Porcine Small Intestinal Mucus Research Articles

Calpain-1: a Novel Antiviral Host Factor Identified in Porcine Small Intestinal Mucus.

ABSTRACTThe thick mucus layer covering of the intestinal epithelium has received increasing attention, owing to its protective role in intestinal infection. However, the exact mechanisms by which the mucus increases intestinal resistance against viral infection remain largely unclear. Here, we identify prominent antiviral activity of the small intestinal mucus and extracted total mucus proteins, as evidenced by their inhibitory effects against porcine epidemic diarrhea virus (PEDV) infection. Of all the extracted mucus proteins, mucin 2 and fraction III (~70 kDa) exhibited potent antiviral activity. We further evaluated the antiviral effects of three candidate factors in fraction III and found that calpain-1 contributed substantially to its antiviral activity. In vivo studies demonstrated that oral administration of calpain-1 provided effective protection against intestinal PEDV infection. As a calcium-activated cysteine protease, calpain-1 inhibited viral invasion by binding to and hydrolyzing the S1 domain of the viral spike protein. The region between amino acids 297 and 337 in the b domain of PEDV S1 protein was critical for calpain-1-mediated hydrolysis. Further investigation indicated that calpain-1 could be produced by goblet cells between intestinal epithelia. Taken together, the results of our study revealed calpain-1 to be a novel antiviral protein in porcine small intestinal mucus, suggesting that calpain-1 has potential for defending against intestinal infections.

Comparing the permeability of human and porcine small intestinal mucus for particle transport studies

The gastrointestinal mucus layer represents the last barrier between ingested food or orally administered pharmaceuticals and the mucosal epithelium. This complex gel structure plays an important role in the process of small intestinal absorption. It provides protection against hazardous particles such as bacteria but allows the passage of nutrients and drug molecules towards the intestinal epithelium. In scientific research, mucus from animal sources is usually used to simulate difficult-to-obtain human small intestinal mucus for investigating the intramucus transport of drug delivery systems or food nanoparticles. However, there is a lack of evidence the human mucus can be reliably substituted by animal counterparts for human-relevant transport models. In this report, a procedure for collecting human mucus has been described. More importantly, the permeability characteristics of human and porcine small intestinal mucus secretions to sub-micron sized particles have been compared under simulated intestinal conditions. Negatively charged, 500 nm latex beads were used in multiple-particle tracking experiments to examine the heterogeneity and penetrability of mucus from different sources. Diffusion of the probe particles in adult human ileal mucus and adult pig jejunal and ileal mucus revealed no significant differences in microstructural organisation or microviscosity between the three mucus types (P > 0.05). In contrast to this interspecies similarity, the intraspecies comparison of particle diffusivity in the mucus obtained from adult pigs vs. 2-week old piglets showed better penetrability of the piglet mucus. The mean Stokes–Einstein viscosity of the piglet jejunal mucus was approx. two times lower than the viscosity of the pig jejunal mucus (P < 0.05). All mucus structures were also visualised by scanning electron microscopy. This work validates the use of porcine small intestinal mucus collected from fully-grown pigs for studying colloidal transport of sub-micron sized particles in mucus under conditions mimicking the adult human small intestinal environment.

Colloidal transport of lipid digesta in human and porcine small intestinal mucus

Small intestinal mucus transport of food-derived particulates has not been extensively studied, despite mucus being a barrier nutrients need to cross before absorption. We used complex dispersions of digesta obtained from simulated, dynamic gastrointestinal digestion of yogurt to examine the penetrability of human and porcine mucus to the particles formed of lipolysis products. Quantitative, time-lapse confocal microscopy revealed a sieve-like behaviour of the pig jejunal and ileal mucus. The digesta diffusivity decreased significantly over the first 30 min of mucus penetration, and then remained constant at ca. 5 × 10-12 m2 s−1 (approx. 70% decrease from initial values). A non-significantly different penetrability was recorded for the ileal mucus of adult humans. The digesta diffusion rates in neonatal, jejunal mucus of 2 week old piglets were 5–8 times higher than in the three different types of adult mucus. This is the first report that validates the mucus of fully-grown pigs as a human-relevant substitute for mucus permeation studies of nutrients/bio-actives and/or complex colloidal dispersions (e.g., post-digestion food particulates, orally-administrated delivery systems).

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion.

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host-bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.

Characterization of Probiotic Properties ofEnterococcus faeciumNHRD IHARA Isolated from Porcine Feces

We examined in vitro the adhesion of Enterococcus faecium NHRD IHARA (NHRD IHARA) to porcine small intestinal mucin (PSIM) and inhibition of the adherence of enteropathogenic bacteria due to pre-incubation of PSIM with NHRD IHARA. NHRD IHARA exhibited an effective barrier function in porcine small intestinal mucus layer.

Purification and Characterization of a Surface Protein from Lactobacillus fermentum 104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.

31] A dot-blot assay for adhesive components relative to probiotics

Publisher Summary This chapter discusses a dot-blot assay for adhesive components. Both Lactobacillus and E. coli have the capacity to adhere to immobilized porcine small intestinal mucus. The dot-blot assay discussed, gives direct evidence when it is used to evaluate whole bacterial cell suspensions of Lactobacillus along with the well-defined adhesion of E. coli K88 fimbriated cells. This chapter develops a qualitative in vitro assay for detection of binding of bacterial cell surface components to mucous extracts. A dot-blot assay is developed, whereby extracts containing bacterial components and fractions were immobilized and then blotted with enzymatically labeled mucus. Results are compared with those obtained using the inhibition assay. In addition, whole cells of Lactobacillus and E. coli are tested in the dot-blot assay and results are compared with a modification of the method of Laux and co-workers. The results obtained with the dot-blot assay provided further information about the binding of Lactobacillus and E. coli to gastrointestinal mucus, not only because adhesionpromoting compounds could be detected in fractionated extracts, but also because porcine gastric mucin along with small intestinal mucus could be used for blotting.

Characterization and purification of porcine small intestinal mucus receptor for Escherichia coli K88ac fimbrial adhesin

The objectives of this study were to investigate the nature of, and to purify K88ac fimbrial adhesin-specific receptors in the mucus from the small intestine of piglet. Adhesion was studied by incubating 3H-labeled Escherichia coli with mucus that were treated with or without pronase, proteinase, trypsin or sodium metaperiodate. The results indicated that treatment with either proteolytic enzymes or sodium metaperiodate (to oxidize sugars) significantly reduced E. coli K88ac or K88+MB adhesion to the mucus, suggesting that the K88ac and K88+MB specific receptors in this preparation were, at least in part, glycoprotein in nature. The K88+MB fimbriae specific receptor was purified using affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified K88+MB specific receptor together with the above data suggested that the receptor from the mucus of the small intestine of the pig was a 80-kDa glycoprotein.

Gel permeation chromatography used to determine the stability of FITC-Dextrans in human saliva and porcine small intestinal mucus

Fluorescein isothiocyante dextrans (FITC-Dextrans) have been used extensively as high molecular weight markers. The stability of FITC-Dextrans has been demonstrated in many situations, but there are instances where dextrans have not been stable. A gel permeation chromatography (GPC) method has been established that can be used to monitor molecular weight, concentration, polydispersity and free label in FITC-Dextrans. This has been applied to stability studies of FITC-Dextrans in human saliva, porcine small intestinal mucus and with human α-amylase. The molecular weight of FITC-Dextran was reduced in human saliva with a half-life of 5 h. The reduction in molecular weight was less rapid in porcine small intestinal mucus and non-existent with human a-amylase; sodium azide helped reduce degradation but did not eliminate it. The degradation of FITC-Dextrans is thought to be mediated by dextranase-producing micro-organisms. The successful use of FITC-Dextrans as high molecular weight markers must be accompanied by careful quality control of FITC-Dextrans, particularly when incubation times are long and microbiological spoilage is a possibility.