was over 10 years.The colonic transit time of 66 cases was between three and five days,of 42 cases was between five and seven days,and of 28 cases was over seven days.Altogether,35 cases received subtotal resection of the colon.At same time,individuals who received partial resection of the colon because of single polypus were set as control group.Fasting blood and urine samples of all subjects were collected.The colonic specimens of STC patients who received surgery and control group were harvested.The urinary lactulose and mannitol ratio (L/M) was detected by high performance liquid chromatography (HPLC).The level of blood Dlactic acid (D-LAC) was tested by enzymatic spectrophotometric.The level of blood diamine oxidase (DAO) was determined by speetrophotometry.The level of endotoxin (ET) was detected by azo chromogenic substrate limulus test.The colonic epithelial cells membrane resistance (TER) and paracellular mannitol permeability (PMP) were measured with Ussing perfusion chamber.t-test was performed for comparison between groups.Results Urinary L/M of STC group and control group was 0.16±0.03 and 0.10±0.02,respectively.The level of blood D-LAC was (1.81±0.19) and (1.04±0.13) mmol/L.The level of blood DAO was (17.07±1.81) and (9.78±1.14) U/L.The level of blood ET was (64.20±6.85) and (51.30±5.90) EU/L.The TER of colonic epithelia cell was (61.23±7.76) and (75.87±9.65) Ω/cm2.The PMP of colonic epithelia cell was (3.17±0.35) ％ and (2.14 ±0.22)％.All the differences were statistically significant (t =3.185,3.378,3.863,3.201,3.125 and 3.543,all P＜0.05).Among patients with disease course between one and six years,six to 10 years and over 10 years,colonic transit time of STC between three and five days,five to seven days and over seven days,urinary L/M,blood D-LAC level and blood DAO level increased along with the disease course and colonic transit time and the differences were statistical significantly compared with control group (urinary L/M:t=1.993,2.311,2.356,2.204,2.347 and 3.673; blood D-LAC level:t=2.023,2.886,4.124,1.999,2.998 and 3.465; blood DAO level:t=1.994,2.995,4.423,2.203,3.673 and 5.211; all P＜0.05).Compared with control group,there were significant differences in blood ET level of course of STC between six and 10 years,over 10 years,colonic transit time of STC between five and seven days and over seven days (t=2.121,4.245,3.241 and 4.657,all P＜0.05).Conclusion The permeability of colonic mucosal barrier increased and which was more significant in longer colonic transit time and long course of disease. Key words: Constipation; Colonic diseases; Intestinal mucosa

Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation

ObjectiveTo determine the role of colonic barrier defects and low-grade inflammation in irritable bowel syndrome (IBS)-like symptoms in quiescent inflammatory bowel disease (IBD).DesignCaecal biopsies were collected from 51 IBS, 49...

Luminal Cysteine-Proteases Degrade Colonic Tight Junction Structure and Are Responsible for Abdominal Pain in Constipation-Predominant IBS

Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies. Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN. We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64. These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.

ACF7 regulates colonic permeability

Colonic paracellular permeability is regulated by various factors, including dynamics of the cytoskeleton. Recently, ACF7 has been found to play a critical role in cytoskeletal dynamics as an essential integrator. To elucidate the physiological importance of ACF7 and paracellular permeability, we conditionally knocked out ACF7 in the intestinal mucosa of mice. Histopathological findings indicated that ACF7 deficiency resulted in significant interstitial proliferation and columnar epithelial cell rearrangement. Decreased colonic paracellular permeability was detected using a Ussing chamber and the FITC-inulin method. In order to clarify the underlying mechanism, we further analyzed the expression levels of three important tight junction proteins. Downregulation of ZO-1, occludin and claudin-1 was identified. Immunofluorescence provided strong evidence that ZO-1, occludin and claudin-1 were weakly stained. We hypothesized that ACF7 regulates cytoskeleton dynamics to alter mucosal epithelial arrangement and colonic paracellular permeability.

Prevention of gut leakiness by a probiotic treatment leads to attenuated HPA response to an acute psychological stress in rats

Intestinal barrier impairment is incriminated in the pathophysiology of intestinal gut disorders associated with psychiatric comorbidity. Increased intestinal permeability associated with upload of lipopolysaccharides (LPS) translocation induces depressive symptoms. Gut microbiota and probiotics alter behavior and brain neurochemistry. Since Lactobacillus farciminis suppresses stress-induced hyperpermeability, we examined whether (i) L. farciminis affects the HPA axis stress response, (ii) stress induces changes in LPS translocation and central cytokine expression which may be reversed by L. farciminis, (iii) the prevention of "leaky" gut and LPS upload are involved in these effects. At the end of the following treatments female rats were submitted to a partial restraint stress (PRS) or sham-PRS: (i) oral administration of L. farciminis during 2 weeks, (ii) intraperitoneal administration of ML-7 (a specific myosin light chain kinase inhibitor), (iii) antibiotic administration in drinking water during 12 days. After PRS or sham-PRS session, we evaluated LPS levels in portal blood, plasma corticosterone and adrenocorticotropic hormone (ACTH) levels, hypothalamic corticotropin releasing factor (CRF) and pro-inflammatory cytokine mRNA expression, and colonic paracellular permeability (CPP). PRS increased plasma ACTH and corticosterone; hypothalamic CRF and pro-inflammatory cytokine expression; CPP and portal blood concentration of LPS. L. farciminis and ML-7 suppressed stress-induced hyperpermeability, endotoxemia and prevented HPA axis stress response and neuroinflammation. Antibiotic reduction of luminal LPS concentration prevented HPA axis stress response and increased hypothalamic expression of pro-inflammatory cytokines. The attenuation of the HPA axis response to stress by L. farciminis depends upon the prevention of intestinal barrier impairment and decrease of circulating LPS levels.

Mo1659 Perinatal Exposure to Low Doses of the Food Endocrine Disruptor Bisphenol A Impairs Oral Tolerance and Sensitization in Offspring Rats at Adulthood

Background: The food contaminant Bisphenol A (BPA) is the building plastic monomer widely used in food packaging and epoxy resins lining metal cans. BPA is a xenoestrogen known to disrupt endocrine function, liver metabolism and intestinal barrier function. We have previously shown that BPA exposure in utero and through lactation at the No Observed Adverse Effect Level (NOAEL) (5mg/kg/j) decreased colonic paracellular permeability and enhanced the TNBS-induced colitis severity in adult female offspring. Herein we aimed to address the consequences of perinatal exposure to 1000 time lower than NOAEL dose of BPA (5μg/kg/day) on the upper intestinal immune homeostasis at adulthood. Material and methods: Gravid and breast feeding rats were treated per os from the 15th day of gravidity to pups weaning (D21) with or without BPA [5μg/kg/d]. Paracellular (4kDa dextran-FITC) and transcellular (Horse Radish Peroxidase) jejunal permeability were addressed by Ussing chamber on female adult offspring aged of 45 days. In parallel, effects of BPA perinatal exposure on oral tolerance and systemic immunisation to ovalbumin (OVA) in adult offspring were tested. Anti-OVA IgG titers were measured by ELISA as well as IFNγ secretion after In Vitro stimulation of either spleen ormesenteric lymph nodes cells culture.Results: Perinatal treatment with BPA induced a decrease of jejunal transcellular permeability compared to controls (0.03±0.0045x10-7 vs 0.09±0.02 x10-7 cm/s; p<0.05) but did not affect jejunal paracellular permeability. Furthermore, BPA perinatally treated adult rats exhibited higher anti-OVA IgG titers following either an oral tolerance protocol (162±88.7x103 compared to 6.2±1.9 x103 in controls; p<0.03) or a sensitization with OVA (783±127x103 compared to 433±100 x103 in controls; p<0.03). Enhanced humoral response in perinatally BPA treated adult rats was associated with a higher production of IFNγ by spleen in sensitized rats (628.2±392 compared to 114.9±72 pg/ml in controls) and by mesenteric lymph nodes in tolerized rats (707±325 compared to 54.6±43 pg/ml in controls). Conclusion: Perinatal exposure to low doses of BPA decreased jejunal transcellular permeability and impaired oral tolerance and sensitization to dietary antigen protein in adult offsprings. These results suggest that the perinatal period is a critical window for BPA exposure that may trigger food intolerance in later life.

Altered colonic barrier function and paracellular permeability in Muc2−/− mice

A single layer of epithelial cells connected by tight junctions (TJ) and an overlying layer of Muc2‐containing mucus serve as the intestinal mucosal barrier. In this study, we investigated whether mice lacking Muc2 mucin (Muc2−/−) have a compromised epithelial barrier, increasing their susceptibility to intestinal inflammation. Full thickness colonic tissues from Muc2−/− and Wt mice were mounted in Ussing chambers to access ion transport, transepithelial resistance (TER) and mucosal to serosal flux of FITC‐dextran. TJ proteins were analyzed by Western blot and qPCR. Site‐specific and temporal permeability alterations were measured in vivo using oligosaccharide probes. Ussing chamber studies revealed Muc2−/− mice had increased colonic TER and decreased FITC‐dextran flux when compared to Wt mice, indicating increased barrier function. Of the TJ proteins tested, both barrier‐forming occludin and cation‐selective claudin‐2 protein levels were increased in Muc2−/− colons. However, in vivo oligosaccharide uptake revealed that Muc2−/− mice had increased colonic permeability. Surprisingly, colonic epithelial barrier functions were enhanced in the absence of Muc2 mucin, but in vivo measurements show alterations in colonic permeability that were not identified using ex vivo techniques. These studies suggest that Muc2 mucin confers both luminal and epithelial barrier functions. Support: CIHR, CCFC, AIHS

Spray-Dried Porcine Plasma Influences Intestinal Barrier Function, Inflammation,and Diarrhea in Weaned Pigs

AbstractThe objective of this study was to evaluate the effects of dietary inclusion levels of spray-dried porcine plasma (SDPP) on postweaning (PW) intestinal barrier function, mucosal inflammation, and clinical indices of gut health in pigs. Ex vivo Ussing chamber studies were conducted to measure Ileal and colonic barrier function in terms of transepithelial electrical resistance and paracellular flux of3H-mannitol and14C-inulin. Intestinal inflammation was assessed by histological analysis and mucosal levels of proinflammatory cytokines. Dietary inclusion of 2.5 and 5% SDPP reduced colonic paracellular permeability of14C-inulin compared with controls (0% SDPP) on d 7 PW. Both 2.5 and 5% dietary SDPP reduced ileal3H-mannitol and14C-inulin permeability on d 14 PW. The 5% SDPP diet reduced colonic short-circuit current, an index of net electrogenic ion transport, and fecal scores when measured on d 7 and 14 PW compared with the control and 2.5% SDPP groups (P< 0.05). Histological analysis revealed fewer lamina propria cells in ileum and colon from pigs fed diets containing 2.5 and 5% SDPP on d 7 and 14 PW. Levels of the proinflammatory cytokine TNFa were reduced in the colon but not ileum from pigs fed the 5% SDPP on d 7 and 14 PW compared with controls (P< 0.05). IFN? levels were lower than in controls in both of the SDPP-fed groups in the ileum and colon on d 7 but not on d 14 PW. Overall, this study demonstrated that dietary inclusion of SDPP had beneficial effects on intestinal barrier function, inflammation, and diarrhea in weaned pigs.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice

Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters. Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.