Gut Mucus Research Articles

Impact of pesticide monocrotophos on microbial populations and histology of intestine in the Indian earthworm Lampito mauritii (Kinberg)

Soil contamination has enlarged over the decades due to intensive use of pesticides and chemical fertilizers in agronomy. Earthworms are significant organisms in the soil community. Earthworms are the major role in soil fertility in most ecological system and the production of biogenic structures. Moreover, earthworm gut mucus enhances the beneficial soil microorganism potential biological activities. They are used as model organisms for assessing the ecological risks of chemicals. Enrichment of essential nutrients in soil through earthworm is a cost-effective and eco-friendly approach. In India, the organophosphorus pesticide monocrotophos is commonly used to control agricultural pests. Hence, it is important to study the effect of monocrotophos on the gut microbiota in Lampito mauritii. A 15-day exposure to a low (1/10th of the LC50 after 96 h i.e., 0.093 ppm kg−1) and high sublethal concentration (1/3rd of the LC50 after 96 h i.e., 0.311 ppm kg−1) of monocrotophos led to reduced proliferation of the gut microbiota in L. mauritii. However, exposure for 30 days led to a recuperation of the microbial populations to near control values. Among the eight bacterial and five fungal species that inhabit the gut of L. mauritii, only six bacterial and three fungal species were able to survive after exposure to monocrotophos. In addition to the study, histopathological changes were observed in the intestine of L.mauritii after application of lower sublethal concentration of monocrotophos. Severe pathological changes such as vacuolization, degenerated nuclei, damaged villi and congestion of the blood sinuses were noticed in the intestine on 1st and, 5th day of the experiment. But in 30th day the damages were slowly recovered due to degradation of monocrotophos by the presence of some pesticides degrading bacterial and fungal species and regenerative capability of chloragogen cells in the intestine. The results suggested that reduced microbial populations and pathological damages in intestine were observed during the application of monocrotophos. So, the monocrotophos have several harmful impacts on earthworms.

Dietary Protein Sources Differentially Affect the Growth of Akkermansia muciniphila and Maintenance of the Gut Mucus Barrier in Mice.

The gut microbiota plays an essential role in linking diet to host health. The specific role of different dietary proteins on the gut microbiota and health is less understood. Here, the impact of proteins derived from chicken and soy on the gut microbiota and host gut barrier in C57BL/6 mice is investigated. Specific-pathogen-free and germ-free mice are assigned to either a chicken- or a soy protein-based diet for 4 weeks. Compared with a chicken-protein-based diet, intake of a soy-protein-based diet reduces the abundance of A. muciniphila and the number of goblet cells, lowers the level of Muc2 mRNA, and decreases the thickness of the mucus layer in the colon of specific-pathogen-free mice. In germ-free mice, colonization with A. muciniphila combined with intake of a chicken-protein-based diet results in a higher expression of the Muc2 mRNA in colon, and surprisingly, an increased potential for oxidative phosphorylation in A. muciniphila compared with colonized mice fed a soy-protein-based diet. These findings suggest possible mutually beneficial interactions between the growth and function of A. muciniphila and host mucus barrier in response to intake of a chicken-protein-based diet contrasting the intake of a soy-protein-based diet.

ADHESION OF PATHOGENIC BACTERIA TO POLYSTYRENE, SKIN AND GUT MUCUS OF GILTHEAD SEABREAM, INFECTIOUS CAPACITY AND ANTIBIOTICS SUSCEPTIBILITY

Linking proprieties of adhesion, infectious capacities and antibiotic resistance of pathogen bacteria could help to treat fish diseases. Adhesions of ten fish pathogenic bacteria were tested in microtiter plates vacant, coated with skin or gut mucus, fixed with methanol, stained with 2% crystal-violet and revealed by colorimetric method. Infectious capacity was performed by exposing gilthead seabream fibroblast cell line (SAF-1) to 107-108 CFUmL-1 of pathogen bacteria. Cell viability was measured 3h, 9h and 20 hours post-infection. The sensitivity to antibiotics was executed by disk diffusion. Data showed that all the bacteria tested adhere to polystyrene. For skin mucus, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta, and Aeromonas bivalvium were moderately adherent. Dietzia maris was strongly adherent. For gut mucus, 60% of tested bacteria were weakly adherent and 40% were non adherent. For infection, D. maris, V. harveyi and A. bivalvium decreased the cells viability to 89% after only 3h. After 20h, the viability percentage ranged between 1% and 32%. All isolates presented resistance to 1000 mg ml-1 of sulphonamide, 60% were resistant to sulfonamide and penicillin G. Present findings could be relevant in fish aquaculture and underscore the importance of the linkage between adhesion, infectious capacity, and antibiotic susceptibility of pathogen bacteria to avoid fish diseases.

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)

BackgroundHirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.ResultsGlycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 109 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 104–105 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 102–103L. lactis cells could still be recovered.ConclusionsHIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

Amazon fish bacterial communities show structural convergence along widespread hydrochemical gradients.

The world's richest freshwater fish community thrives in gradients of contrasting environments in Amazonia, ranging from ion-poor acidic black waters, to ion-rich circumneutral white waters. These hydrochemical gradients structure Amazonian fish assemblages via ecological speciation events. Fish bacterial communities contain an important genetic heritage essential for their hosts' survival and are also involved in adaptive divergence via niche adaptation processes, but the extent to which they evolve in response to hydrochemical gradients in Amazonia is unknown. Here we investigated bacterial communities (gut and skin mucus) of two ecologically and phylogenetically divergent host species (Mesonauta festivus and Serrasalmus rhombeus) distributed throughout these hydrochemical gradients. The goal was to characterize intra- and interspecific Amazonian fish microbiome variations across multiple scales. Using a 16S metabarcoding approach, we investigated the microbiota of 43 wild M.festivus, 32 S.rhombeus and seven water samples, collected at seven sampling sites encompassing both water colours. Taxonomical structures of bacterial communities from both host species were significantly correlated to the environmental continua of magnesium, sodium, dissolved organic carbon, calcium, dissolved O2 , pH, potassium, hardness and chloride. Analysis of discriminating features in community structures across multiple scales demonstrated intra- and interspecific structural parallelisms in the response to the hydrochemical gradients. Together, these parallelisms suggest the action of selection on bacterial community structures along Amazonian hydrochemical gradients. Functional approaches along with reciprocal transplant experiments will provide further insights on the potential contribution of Amazonian fish microbiomes in host adaptation and ecological speciation events.

Administration of antibiotics can cause dysbiosis in fish gut

Compositions of gastrointestinal microorganisms are known to vary depending on surrounding environment, health status and diet. However, comprehensive analysis of composition and diversity of gut microbiota of fish after treatment with antibiotics has been limited. In this study, therefore, effects of oxytetracycline (OTC) or amoxicillin (AMX) administration on intestinal microbiota of olive flounder, Paralichthys olivaceus, were investigated. Fish were administered with AMX and OTC by oral (80 mg/kg of BW/day for 10 days) or intramuscular injection (12.5 mg/kg bw and 50 mg/kg bw) route. Culture-dependent method and metagenome analysis were performed using samples of gut mucus taken from fish at day 0, 5 and/or 10. A total of 23 and 26 species were isolated from gut mucus of fish in oral and injection groups, respectively. Virbio scophthalmi, V. harveyi and Photobacterium damselae, and V. scophthalmi and Streptococcus parauberis were dominantly isolated from oral and injection treatment groups, respectively. In metagenomics analysis, operating taxonomic unit (OTU) counts obtained from AMX and OTC oral treatment group were 42 and 78, respectively, which are much lower than 178 OTUs retrieved from the control group. OTU counts obtained from fish injected with AMX at day 5 were 22, remarkably lower than those of other samples in injection treatment groups. These results indicate that administration of antibiotics to fish can have great impact on gut microbial community by reducing its diversity and number. Our data also show that AMX might cause changes in microbial community greater than OTC. In some cases, it can lead to significant increase of opportunistic pathogen(s), causing dysbiosis.

A37 DEFECTS IN INTESTINAL MUCUS DISRUPTS THE REPERTOIRE OF DEVELOPING T CELLS IN THE THYMUS

Severe, chronic inflammation of the intestinal tract is a defining hallmark of inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease. IBD is postulated to result from a complex combination of environmental and genetic factors that lead to excessive bacterial stimulation of the mucosal immune system. An overlying layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium and the underlying immune system. Importantly, decreased mucus production and impaired mucus function have been associated with UC pathogenesis. MUC2 is the primary intestinal mucin and acts as a structural scaffold for mucus formation - forming a physical barrier to shield the intestinal wall. However, recent work suggests that mucus may also function to maintain immunological tolerance towards luminal antigens by promoting the regulatory activity of intestinal antigen-presenting cells. Currently, it is unclear what role mucus plays in influencing the T cell repertoire and modulating T cell immunity both at local and distal sites. We hypothesize that mucus is essential for maintaining T cell tolerance towards intestinal luminal antigens and preventing disease caused by constitutive T cell activation. To investigate whether peripheral T cells become highly activated in young, mildly colitic Muc2-/- mice following oral antigen gavage. To test our hypothesis, young (6–10 week old) wild type and MUC2-deficient (Muc2-/-) mice were gavaged with the model antigen ovalbumin (OVA) and the elicited OVA-specific T cell responses monitored at various time points post-treatment. We found that oral administration of OVA resulted in the antigen rapidly escaping the gut and disseminating throughout the blood and lymphoid tissues of Muc2-/- but not wild type mice. Given the systemic spread of antigen in the Muc2-/- mice, we next sought to investigate whether the selection of developing T cells in the thymus of Muc2-/- mice was impacted by dietary antigens. Remarkably, we found that gavage of Muc2-/- mice led to presentation of OVA by thymic dendritic cells and the deletion of OVA-specific thymocytes whereas T cell development in wild type mice appeared unaffected. Based on our results, we concluded that oral antigens are rapidly disseminated throughout Muc2-/- mice. The heightened concentration of oral antigens induced rapid depletion of oral antigen-specific double positive (DP) thymocytes, and altered the T cell repertoire. Future studies will explore whether IBD patients suffering from impaired gut mucus structure/function also suffer defects in their T cell repertoires. CIHR

Polymeric immunoglobulin receptor and mucosal IgM responses elicited by immersion and injection vaccination with inactivated Vibrio anguillarum in flounder (Paralichthys olivaceus)

Polymeric immunoglobulins (Igs) and polymeric immunoglobulin receptor (pIgR) play crucial roles in teleost mucosal surface defenses, but their mucosal immune responses, especially under different routes of vaccine administration, needs to be better clarified. In the present study, the flounder (Paralichthys olivaceus) was immunized with inactivated Vibrio anguillarum by intraperitoneal injection and immersion, and pIgR and IgM responses were analyzed. Real-time PCR showed that pIgR and IgM mRNA expression were up-regulated with a similar variation trend, but pIgR mRNA responded earlier than IgM; the pIgR mRNA levels were higher in spleen, gills, skin and hindgut in both immunized groups, while IgM mRNA response was higher in skin, gills, hindgut and liver in immersion group, or head kidney, spleen, hindgut and liver in injection group. Immunohistochemical results revealed that pIgR and IgM were localized in the epithelium of skin, gills and hindgut, and biliary epithelium of liver. Western blot demonstrated that a ~800 kDa protein band in skin, gill and gut mucus and bile reacted with anti-pIgR and anti-IgM antibody, indicating that secretory component (SC) of pIgR associated with polymeric IgM as a complex in these secretions. ELISA assay revealed that the vaccine-specific IgM and SC of pIgR levels were up-regulated in skin, gill and gut mucus and bile, reaching a higher peak level at earlier time in skin and gill mucus in immersion than injection group, and a higher peak level in bile and gut mucus in injection group. These results illustrated that immersion and injection vaccination stimulated pIgR and secretory IgM production in mucus and bile, and a disparity in pIgR and IgM response was observed under different administration routes, which provided better understanding for fish mucosal immunity and in turn helped to develop vaccination strategies aimed at maximizing mucosal and consequently fish health.

Analysis of immunoglobulin and T cell receptor gene expression in ballan wrasse (Labrus bergylta) revealed an extraordinarily high IgM expression in the gut

The serum IgM concentration of ballan wrasse is relatively high, estimated to approximately 13 mg/ml in adult wild fish of 800 g. The present study revealed an unusual high abundance of IgM mRNA in the gut of ballan wrasse. Initially, transcripts encoding IgM, IgT, IgD, TCRα, TCRδ and CD3ε were quantified by RT-qPCR in several tissues of wild caught fish (approx. 800 g), indicating an elevated immune activity in hindgut and an extraordinarily high expression of IgM. Subsequently, a new RT-qPCR analysis was performed on the entire intestine, cut into four different segments, of reared fish (32–100 g). The analysis indicated immune activity along the entire intestine, but not as strong as in the hindgut. Furthermore, similar to the larger fish, the relative abundance of IgM transcripts was higher in the hindgut than in kidney and spleen, although the absolute level of IgM was in general higher in the larger fish. The secreted form of IgM was completely dominant in comparison to the membrane bound form of IgM and the other analysed genes. IgM was purified from gut mucus and external mucosal surfaces by magnetic beads coated with protein A. Mucus IgM reacted with rabbit antisera raised against serum IgM and contained subunits of the same size. Regarding the elevated immune activity in the intestine it is tempting to speculate on a possible compensatory strategy in this lineage of stomach-less fish, and that natural antibodies have an important role in the first line defence.

Abstract 130: Histamine Increase After Ischemic Stroke Leads to Gut Barrier Breakdown Related Post-Stroke Inflammation in Aging

Human aging is characterized by chronic, low-grade inflammation, a phenomenon termed “inflammaging”. Aging is a non-modifiable risk factor for stroke and inflammatory processes are a major contributor to ischemic brain damage. Mast cell (MC) degranulation and the subsequent release of histamine (HA) may contribute to chronic inflammation, BBB leakage and neutrophil accumulation. Over the past decade, the histaminergic system has been implicated in the modulation of cell death in brain disorders including stroke. However, the exact mechanisms underlying HA actions on ischemia-induced damage have not yet been defined. Stroke leads to gut dysfunction and dysbiosis, which might trigger MC infiltration. Since previous studies have shown that the gut/microbiome has a significant impact on stroke outcomes, we hypothesized that stroke increases HA levels, histamine receptors (HR) and MC infiltration in the gut leading to gut barrier breakdown further contributing to post-stroke inflammation (PSI) and damage. Methods: Aged (Ag) C57BL/6 (20-22 months old) and young (Yg) (3 months old) male mice were subjected to 60 min transient middle cerebral artery occlusion (MCAO) or sham surgery. Mice were sacrificed after 6-hours (h), 12-h, 24-h, 3-days (d) and 7-d post-MCAO. Brain, blood, gut and spleen were harvested and used for flow cytometry, mRNA and immunohistochemistry (IHC) before behavior analysis. Luminal contents were collected and subjected to 16s sequencing and metabolomics analysis. Results: Stroke increased HR2 expression in the intestine of 7-d PS Ag mice compared to the Ag controls. HA levels in Ag brain and blood was 2-fold higher than in Yg mouse at 6-h after stroke and remained high at 24-h. In addition, locomotor activity was significantly reduced in post stroke mice. Increased MCs were seen in the gut of PS compared to control mice. The protective gut mucus layer was depleted with increased HR activation and with a pro-inflammatory bio-marker elevation. This was correlated with imbalanced gut microbiota. This work suggests that there is a role of gut HR and systemic HA in modulating brain health. Controlling HA release and suppressing HR activation in the periphery especially in the gut PS might improve recovery after stroke.

Abstract TMP30: Mucin is a Novel Target for Gut Protective Immunity in Stroke

Introduction: Infections are a common cause of post-stroke morbidity and mortality. Recent studies suggest that gut microbiota are a major source of post-stroke infection. Gut mucus layers are a major area of communication between gut microbiota and host immune cells. To our knowledge there have been no studies evaluating the role of colonic epithelial mucins, where most of the gut bacteria reside, after ischemic stroke. Hypothesis: Epithelial mucin plays a protective role in stroke-induced colonic disruption. Methods: Mice were subjected to either sham or 60-minute middle cerebral artery occlusion (MCAO). Neurological deficit score (NDS) and levels of lipopolysaccharide-binding protein (LBP) in plasma were assessed at day 7 after stroke. Mice were sacrificed at day 7 after stroke and colonic epithelial cells (ECs) were isolated for a gene expression study. Tissues were used for histology and bacterial in situ hybridization. Fecal samples were collected for 16S sequencing and IgA ELISA. Results: Young female mice had lower NDS and LBP levels, with intact colonic structures (n=4/group, P &lt;0.05). Young mice ECs significantly increased 1) regenerating islet-derived protein (Reg) family-specific antimicrobial proteins in males (24.3, 21.3 and 2.5 fold change in Reg3β, -γ and -4, respectively) and 2) mucin genes in females (1.8 and 1.4 fold change in Muc2 and -4, respectively, n=4-5/group, P &lt;0.05). Young ovariectomized mice switched their protective strategies from enhanced expression of mucin genes to a “male-like” pattern in which Reg genes were increased after stroke (n=3-5/group, P &lt;0.05). Aged mice failed to express these sex-specific genes after stroke. Interestingly, after stroke, bacteria from young female but not male mice were found at a farther distance from the colonic epithelium secondary to intact mucus layers. Finally, young female mice increased mucin-regulating Akkermansia bacteria and IgA in the feces after stroke (n=4/group, P &lt;0.05). Conclusions: Our data demonstrates that 1) stroke provokes a colonic epithelial response and 2) mucin is critical for colonic protection in stroke. In conclusion, mucin should be considered as a novel target to improve post-stroke recovery as well as an important element in maintaining colonic structure.

Molecular cloning of polymeric immunoglobulin receptor-like (pIgRL) in flounder (Paralichthys olivaceus) and its expression in response to immunization with inactivated Vibrio anguillarum

In the present work, the polymeric immunoglobulin receptor-like (pIgRL) from flounder (Paralichthys olivaceus) was firstly cloned and identified. The full length cDNA of flounder pIgRL was of 1393 bp including an open reading frame of 1053 bp, and the deduced pIgRL sequence encoded 350 amino acids, with a predicted molecular mass of 39 kDa. There were two immunoglobulin-like domains in flounder pIgRL. In healthy flounder, the transcriptional level of pIgRL was detected in different tissues by real-time PCR, showing the highest level in the skin and gills, and higher levels in the spleen and hindgut. After flounders were vaccinated with inactivated Vibrio anguillarum via intraperitoneal injection and immersion, the pIgRL mRNA level increased firstly and then declined in all tested tissues during 48 h, and the maximum expression levels in the gills, skin, spleen and hindgut in immersion group, or in the spleen, head kidney, skin and gills in injection group, were higher than in other tested tissues. In addition, recombinant protein of the extracellular region of flounder pIgRL was expressed in Escherichia coli BL21 (DE3), and rabbit anti-pIgRL polyclonal antibodies were prepared, which specifically reacted with the recombinant pIgRL, and a 39 kDa protein confirmed as natural pIgRL by liquid chromatography-mass spectrometry in skin mucus of flounder. Co-immunoprecipitation assay and western-blotting demonstrated that the pIgRL, together with IgM, could be immunoprecipitated by anti-pIgRL antibody in gut, skin and gill mucus of flounder, suggesting the existence of pIgRL-IgM complexes. These results indicated that the flounder pIgRL was probably involved in the mucosal IgM transportation and played important roles in mucosal immunity.

Specific immune response in mucosal and systemic compartments of Cirrhinus mrigala vaccinated against Edwardsiella tarda: In vivo kinetics using different antigen delivery routes

Mucosal immune barriers confer protection against invading fish pathogens. Here, we conducted an experiment for 60 days to assess the mucosal and systemic immune response in Mrigal (Cirrhinus mrigala), an Indian major carp. Fish were immunized with inactivated Edwardsiella tarda by four different routes, namely, oral, immersion, injection, and anal intubation. An indirect enzyme‐linked immunosorbent assay (ELISA) was used to measure the specific immune response (antibody) in serum and mucus (collected from skin, gill, and gut) of the fish on 0, 15, 30, 45, and 60 days postimmunization. For specific immune response in the serum, significantly higher (p &lt; 0.05) optical density (OD) values were obtained in the anal group (0.52 ± 0.03) and in the oral group (0.48 ± 0.03). In the skin mucus, significantly higher OD values were obtained in the oral group (0.48 ± 0.04) and immersion group (0.32 ± 0.03). In the gill mucus, significantly higher OD values were obtained in the oral group (0.82 ± 0.08) and the immersion group (0.73 ± 0.03). In the gut mucus, significantly higher OD values were obtained in the immersion group (0.080 ± 0.007) compared to the rest of the treatments. Fish from all the groups were challenged with LD50 dose of E. tarda at the end of the experiment. We conclude that oral and immersion immunization routes offer better protection of C. mrigala compared to other antigen delivery routes.

Effects of Stress on the Mucus-microbial Interactions in the Gut.

Stress shows both direct- and indirect-effects on the functions of the gastrointestinal tract, in particular on the mucus physiology and the composition of microbiota. Mucus mainly consists of heavily glycosylated proteins called mucins, which are secreted by goblet cells. The gut mucus layer is a pivotal part of the intestinal protection and colonized by commensal microbes, essential for the development and health of the host. There is a symbiotic interaction between intestinal microbiota and the host cells. On the one hand, mucus provides nutrients for the growth and adhesion of microbes; on the other hand, mucin-degrading bacteria generate energy sources for the host epithelium. However, the mucusmicrobial interaction has rarely been considered in the context of stress exposure. Therefore, this paper principally reviews the effects of stress on both mucus secretion and gut microbiota and is hoped to provide a new perspective for future study.

Using human iPS cell-derived enterocytes as novel in vitro model for the evaluation of human intestinal mucosal damage.

The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells. Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated. Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase. The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.

Role of food-derived opioid peptides in the central nervous and gastrointestinal systems.

Opioid receptors are widely distributed in central nervous system and peripheral tissues. Endogenous opioid receptor ligands are involved in many physiological processes. Exogenous peptides, derived from food proteins with gastrointestinal proteases, also exert opioid-like activities, and they include gluten exorphins (wheat), casomorphins (milk), rubiscolins (spinach), and soymorphins (soybean). Milk-derived opioid peptides play both agonistic and antagonistic roles, and most of the opioid peptides exert regulatory functions in the central nervous system, related to nociception, emotion and memory after oral, intracerebroventricular, or intraperitoneal administration. This indicates that the peptides may have crossed the blood-brain barrier or acted peripherally. Furthermore, some food-derived opioid peptides influence gastrointestinal functions such as gut motility, hormone release, appetite, mucus production, and local immunity. In healthy states, food-derived opioid peptides could benefit both the nervous and digestive systems, whereas in pathological conditions, the gastrointestinal permeability change and opioid excess may contribute to pathogenesis of some disorders. PRACTICAL APPLICATIONS: Opioid receptors are important biological targets for the treatment of multiple diseases. Traditional opiate compounds, such as alkaloids, are demonstrated to exert numerous side effects, thereby limiting their clinical effectiveness. It is thought that food-derived opioid peptides may be safer than the alkaloids, and therefore can be applied in functional food development. In this review, we summarized the already discovered food opioid peptides from different sources, and elaborated their physiological functions on the central nervous and gastrointestinal systems. These effects support further exploration of the opioid peptides as therapeutic agents or as functional food ingredient for human health promotion.

Polymeric Immunoglobulin Receptor Mediates Immune Excretion of Mucosal IgM-Antigen Complexes Across Intestinal Epithelium in Flounder (Paralichthys olivaceus).

Polymeric immunoglobulin receptor (pIgR) is one important player of mucosal defenses, but very little is known on pIgR-mediated immune excretion of the antigens that penetrate mucosal surface in fish. Previously, we cloned the pIgR of flounder (Paralichthys olivaceus) and developed anti-pIgR antibody. In this study, the flounders were immunized intraperitoneally with the chicken ovalbumin (OVA) and the control protein bovine serum albumin (BSA) to elicit mucosal IgM antibody and pIgR response, and then challenged with OVA via caudal vein injection after the immunized OVA was absent from fish body at the fourth week after immunization. After OVA challenge, strong OVA-positive fluorescence signals were observed in lamina propria (LP) submucosa and epithelial cells of the hindgut at 30 min, increased proceeding toward the distal portion of intestinal folds, reached a peak at 2–3 h, and then weakened and disappeared at 12 h, indicating that the OVA rapidly diffused from bloodstream into LP submucosa and excreted across intestinal epithelium. Whereas in BSA-immunized and OVA-challenged control fish, the OVA was detected in LP submucosa but not in intestinal epithelium due to the lack of OVA-specific antibody. Accordingly, in intestinal epithelium, the transepithelial transport of OVA was confirmed by immunogold electron microscopy, and co-localization of OVA, IgM, and pIgR was illuminated by multiple-label immunofluorescence confocal microscopy and analyzed using Image J software. Furthermore, in gut mucus but not in serum, an ~800-kDa protein band showed IgM-positive, OVA-positive, and pIgR-positive simultaneously, and the OVA, together with IgM and secretory component (SC) of pIgR, could be immunoprecipitated by anti-OVA antibody, demonstrating the existence of SC–polymeric IgM–OVA complexes. All these results collectively revealed that the pIgR could transport mucosal IgM–OVA complexes from LP across intestinal epithelium into gut mucus via the transcytosis in flounder. These new findings provided direct evidences for pIgR-mediated immune excretion of IgM–antigen complexes, and better understanding the role of pIgR in mucosal immunity in teleost fish.

Anti-diabetic effect of baicalein is associated with the modulation of gut microbiota in streptozotocin and high-fat-diet induced diabetic rats

Baicalein, a dietary flavonoid, is the major constituent of Oroxylum indicum and Scutellaria baicalensis which are consumed as teas or dietary supplements in Asia, European, and the United States. The compound exhibits a variety of bioactivities despite its limited bioavailability. In the present work, 4-week treatment of baicalein significantly decreased the levels of blood glucose and circulating lipopolysaccharide (LPS) and improved insulin resistance, inflammation, and lipid profile in diabetic rats induced by STZ and high-fat-high-sugar-diet. These anti-diabetic effects were attributed to the increased SCFAs content and the increased thickness of gut mucus layer, and were associated with the modulation of gut microbiota. Among the key phylotypes, Bacteroides and Bacteroidales S24-7 had the highest relative abundance in the rats receiving high-dose baicalein and showed positive correlation with the phenotypes related to the improvement of type 2 diabetes (T2DM). Our study supports the use of baicalein as a dietary supplement and a potential prebiotic for its ability to modify gut microbiota and to improve T2DM-related biochemical abnormalities.

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

AIMTo demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine.METHODSExtracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro.RESULTSTOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut.CONCLUSIONOur results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

Dietary Pea Fiber Supplementation Improves Glycemia and Induces Changes in the Composition of Gut Microbiota, Serum Short Chain Fatty Acid Profile and Expression of Mucins in Glucose Intolerant Rats.

Several studies have demonstrated the beneficial impact of dried peas and their components on glucose tolerance; however, the role of gut microbiota as a potential mediator is not fully examined. In this study, we investigated the effect of dietary supplementation with raw and cooked pea seed coats (PSC) on glucose tolerance, microbial composition of the gut, select markers of intestinal barrier function, and short chain fatty acid profile in glucose intolerant rats. Male Sprague Dawley rats were fed high fat diet (HFD) for six weeks to induce glucose intolerance, followed by four weeks of feeding PSC-supplemented diets. Cooked PSC improved glucose tolerance by approximately 30% (p < 0.05), and raw and cooked PSC diets reduced insulin response by 53% and 56% respectively (p < 0.05 and p < 0.01), compared to HFD (containing cellulose as the source of dietary fiber). 16S rRNA gene sequencing on fecal samples showed a significant shift in the overall microbial composition of PSC groups when compared to HFD and low fat diet (LFD) controls. At the family level, PSC increased the abundance of Lachnospiraceae and Prevotellaceae (p < 0.001), and decreased Porphyromonadaceae (p < 0.01) compared with HFD. This was accompanied by increased mRNA expression of mucin genes Muc1, Muc2, and Muc4 in ileal epithelium (p < 0.05). Serum levels of acetate and propionate increased with raw PSC diet (p < 0.01). These results indicate that supplementation of HFD with PSC fractions can improve glycemia and may have a protective role against HFD-induced alterations in gut microbiota and mucus layer.