Tight Junction mRNA Expression Research Articles

Effects of embryo injected with ochratoxin A on growth performance, jejunal morphology and barrier of ducklings

ABSTRACT 1. This trial investigated the effect on embryo injected with ochratoxin A (OTA) and the growth performance, jejunal morphology and barrier of ducklings to 21 d old. 2. Two hundred forty, fertilised eggs were individually weighed and randomly assigned to two groups, a control (CON) and the OTA treatment, according to average egg weight. On d 13 of embryonic development, the treatment group was injected with 8 ng OTA/g egg and the CON group was injected with NaHCO3 solution as a placebo. All newly hatched ducklings were assigned to the CON or OTA group based on the different treatments. Each treatment consisted of six replicates and each included 10 ducklings and the experiment lasted until 21 d of age. 3. The results showed that embryos injected with OTA affected the 21 d body weight (BW) and average daily gain (ADG) of ducklings (p < 0.05). OTA exposure increased the relative weights of the liver, pancreas, gizzard, proventriculus and jejunum (p < 0.05); and decreased the relative length of the jejunum of ducklings (p < 0.05). Moreover, jejunal crypt depth increased (p < 0.05) and the villus height-to-crypt depth ratio (Vh/Cd) decreased in the OTA-injected group (p < 0.05). Compared with those in the CON group, the mRNA expression of Zonula Occludens-1; (ZO-1) (p = 0.0582) and Occludin; (p = 0.0687) in the OTA treatment group was downregulated. 4. The findings demonstrated that a single low-dose injection of OTA increased body weight and daily gain in ducklings. Moreover, embryo exposure to OTA had negative effects with increased relative weight of organs and the jejunal crypt depth, decreased relative length of the intestine and mRNA expression of tight junctions (ZO-1, Occludin).

Extracellular vesicles from PBDE-47 treated M(LPS) THP-1 macrophages modulate the expression of markers of epithelial integrity, EMT, inflammation and muco-secretion in ALI culture of airway epithelium

AimsThe lung epithelial cells form a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli. These cells interact with several other cellular components, of which macrophages are some of the most relevant. We analysed the effects of the PBDE-47 on the microRNA cargo of THP-1 macrophage like derived small Extracellular Vesicles (sEVs) and the effects on A549 lung epithelial cells. Main methodssEVs from M(LPS) THP-1 macrophage-like cells after PBDE-47 treatment (sEVsPBDE+LPS) were characterized by nanoparticle tracking analysis and their microRNA cargo studied by qPCR. Confocal microscopy was applied to study sEVs cellular uptake by A549 cells. The expression of tight junctions (TJs), adhesion molecules, inflammation markers and mucus production in A549 cultured in air liquid interface (ALI) conditions were studied by Real Time PCR and confocal microscopy. Key findingssEVsPBDE+LPS microRNA cargo analysis showed that the PBDE-47 modulated the expression of the miR-15a-5p, miR29a-3p, miR-143-3p and miR-122-5p. Furthermore, ALI cultured A549 cells incubated with sEVsPBDE+LPS showed that zonula occludens-1 (p ≤ 0.04), claudin (p ≤ 0.02), E-cadherin (p ≤ 0.006) and Vimentin (p ≤ 0.0008) mRNAs were increased in A549 cells after sEVsPBDE+LPS treatment. Indeed, Interleukin (IL)-8 (p ≤ 0.008) and mucin (MUC5AC and MUC5B) (p ≤ 0.03 and p ≤ 0.0001) mRNA expression were up- and down-regulated, respectively. SignificancePBDE-47 treated macrophages secrete sEVs with altered microRNA cargo that affect the mRNA expression of TJs, adhesion molecules, cytokines and EMT markers damaging the normal function of the lung epithelium, potentially contributing to the development of lung diseases.

Novel Phocaeicola Strain Ameliorates Dextran Sulfate Sodium-induced Colitis in Mice.

Previously, we isolated a novel Phocaeicola strain, Phocaeicola faecalis FXJYN30E22, from the feces of a healthy human from China. Metagenomic analysis revealed that the distribution of FXJYN30E22 differed in the intestinal tract of different hosts. We aimed to determine whether FXJYN30E22 protects against ulcerative colitis by employing a mouse model. In this study, dextran sulfate sodium was used to construct the UC model. The disease activity index, colon length, body weight changes, and histological scores were used as the pathological indicators to assess the anti-inflammatory effect of P. faecalis FXJYN30E22. Further, cytokine levels, tight junction mRNA expression levels, and short-chain fatty acid (SCFA) concentrations were also analyzed. Phocaeicola faecalis FXJYN30E22 could reduce the DSS-induced increase in DAI score, and enhance the colon length and body weight. Phocaeicola faecalis FXJYN30E22 could enhance TJ protein concentration and modulate the level of cytokines to reach levels close to those of the control group. FXJYN30E22 could also upregulate the concentrations of SCFA, which include acetate and butyrate. Based on the correlation analysis, four factors, including interleukin (IL)-6, IL-10, IL-1β levels, and propionate concentration, were related to the protective roles of FXJYN30E22 in UC mice to different degrees. According to an analysis of the genomic information, the potential protective effects of strain FXJYN30E22 may be associated with the secretion of SCFA by specific genes. These findings suggest that oral P. faecalis FXJYN30E22 could help maintain the epithelial barrier by regulating cytokine levels and secreting SCFA.

Dietary supplementation with fermented plant product modulates production performance, egg quality, intestinal mucosal barrier, and cecal microbiota in laying hens.

Fermented plant product (FPP) is a kind of functional complex containing probiotics and a variety of bioactive substances, which has multiple physiological functions. However, there is no systematic appraisal of FPP as a feed additive for laying hens. This study was conducted to evaluate the utilization of FPP in laying hens. A total of 120 healthy 34-week-old Xianju layers with similar body weight and egg production were randomly allocated into two dietary treatments with four replicates per treatment and 15 birds per replicate for 8 weeks. The dietary treatments included the basal diet without FPP (CON group) and CON diet supplemented with 500 mg/kg of FPP (FPP group). Compared with the CON group, the egg production and egg mass were significantly increased in the FPP group from 38 to 42 and 34 to 42 weeks of age (P < 0.05). Birds fed with the diet containing 500 mg/kg FPP had higher albumen height (P < 0.01) and Haugh unit (P < 0.05) than those of the controls. FPP supplementation significantly increased the villus height (VH) and crypt depth (CD) in the jejunum of laying hens (P < 0.01), as well as the ratio of VH to CD (P < 0.05). The mRNA expression of tight junctions showed that dietary supplementation with FPP significantly increased the expression levels of Occludin (P < 0.01) and ZO-1 (P < 0.05) in jejunum of hens compared to the control group. In addition, dietary supplementation with FPP influenced cecal microbiota of laying hens, which was characterized by the changes in the microbial community composition, including the increased abundances of Firmicutes, Faecalibacterium, Oscillospira, Clostridium, Ruminococcus, and Coprococcus, along with the decreased abundance of Bacteroidetes, Proteobacteria, Phascolarctobacterium, Odoribacter, Desulfovibrio, and Mucispirillum. Spearman's correlation analysis revealed that bacteria such as Faecalibacterium, Ruminococcus, Coprococcus, and Blautia were significantly and positively correlated with the intestinal barrier markers (P < 0.05), with extremely significant correlations between Ruminococcus and ZO-1, and Coprococcus and Occludin (P < 0.01), whereas Desulfovibrio had a negative correlation with the expression of Occludin (P < 0.05). As it can be concluded, FPP supplementation increased the egg production, egg mass, albumen height, and Haugh unit of laying hens, and improved intestinal health by ameliorating intestinal barrier function, which may be partially attributed to the regulation of cecal microbiota. Our findings suggest that FPP has the potential to be used as a feed additive to promote the performance of layers.

Chlorine exposure and intensive exercise induces airway hyperreactivity in a 3-week murine exercise model

RationaleExercise-induced bronchoconstriction (EIB) is defined as acute narrowing of the airways during or immediately after exercise. EIB has a high prevalence in elite swimmers probably due to the high ventilation rate and exposure to the chlorine by-products. It is still puzzling which pathophysiological mechanisms drive EIB. ObjectiveIn this study, we evaluated airway hyperreactivity, permeability, integrity and inflammation in a murine swimmers EIB model with and without chlorine exposure. MethodsMice performed a 3-week swimming protocol in a swimming pool with counter current. Three hours after the last swimming session, airway hyperreactivity to methacholine was assessed. Cytokine levels and cellular differential analysis was performed in BAL fluid. Airway permeability and tight junction expression was measured in serum and lung tissue. T-, B-, dendritic and innate lymphoid cells were determined in lung tissue via flow cytometry. ResultsA significant higher airway resistance (Rn; P < 0.0001) was observed in mice swimming in chlorinated water (mean Rn = 1.26 cmH2O.s/ml) compared to mice swimming in tap water (mean Rn = 0.76 cmH2O.s/ml) and both inhalation groups in the absence of cellular inflammation. No significant differences were found in lung immune cell populations or in lung tight junction mRNA expression. Experiments in SCID, Rag2−/−γc−/− or Cpa3cre/+ mice showed a limited involvement of the innate, adaptive immune system or the mast cells. ConclusionOur 3-week swimming murine model mimics intensive swimming in chlorinated water with the presence of airway hyperreactivity in mice swimming in chlorinated water in the absence of airway inflammation and airway epithelial damage.

Paracellular intestinal permeability of chickens induced by DON and/or C. jejuni is associated with alterations in tight junction mRNA expression

Toxins, antigens, and harmful pathogens continuously challenge the intestinal mucosa. Therefore, regulation of the intestinal barrier is crucial for the maintenance of mucosal homeostasis and gut health. Intercellular complexes, namely, tight junctions (TJs), regulate paracellular permeability. TJs are mainly composed of claudins (CLDN), occludin (OCLN), tight junction associated MARVEL-domain proteins (TAMPS), the scaffolding zonula occludens (ZO) proteins and junction-adhesion molecules (JAMs). Different studies have shown that a Campylobacter infection can lead to a phenomenon so-called “leaky gut”, including the translocation of luminal bacteria to the underlying tissue and internal organs. Based on the effects of C. jejuni on the chicken gut, we hypothesize that impacts on TJ proteins play a crucial role in the destructive effects of the intestinal barrier. Likewise, the mycotoxin deoxynivalenol (DON) can also alter gut permeability in chickens. Albeit DON and C. jejuni are widely distributed, no data are available on their effect on the tight junctions’ barrier in the broiler intestine and consequences for permeability. Therefore, the aim of this study was to analyze the interaction between DON and C. jejuni on the gut barrier by linking permeability with gene expression of TJ proteins and to determine the relationships between the measurements. Following oral infection of birds with C. jejuni NCTC 12744 at 14 days of age, we demonstrate that the co-exposure with DON has considerable consequences on gut permeability as well as on gut TJ mRNA expression. Co-exposure of DON and C. jejuni enhanced the negative effect on paracellular permeability of the intestine, which was also noticed for the bacteria or the mycotoxin alone by the Ussing chamber technique at certain time points in both jejunum and caecum. Furthermore, the increased paracellular permeability was associated with significant changes in TJ mRNA expression in the small and large intestine. The actual study demonstrates that co-exposure of broiler chickens to DON and C. jejuni resulted in a decreased barrier function via up-regulation of pore-forming tight junctions (CLDN7 and CLDN10), as well as the cytosolic TJ protein occludin (OCLN) that can shift to various paracellular locations and are therefore able to alter the epithelial permeability. These findings indicate that the co-exposure of broiler chickens to DON and C. jejuni affects the paracellular permeability of the gut by altering the tight junction proteins. Furthermore, analysing of correlations between TJs revealed that the mRNA expression levels of most tight junctions were correlated with each other in both jejunum and caecum. Finally, the findings indicate that the molecular composition of tight junctions can be used as a marker for gut health and integrity.

Differential effects of intense exercise and pollution on the airways in a murine model

BackgroundExercise-induced bronchoconstriction (EIB) is a transient airway narrowing, occurring during or shortly after intensive exercise. It is highly prevalent in non-asthmatic outdoor endurance athletes suggesting an important contribution of air pollution in the development of EIB. Therefore, more research is necessary to investigate the combination of exercise and pollutants on the airways.MethodsBalbc/ByJ mice were intranasally challenged 5 days a week for 3 weeks with saline or 0.2 mg/ml diesel exhaust particles (DEP), prior to a daily incremental running session or non-exercise session. Once a week, the early ventilatory response was measured and lung function was determined at day 24. Airway inflammation and cytokine levels were evaluated in bronchoalveolar lavage fluid. Furthermore, innate lymphoid cells, dendritic cells and tight junction mRNA expression were determined in lung tissue.ResultsSubmaximal exercise resulted in acute alterations of the breathing pattern and significantly improved FEV0.1 at day 24. DEP exposure induced neutrophilic airway inflammation, accompanied with increased percentages of CD11b+ DC in lung tissue and pro-inflammatory cytokines, such as IL-13, MCP-1, GM-CSF and KC. Occludin and claudin-1(Cldn-1) expression were respectively increased and decreased by DEP exposure. Whereas, exercise increased Cldn-3 and Cldn-18 expression. Combining exercise and DEP exposure resulted in significantly increased SP-D levels in the airways.ConclusionDEP exposure induced typical airway neutrophilia, DC recruitment and pro-inflammatory cytokine production. Whereas, intensive exercise induced changes of the breathing pattern. The combination of both triggers resulted in a dysregulation of tight junction expression, suggesting that intensive exercise in polluted environments can induce important changes in the airway physiology and integrity.

Sodium propionate protect the blood-milk barrier integrity, relieve lipopolysaccharide-induced inflammatory injury and cells apoptosis

AimsSodium propionate (SP) has been reported to possess an anti-inflammatory and anti-apoptotic potential by inhibiting certain signaling pathways and helps in reducing the pathological damages of the mammary gland. However, the effects of sodium propionate on attenuating Lipopolysaccharide (LPS)-induced inflammatory condition and cell damage in bovine mammary epithelial cells (bMECs) are not comprehensively studied yet. Therefore, the aim of the current investigation was to evaluate the protective effects of sodium propionate on LPS-induced inflammatory conditions and to clarify the possible underlying molecular mechanism in bMECs. Main methodsThe effects of increasing doses of SP on LPS-induced inflammation, oxidative stress and apoptosis was studied in vitro. Furthermore, the underlying protective mechanisms of SP on LPS-stimulated bMECs was investigated under different experimental conditions. Key findingsThe results reveled that increased inflammatory cytokines, chemokines and those of tight junction's mRNA expression was significantly attenuated dose-dependently by propionate. Biochemical analysis revealed that propionate pretreatment modulated the LPS-induced intercellular reactive oxygen species (ROS) accumulation, oxidative and antioxidant factors and apoptosis rate. Furthermore, we investigated that the LPS activated nuclear factor-kB (NF-kB), caspase/Bax apoptotic pathways and Histone deacetylases (HDAC) was significantly attenuated by propionate in bMECs. SignificanceOur results suggest that sodium propionate is a potent agent for ameliorating LPS-mediated cellular disruption and limiting detrimental inflammatory responses, partly via maintaining blood milk barrier integrity, inhibiting HDAC activity and NF-kB signaling pathway.

The Laetiporus sulphureus Fermented Product Enhances the Antioxidant Status, Intestinal Tight Junction, and Morphology of Broiler Chickens.

Simple SummaryThis study investigated the effects of the Laetiporus sulphureus fermented product (FL) as a feed supplement on antioxidant activities, intestinal Tight Junction (TJ) mRNA expression, and the intestinal morphology of broiler chickens. FL supplementation could potentially enhance the feed conversion ratio in broilers by improving their antioxidative status, TJ mRNA expression, and intestinal morphology. Broilers supplemented with 5% FL exhibited the best overall results on improving antioxidant status, TJ mRNA expression, and intestinal morphology.The Laetiporus sp. is a fungal species that is traditionally used for medicinal purposes. This study investigated the effects of the Laetiporus sulphureus fermented product (FL) as a feed supplementation on the antioxidant activities, the intestinal Tight Junction (TJ) mRNA expression, and the intestinal morphology of broiler chickens. Four-hundred one-day-old male broilers (Ross 308) were randomly allocated to five experimental diets: (1) a corn-soybean meal basal diet (control), (2) a basal diet replaced with 5% Wheat Bran (5% WB), (3) a basal diet replaced with 10% WB (10% WB), (4) a basal diet replaced with 5% FL (5% FL), and (5) a basal diet replaced with 10% FL (10% FL). The FL-supplemented groups exhibited a better feed conversion ratio in the overall experimental period compared to the WB and control groups. The serum antioxidant profiles of 35-day-old broilers showed that, compared to the control and 10% WB groups, the 5% FL supplementation group had a significantly increased superoxide dismutase activity, while it down-regulated the concentration of malondialdehyde in the serum (p < 0.05). The assessment of selected antioxidant gene expression showed that the 5% FL group significantly elevated heme oxygenase-1 and nuclear factor erythroid 2–related factor 2 expression, compared to the control and WB groups (p < 0.05). Furthermore, both of the FL supplemented groups had a significantly higher expression of glutathione peroxidase and catalase, compared to that of the WB and control groups in the jejunum (p < 0.05). The TJ mRNA expression in the jejunum showed that 5% FL significantly elevated the zonula occludens-1, claudin-1, and mucin-2 expression (p < 0.05), while 5% and 10% FL supplementation significantly improved OCLN expression in both the jejunum and ileum, compared to control group (p < 0.05). The intestinal morphology of 35-day-old broilers showed that a 5% FL supplementation significantly increased the villus height in the ileum and jejunum, compared to the WB and control groups (p < 0.05). Moreover, the 5% and 10% FL supplementation groups had a significantly higher villi:crypt ratio in the ileum, compared to the WB and control groups (p < 0.05). To conclude, FL supplementation improved the antioxidative status, the TJ mRNA expression, and the intestinal morphology, and it was accompanied by a lowered feed conversion ratio in broilers. Finally, 5% supplementation had the overall best results in improving the antioxidant status, TJ mRNA expression, and intestinal morphology of broilers.

Effects of Saccharomyces cerevisiae and phytase co-fermentation of wheat bran on growth, antioxidation, immunity and intestinal morphology in broilers

ObjectiveThe aim of this study was to investigate the effects of different amounts of wheat bran (WB) inclusion and postbiotics form by Saccharomyces cerevisiae and phytase co-fermented wheat bran (FWB) on the growth performance and health status of broilers.MethodsStudy randomly allocated a total of 300 male broilers to a control and 4 treatment groups (5% WB, 5% FWB, 10% WB, and 10% FWB inclusion, respectively) with each pen having 20 broilers and 3 pens per treatment.ResultsThe WB does not contain enzymes, but there are 152.8, 549.2, 289.5, and 147.1 U/g dry matter xylanase, protease, cellulase and β-glucanase in FWB, respectively. Furthermore, FWB can decrease nitric oxide release of lipopolysaccharide stimulated chicken peripheral blood mononuclear cells by about two times. Results show that 10% FWB inclusion had significantly the highest weight gain (WG) at 1 to 21 d; 5% FWB had the lowest feed conversion rate at 22 to 35 d; 10% WB and 10% FWB inclusion have the highest villus height and Lactobacillus spp. number in caecum; and both 5% and 10% FWB can increase ash content in femurs. Compared to control group, all treatments increase mucin 2, and tight junction (TJ), such as occludin, claudin-1, zonula occludens-1, and mRNA expression in ileum by at least 5 folds. In chicken peripheral blood mononuclear cells, nicotinamide adenine dinucleotide phosphate-oxidase-1 mRNA expression decreases from 2 to 5 times, and glutamate-cysteine ligase catalytic subunit mRNA expression also increases in all treatment groups compared to control group. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 (IL-6), nuclear factor-κB, and IL-1β, decreases in 5% and 10% FWB groups compared to control group.ConclusionTo summarize, both WB and FWB inclusion in broilers diets increase TJ mRNA expression and anti-oxidation and anti-inflammation, but up to 10% FWB groups have better WG in different stages of broiler development.

Nature-Identical Compounds and Organic Acids Ameliorate and Prevent the Damages Induced by an Inflammatory Challenge in Caco-2 Cell Culture.

Bioactive compounds, such as organic acids (OA) and nature-identical compounds (NIC), can exert a role in the protection of intestinal mucosa functionality due to their biological properties. The aim of this study was to understand the role of 2 OA (citric and sorbic acid) and 2 NIC (thymol and vanillin), alone or combined in a blend (OA + NIC), on intestinal barrier functionality, either during homeostatic condition or during an inflammatory challenge performed with pro-inflammatory cytokines and lipopolysaccharides (LPS). The study was performed on the human epithelial cell line Caco-2, a well-known model of the intestinal epithelial barrier. The results showed how OA and NIC alone can improve transepithelial electrical resistance (TEER) and mRNA levels of tight junction (TJ) components, but OA + NIC showed stronger efficacy compared to the single molecules. When an inflammatory challenge occurred, OA + NIC blend was able both to ameliorate, and prevent, damage caused by the pro-inflammatory stimulus, reducing or preventing the drop in TEER and improving the TJ mRNA expression. The data support the role of OA + NIC in modulating gut barrier functionality and reducing the negative effects of inflammation in intestinal epithelial cells, thereby supporting the gut barrier functionality.

Dietary corn resistant starch regulates intestinal morphology and barrier functions by activating the Notch signaling pathway of broilers.

ObjectiveThis study was conducted to investigate the effects of dietary corn resistant starch (RS) on the intestinal morphology and barrier functions of broilers.MethodsA total of 320 one-day-old broilers were randomly allocated to 5 dietary treatments: one normal corn–soybean (NC) diet, one corn–soybean–based diet supplementation with 20% corn starch (CS), and 3 corn–soybean–based diets supplementation with 4%, 8%, and 12% corn resistant starch (RS) (identified as 4% RS, 8% RS, and 12% RS, respectively). Each group had eight replicates with eight broilers per replicate. After 21 days feeding, one bird with a body weight (BW) close to the average BW of their replicate was selected and slaughtered. The samples of duodenum, jejunum, ileum, caecum digesta, and blood were collected.ResultsBirds fed 4% RS, 8% RS and 12% RS diets showed lower feed intake, BW gain, jejunal villus height (VH), duodenal crypt depth (CD), jejunal VH/CD ratio, duodenal goblet cell density as well as mucin1 mRNA expressions compared to the NC group, but showed higher concentrations of cecal acetic acid and butyric acid, percentage of jejunal proliferating cell nuclear antigen-positive cells and delta like canonical Notch ligand 4 (Dll4), and hes family bHLH transcription factor 1 mRNA expressions. However, there were no differences on the plasma diamine oxidase activity and D-lactic acid concentration among all groups.ConclusionThese findings suggested that RS could suppress intestinal morphology and barrier functions by activating Notch pathway and inhibiting the development of goblet cells, resulting in decreased mucins and tight junction mRNA expression.

Necrotic Enteritis in Broiler Chickens: The Role of Tight Junctions and Mucosal Immune Responses in Alleviating the Effect of the Disease.

Necrotic enteritis (NE) continues to present major challenges to the poultry industry, and the etiologic agent Clostridium perfringens is the fourth leading cause of bacterially-induced food- borne illnesses in the US. This study was designed to evaluate the effects of a probiotic during naturally occurring NE. On day of hatch, 1080 Cobb 500 male broilers were randomly allocated to three groups (12 replicate pens/treatment, 30 birds/pen) including (1) negative control (NC): corn-soybean meal diet; (2) positive control (PC): NC + 20 mg virginiamycin/kg diet (0.450 kg Stafac®20/ton); and (3) NC + PrimaLac (1.36 and 0.91 kg/ton from 1–21 and 22–42 days, respectively). One day (d) post placement, all birds were challenged by a commercial live oocyst coccidia vaccine as a predisposing factor to NE. Body weight and feed intake were measured at the onset of NE (d 8) and end of each feeding phase. On d 8, small intestines of two birds/pen were examined for NE lesions, and jejunum samples from one bird were collected for mRNA gene expression analysis of tight junction proteins, cytokines, and nutrient transporters. Data were analyzed using the JMP software and significance between treatments identified by LSD (p < 0.05). Compared to NC, supplementation of probiotic reduced d 1–42 mortality; however, PC was the only group with significantly lower mortality. Despite significantly improved feed conversion ratio (FCR) in PC and probiotic groups during d 1–42, average daily gain was only higher in PC (77.69 g/bird) compared with NC (74.99 g/bird). Furthermore, probiotic and PC groups had significantly reduced lesion scores in the duodenum and jejunum compared to NC. Expression of claudin-3 was higher, while expression of zonula occluden-2 tended (p = 0.06) to be higher in probiotic-supplemented birds compared to NC. Moreover, birds fed the probiotic diet had significantly higher expression of IL-10, IL-17, AMPK-α1, and SGLT1 mRNA compared to NC birds. The expression of PepT1 was higher for the probiotic-supplemented group compared to PC. IFN-γ expression was lower in PC compared to NC, while there was no difference between probiotic and NC. There were no differences in gene expression of sIgA, TNF-α, IL-1β, and IL-22 among treatments. Collectively, these data indicate that in a naturally occurring NE model, supplementation of a probiotic helps to improve FCR and reduce lesions, potentially due to the improvements in mRNA expression of tight junctions, cytokines, and nutrient transporters.

72 Use of aspirin to intentionally induce gastrointestinal tract barrier dysfunction in feedlot cattle

Abstract Stress negatively effects gastrointestinal tract (GIT) barrier function, resulting in compromised animal health. The objective of this study was to evaluate the effectiveness of aspirin to intentionally induce GIT barrier dysfunction in beef cattle. In experiment 1, sixteen crossbred heifers (425 ± 8.6 kg) were enrolled in 2 experimental periods and allotted by BW to 0, 50, 100, or 200 mg/kg BW aspirin. Four heifers per treatment received the same aspirin dose during each period, which were separated by 4 wks. Heifers were fed a 49.4% corn silage, 50.6% concentrate diet. Aspirin was delivered to animals as an oral bolus. The 200 aspirin treatment was dosed as 100 mg/kg BW aspirin 36 and 24 h prior to Cr-EDTA dosing (1 L; 180 mM). The 50 and 100 aspirin treatments were dosed 24 h prior to Cr-EDTA dosing. Urine was collected every 3 h for 48 h and analyzed for Cr using atomic absorption spectrometry. Serum was collected at 0, 24, and 48 h and analyzed for lipopolysaccharide binding protein (LBP) and interleukin-6 (IL-6) using ELISA. In experiment 2, sixteen crossbred steers (576 ± 14.2 kg) fed the same diet were allotted by BW to the 0 and 200 mg/kg BW aspirin treatments (8 steers/treatment) and were slaughtered 24 h after the last dose. Jejunal tissues were collected and tight junction mRNA expression was determined. Data were analyzed using the MIXED procedure of SAS. Aspirin linearly increased Cr absorption (P = 0.02) and elimination (P = 0.04) rate and linearly decreased mean retention time of Cr (P = 0.02). Aspirin tended to increase jejunal claudin-1 mRNA expression (P = 0.10) but did not affect serum IL-6 or LBP or expression of other jejunal tight junction mRNA (P ≥ 0.20). This study indicates that aspirin disrupts GIT barrier function in beef cattle and has potential as a model in GIT permeability research.

Histamine and T helper cytokine–driven epithelial barrier dysfunction in allergic rhinitis

Allergic rhinitis (AR) is characterized by mucosal inflammation, driven by activated immune cells. Mast cells and TH2 cells might decrease epithelial barrier integrity in AR, maintaining a leaky epithelial barrier. We sought to investigate the role of histamine and TH2 cells in driving epithelial barrier dysfunction in AR. Air-liquid interface cultures of primary nasal epithelial cells were used to measure transepithelial electrical resistance, paracellular flux of fluorescein isothiocyanate-dextran 4kDa, and mRNA expression of tight junctions. Nasal secretions were collected from healthy control subjects, AR patients, and idiopathic rhinitis patients and were tested invitro. In addition, the effect of activated TH1 and TH2 cells, mast cells, and neurons was tested invitro. The effect of IL-4, IL-13, IFN-γ, and TNF-α on mucosal permeability was tested invivo. Histamine as well as nasal secretions of AR but not idiopathic rhinitis patients rapidly decreased epithelial barrier integrity invitro. Pretreatment with histamine receptor-1 antagonist, azelastine prevented the early effect of nasal secretions of AR patients on epithelial integrity. Supernatant of activated TH1 and TH2 cells impaired epithelial integrity, while treatment with anti-TNF-α or anti-IL-4Rα monoclonal antibodies restored the TH1- and TH2-induced epithelial barrier dysfunction, respectively. IL-4, IFN-γ, and TNF-α enhanced mucosal permeability in mice. Antagonizing IL-4 prevented mucosal barrier disruption and tight junction downregulation in a mouse model of house dust mite allergic airway inflammation. Our data indicate a key role for allergic inflammatory mediators in modulating nasal epithelial barrier integrity in the pathophysiology in AR.

Respiratory syncytial virus infection influences tight junction integrity.

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air-liquid interface (ALI) and infected with RSV and ultraviolet (UV)-irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV-irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post-infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down-regulated claudin-1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin-1 and claudin-7 at protein level.

Jian carp (Cyprinus carpio var. Jian) intestinal immune responses, antioxidant status and tight junction protein mRNA expression are modulated via Nrf2 and PKC in response to dietary arginine deficiency.

This study investigated the effect of dietary arginine on the immune response, antioxidant status and tight junction mRNA expression in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with an average initial weight of 6.33±0.03g were fed graded levels of arginine (9.8-24.5gkg(-1) diet) for nine weeks. The study showed that arginine deficiency up-regulated interleukin 1, interleukin 8 and transforming growth factor-β and down-regulated tumour necrosis factor α gene expression (P<0.05). Additionally, arginine deficiency increased malondialdehyde (MDA), protein carbonyl (PC) and glutathione contents and decreased the activities of copper/zinc superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR) and glutathione-S-transferase (GST) (P<0.05). Meanwhile, arginine deficiency significantly increased claudin 7, occludin, protein kinase C, NF-E2-related factor 2 and Kelch-like-ECH- associated protein 1 mRNA expression and decreased SOD1, CAT and GR mRNA expression (P<0.05). All of these results indicated that arginine deficiency impaired intestinal immune function via the regulation of mRNA expression of cytokines, tight junction proteins, antioxidant enzymes, Nrf2/Keap1 and PKC in fish intestine.

Copper-induced tight junction mRNA expression changes, apoptosis and antioxidant responses via NF-κB, TOR and Nrf2 signaling molecules in the gills of fish: Preventive role of arginine

This study explored the possible preventive effects of dietary arginine on copper (Cu)-induced tight junction mRNA expression changes, apoptosis and antioxidant responses in the gills of young grass carp (Ctenopharyngodon idella). The results indicated that exposure to 0.7mg/L (11.01μmol/L) Cu for 96h induced the production of reactive oxygen species (ROS), thereby increasing protein oxidation, lipid peroxidation and DNA damage in the gills of fish. However, these oxidative effects were prevented by arginine supplementation. Arginine also prevented the toxic effects of Cu on the activities of copper/zinc superoxide dismutase (SOD1), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) and the glutathione (GSH) content (P<0.05). However, Cu induced an adaptive increase in the activity of catalase (CAT), and arginine supplementation further increased CAT activity (P<0.05). Moreover, Cu induced increases in the relative mRNA expressions of SOD1, CAT, GPx, GST, caspase-3, caspase-9, NF-E2-related factor 2 (Nrf2), Kelch-like-ECH-associated protein 1a (Keap1a), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), transforming growth factor-β (TGF-β) and nuclear transcription factor-κB p65 (NF-κB p65) in the gills of grass carp (P<0.05). In contrast, the relative mRNA expression levels of occludin, zonula occludens-1 (ZO-1), claudin b, claudin 3, claudin 12, target of rapamycin (TOR) and inhibitor factor κBα (IκBα) in the gills were decreased by Cu (P<0.05). However, pre-treatment of fish with arginine prevented Cu-induced relative mRNA expression decrease. Interestingly, Cu exposure resulted in increases in claudin 15a mRNA expression (P<0.05) but could not induce claudin c, caspase-8 and interleukin-10 (IL-10) mRNA expression changes in the gill of fish (P>0.05). These results indicated that Cu exposure induced apoptosis and antioxidant system and tight junction mRNA changes in the fish gills, which could be completely blocked by dietary arginine pre-supplementation.

Association of HLA-DQ gene with bowel transit, barrier function, and inflammation in irritable bowel syndrome with diarrhea

Patients with irritable bowel syndrome (IBS) with diarrhea (IBS-D) carrying human leukocyte antigen (HLA)-DQ2/8 genotypes benefit from gluten withdrawal. Our objective was to compare gastrointestinal barrier function, mucosal inflammation, and transit in nonceliac IBS-D patients and assess association with HLA-DQ2/8 status. In 45 IBS-D patients who were naive to prior exclusion of dietary gluten, we measured small bowel (SB) and colonic mucosal permeability by cumulative urinary lactulose and mannitol excretion (0-2 h for SB and 8-24 h for colon), inflammation on duodenal and rectosigmoid mucosal biopsies (obtained in 28 of 45 patients), tight junction (TJ) protein mRNA and protein expression in SB and rectosigmoid mucosa, and gastrointestinal and colonic transit by validated scintigraphy. SB mucosal biopsies were stained with hematoxylin-eosin to assess villi and intraepithelial lymphocytes, and immunohistochemistry was used to assess CD3, CD8, tryptase, and zonula occludens 1 (ZO-1); colonic biopsy intraepithelial lymphocytes were quantitated. Associations of HLA-DQ were assessed using Wilcoxon's rank-sum test. Relative to healthy control data, we observed a significant increase in SB permeability (P < 0.001), a borderline increase in colonic permeability (P = 0.10), and a decrease in TJ mRNA expression in rectosigmoid mucosa in IBS-D. In HLA-DQ2/8-positive patients, ZO-1 protein expression in the rectosigmoid mucosa was reduced compared with that in HLA-DQ2/8-negative patients and colonic transit was slower than in HLA-DQ2/8-negative patients. No other associations with HLA genotype were identified. There is abnormal barrier function (increased SB permeability and reduced mRNA expression of TJ proteins) in IBS-D relative to health that may be, in part, related to immunogenotype, given reduced ZO-1 protein expression in rectosigmoid mucosa in HLA-DQ2/8-positive relative to HLA-DQ2/8-negative patients.

Blockade of AT1 Receptors Protects the Blood-Brain Barrier and Improves Cognition in Dahl Salt-Sensitive Hypertensive Rats

The present study tested the hypothesis that inappropriate activation of the brain renin-angiotensin system (RAS) contributes to the pathogenesis of blood-brain barrier (BBB) disruption and cognitive impairment during development of salt-dependent hypertension. Effects of an angiotensin II (AngII) type-1 receptor blocker (ARB), at a dose that did not reduce blood pressure, were also examined. Dahl salt-sensitive (DSS) rats at 6 weeks of age were assigned to three groups: low-salt diet (DSS/L; 0.3% NaCl), high-salt diet (DSS/H; 8% NaCl), and high-salt diet treated with ARB, olmesartan at 1 mg/kg. DSS/H rats exhibited hypertension, leakage from brain microvessels in the hippocampus, and impaired cognitive functions, which were associated with increased brain AngII levels, as well as decreased mRNA levels of tight junctions (TJs) and collagen-IV in the hippocampus. In DSS/H rats, olmesartan treatment, at a dose that did not alter blood pressure, restored the cognitive decline, and ameliorated leakage from brain microvessels. Olmesartan also decreased brain AngII levels and restored mRNA expression of TJs and collagen-IV in DSS/H rats. These results suggest that during development of salt-dependent hypertension, activation of the brain RAS contributes to BBB disruption and cognitive impairment. Treatment with an ARB could elicit neuroprotective effects in cognitive disorders by preventing BBB permeability, which is independent of blood pressure changes.