Human Intestinal Mucus Research Articles

Inhibition of S-fimbria-mediated adhesion to human ileostomy glycoproteins by a protein isolated from bovine colostrum.

The aim of this study was to isolate and purify the component in bovine colostrum which is responsible for the inhibition of S-fimbria-mediated adhesion of Escherichia coli. Whey from defatted colostrum was fractionated by ultrafiltration, and the < 100K, < 30K, and < 10K fractions and the colostral whey were tested for inhibition of in vitro adhesion of radiolabelled S-fimbria-bearing E. coli to human ileostomy glycoproteins, which provide a model for human intestinal mucus. The inhibiting compound was purified from a dialyzed < 30K fraction with an anion exchange column which was eluted with a NaCl gradient (0 to 1.0 M). The compound was found to be a heat-resistant but pepsin-sensitive protein with an Mr of approximately 18,000 and an isoelectric point of approximately 5.75. The protein appears to block receptor sites for S-fimbriae on ileostomy glycoproteins, with steric hindrance being the most likely mechanism. Analysis of the amino acid sequence of the amino terminus of the 18K protein showed similarity with the sequence of beta-lactoglobulin.

Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture.

Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations. These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively. Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed. All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity. Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation. The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity. Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border. Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins.

The mucus layer as a barrier to drug absorption in monolayers of human intestinal epithelial HT29-H goblet cells

The efficacy of the intestinal mucus layer as a barrier to drug absorption was investigated in monolayers of a mucus-producing human goblet cell line (HT29-H) using testosterone as a model drug. The investigation included comparison of the permeability of the HT29-H monolayers to testosterone before and after removal of the mucus layer, and comparison of the permeability of HT29-H and mucus-free Caco-2 cell monolayers. The apparent permeability coefficients of testosterone in HT29-H monolayers were 6.8 ± 1.0 × 10−6 cm/s before and 21.5 ± 5.9 × 10−6 cm/s after removal of the mucus layer p < 0.02). The corresponding permeability coefficients in Caco-2 monolayers were 31.5 ± 5.3 × 10−6 and 32.1 ± 9.3 × 10−6 cm/s (p < 0.05) or approx. 50% higher than that of ‘mucus-free’ HT29-H monolayers. A comparison of the relative importance of the mucus layer, the unstirred water layer and the glblet cell monolayer as barriers to testosterone absorption showed that the mucus layer contributes 78% of the total resistance to absorption. We conclude that the intestinal mucus layer produced by HT29-H goblet cells is a significant barrier to testosterone absorption and that HT29-H monolayers can be used to study the barrier functions of an intact human intestinal mucus layer in cell culture.

Use of the ileal conduit as a model for studying human small intestinal mucus glycoprotein secretion

To establish a human model system for investigating control of mucus secretion by the small intestine, we have studied the output of mucus glycoprotein in the urine of patients with ileal conduits. A highly specific enzyme-linked immunosorbent assay (ELISA) was used to measure mucus glycoprotein with a sensitivity down to 0.3 ng protein of mucus glycoprotein. The assay detects human ileal mucus glycoprotein in the urine of ileal conduit patients; for an individual subject, the amount hardly varied from day to day, although there were substantial differences between individuals in the amounts secreted. Control urine from persons with normal bladders did not contain any mucus glycoprotein that reacted in the ELISA. The model has many advantages over other experimental systems for studying the rate of mucus glycoprotein secretion from the human ileum and can also be used as a reproducible resource of undegraded mucus glycoprotein.

Giardia lamblia: Stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold ( P < 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.

An immunohistochemical study of a colonic mucus antigen in normal and neoplastic gastrointestinal tissues.

A monoclonal antibody YPC 44.3 which reacts with human large intestinal goblet cell mucus has been used to examine normal gastrointestinal mucosa and a series of gastric and colorectal carcinomas in an immunohistochemical study. In normal colonic mucosa the antibody is shown to detect an antigen which is expressed polymorphically and depends on the presence of the active allele at the Lewis locus for its expression. However the antigen appears distinct from regular Lewis antigens on the basis of immunoabsorption studies and organ distribution. Results of immunostaining tumours show no correlation with the site, classification or grade of tumour or the type of metaplasia adjacent to gastric cancers. The relationship of YPC 44.3 to other mucus antibodies and the importance of screening tissues with a wide range of blood group phenotypes is discussed.

Reserpine-induced alterations in mucus production and calmodulin-binding proteins in a human epithelial cell line

The characterization of a mucus-producing human cell line (HC-84) derived from a colon carcinoma and its response to in vitro reserpine treatment is reported. Mucous granules were demonstrated within these cells on the basis of electron microscopic examination and incorporation of [ 3 H] glucosamine with subsequent autoradiographic analysis. Fluorographic analysis of total HC-84 cell protein after incubation with [ 3 H] glucosamine indicated that the majority of tritium was incorporated into two proteins with molecular weights of 115 and 120 kD. When total HC-84 protein was subjected to immuno-blot analysis utilizing rabbit antibody against human intestinal mucus, only these two proteins (115K and 120K) reacted positively, indicating a direct correlation between [ 3 H] glucosamine incorporation and mucus production. Immunofluorescence localization of mucus within HC-84 cells utilizing this same antibody resulted in a punctate pattern of fluorescence within the cytoplasm. Treatment of HC-84 cells with 30 μM reserpine for 7 days resulted in a three-fold increase in mucus production compared with controls. There was also a concomitant loss of a 30K calmodulin-binding protein in cells treated with reserpine. These cells represent a useful system for studying the effect of reserpine on the processes of mucus synthesis and secretion.