Changes in skin mucus of common carp, Cyprinus carpio L., after exposure to water with a high bacterial load.

Water in aquaculture systems may contain a high load of microorganisms. Reduction in overall bacterial tank water load improves fish health and growth parameters. In this study, the effect of an increase of overall bacterial load in tank water on carp skin mucus was assessed. Intracellular and released high molecular weight glycoproteins (HMGs) of carp skin mucus were analysed for changes using histological, histochemical and biochemical techniques. Increase of bacterial load did not induce obvious clinical responses in carp, but the skin of exposed carp responded quickly. The amount of skin mucus HMGs isolated increased as well as their total glycosylation. An increased goblet cell number was observed for all carbohydrate stainings, but most clearly for acidic glycoconjugates. A change in the terminal presence of some sugars was also seen. After the initial response of carp, an adaptation to the higher bacterial load in the water appeared to occur as mucins had a higher glycosylation. The changes observed suggest that these skin mucus adaptations are part of a primary defence mechanism of mucosal epithelia, even at a low pathogenic pressure.

Saccharomyces boulardii and bacillus cereus var. Toyoi influence the morphology and the mucins of the intestine of pigs.

The mode of action of probiotics is still incompletely understood. To study the interactions between probiotic micro-organisms and the host their effects on morphology and mucins of the intestinal mucosa were investigated. Fifteen clinically healthy weaned pigs were divided into three groups and received either Saccharomyces boulardii or Bacillus cereus var. toyoi or were left untreated. Sections of duodenum, proximal and mid jejunum, ileum, caecum, and colon were examined. An increase of villus length in the small intestine and a decrease in the number of goblet cells with 2.6-sialylated mucins in the large intestine were observed in both treatment groups. There were no differences in crypt morphology, number of Ki67-positive cells, total number of goblet cells and number of goblet cells with acidic, neutral, sulphated, or 2.3-sialylated mucins between groups. The results indicate an effect of Saccharomyces boulardii and Bacillus cereus var. toyoi on the intestinal architecture of pigs.

[Differential mucin expression in the gastrointestinal tract]. / Differenzielle Muzinexpression im Gastrointestinaltrakt.

Under physiological conditions mucins display a tissue specific expression. MUC2, MUC3, MUC5AC and MUC6 are the major mucins in the gastrointestinal tract. In the intestinal tissue MUC2 and MUC3 are the predominant mucins, whereas MUC5AC and MUC6 are the most important mucins in the stomach. Pathophysiological conditions are characterized by an aberrant gene expression. This includes the expression of non-tissue specific mucins as well as a reduced expression of the specific mucins. During inflammation a co-expression of non-tissue specific mucins was observed. Neoplastic transformations are also associated with an aberrant mucin expression. Obviously, an increased expression of non-specific mucins is related with a more favorable prognosis, while a decreased mucin expression is indicative for an increased cellular dedifferentiation and a poor prognosis. This modified mucin expression may result from a modified gene expression as well as from modifications in the posttranscriptional processing. A modified mucin expression reflects pathophysiological conditions and may support the pathohistological diagnosis.

Role of vacA and cagA in Helicobacter pylori inhibition of mucin synthesis in gastric mucous cells.

The aim of this study was to investigate the effect of Helicobacter pylori on the function of gastric mucous cells. H. pylori (10(4) to 10(7) CFU/well) was incubated with the mucin-producing gastric cell line HM02 for 12 and 24 h. Mucin synthesis and secretion were determined by the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular and released high-molecular-weight glycoproteins. cagA-positive, cytotoxin-producing and non-cytotoxin-producing H. pylori strains impaired the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular glycoproteins. Significant inhibition of mucin synthesis was noted after 12 and 24 h of cocultivation with a bacterial load of >/=10(5) bacteria (bacterium/cell ratio = 0.25). The cagA-positive, cytotoxin-producing strains (HP64, HP57, and HP87) caused significantly stronger inhibition of intracellular mucin synthesis than the cagA-positive, non-cytotoxin-producing strains (HP05, HP83, and HP84). The cagA-negative, non-cytotoxin-producing strains (HP01, HP04, and HP85) did not affect intracellular mucin synthesis. The results indicate that H. pylori directly impairs mucin synthesis in gastric mucous cells and that cytotoxic cagA-positive strains cause more profound inhibition of mucin synthesis. We suggest that the increased inhibitory effect of cagA-positive, cytotoxin-producing strains on mucin synthesis can be considered one possible factor responsible for the increased risk of developing peptic ulceration with these H. pylori strains.

Proinflammatory cytokines trigger MUC gene expression and mucin release in the intestinal cancer cell line LS180.

OBJECTIVES AND DESIGN: Proinflammatory cytokines and a defective mucus layer are involved in the pathogenesis of colitis. Therefore, we determined cytokine effects on MUC gene expression and mucin secretion. MATERIALS AND METHODS: LS180 cells were characterized by light and electron microscopy and subsequently exposed to interleukin 1 (IL-1, 1 ng/ml), interleukin 6 (IL-6, 10 ng/ml), or tumor necrosis factor-alpha (TNFalpha, 10 ng/ml). MUC gene (MUC2, MUC5AC, MUC5B, MUC6) mRNA expression was assessed by RT-PCR, the encoded proteins were identified by immunocytochemistry and Western blotting, and the released mucins were isolated and chromatographically characterized. RESULTS: Thirty to 40% of the cells contained intracellular mucin granules. Incubation with IL-1 transiently stimulated the mRNA expression of MUC2 and MUC5AC, whereas IL-6 induced an early response of MUC2, MUC5B and MUC6. TNFalpha upregulated the expression of MUC2 and MUC5B for 3 hours, and had no effect on the expression of MUC 5AC and MUC6. Immunocytochemistry and Western blotting confirmed TNFalpha effects on MUC2 and MUC5AC on the protein levels. All cytokines stimulated the release of less glycosylated mucins and considerably modulated their carbohydrate composition. CONCLUSION: Our data demonstrate differential cytokine effects on mucin synthesis, secretion and composition. These alterations may contribute to the defective mucus layer in colitis.

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures.

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.

Morphological and molecular characterization of human gastric mucous cells in long-term primary culture.

A primary cell culture of human gastric mucous cells was developed using enzymatic treatment of surgically obtained gastric mucosal specimens. Preferential attachment of gastric mucous cells during a preincubation step resulted in the enrichment of mucous cells [over 90% stained with periodic acid-Schiff (PAS) and mucin-type lectins] in the primary cell culture. Gastric mucous cells could be maintained in culture for 10 days. DNA synthesis peaked during the first 2 days in culture (8+/-1% bromodeoxyuridine-positive cells). During the entire culture period gastric mucous cells released high-molecular-weight glycoproteins into the medium, as determined by gel chromatography on a Sepharose CL-4B column and by metabolic labelling with [14C]-N-acetylglucosamine. Gastric mucin was verified by gas chromatographic analysis of the carbohydrate composition and fractionation of the void-volume fraction by density gradient centrifugation. Determination of the terminal glycosylation of the secreted glycoproteins by a lectin-ELISA revealed that there was a high quantity of alpha-l-fucose. Prostaglandin E2 significantly stimulated glycoprotein secretion during the entire cultivation period by 29-60%. Analysis of mucin-encoding MUC mRNA expression by reverse transcriptase polymerase chain reaction revealed that gastric mucous cells predominantly express MUC1 and MUC5AC, and to a lesser extent MUC6, which reflects the expression pattern obtained following analysis of biopsied samples of gastric mucosa. This primary cell culture model enables the regulation of mucin secretion and mucin gene expression in man to be investigated.

Effects of PGE2 and of different synthetic PGE derivatives on the glycosylation of pig gastric mucins.

The glycosylation of pig gastric mucins, discharged in response to prostaglandin (PG) E2 and to three synthetic PGE-derivatives (misoprostol, nocloprost, rioprostil) was compared. After a 20 h culture period in the absence or presence of 1 micromol/l of one of the PGs, mucins were isolated by gel chromatography and their glycosylation characterized by their linkage to a panel of lectins. For all tested PGs, a significantly increased lectin linkage to mucin glycoproteins of high molecular weight was detected; no significant effects were observed for low molecular weight glycoproteins. Within the stimulatory pattern, major effects were found for the linkage of peanut agglutinin and soybean agglutinin, suggesting predominant effects on the expression of galactose and N-acetyl-galactosamine. Only minor effects were found for sialic acid, mannose, N-acetyl-glucosamine and fucose expression, as evidenced by the linkage of Sambucus nigra agglutinin, Concanavalin A, Datura stramonium agglutinin and Ulex europaeus I agglutinin. All PGs exerted a similar stimulatory pattern. However, at the indicated concentration, misoprostol (281 +/- 36% of control) rendered a significantly higher overall effect than PGE2 (208 +/- 31%), whereas the increases induced by nocloprost (237 +/- 35%) and rioprostil (202 +/- 35%) were not significantly different from the PGE2 effects. These results, suggesting similar stimulatory effects of PGE2 and of the tested synthetic PGs on glycosylation of mucin oligosaccharides, discharged from mucous cells during an in vitro culture, may, at least in part, explain clinical findings that during an impairment of the endogenous PG synthesis, the tested synthetic PGs are effective exogenous substitutes for endogenous E-type prostaglandins and act as anti-ulcer drugs.

Comparative lectin-histochemistry on the pre-epithelial mucus layer in the distal colon of conventional and germ-free rats.

The mucin composition of the rat distal colonic pre-epithelial mucus layer (PML) was studied by lectin histochemistry in conventional (CV), and germ-free (GF) rats to define effects exerted by the gut flora. No peanut agglutinin (PNA) binding was observed in the PML of GF rats, while the PML of their CV counterparts showed a considerable PNA linkage, indicating terminal Gal-beta1,3-GalNAc residues. Soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) stained the PML mucins in CV and in GF rats, indicating terminal GalNAc moieties. A quantitative difference in the Limax flavus agglutinin (LFA) binding capacity was found between CV and GF rats, indicating terminal sialic acid moieties: the staining intensity of bound LFA/ FiTC was higher in CV rats than in GF rats. No linkage of Datura stramonium agglutinin (DSA) and of wheat germ agglutinin (WGA) was found in the PML of GF rats, indicating the absence of terminal GlcNAc, while in CV rats, a clearly marked border was visible next to the luminal content as a "nipple edge" when stained with DSA or WGA. Canavalia ensiformis agglutinin (ConA), indicative for branched mannose, stained PML mucins and goblet cell mucins of GF rat distal colon. In CV rats, both locations were free of ConA binding sites. These results suggest degrading effects, exerted by the gut flora on the rat colonic pre-epithelial mucus layer.

Effects of PGE2 on amount and composition of high molecular weight glycoproteins released by human gastric mucous cells in primary culture.

The aim of this study was to determine effects of prostaglandin E2 (PGE2) on amount and composition of high molecular weight glycoproteins (HMG), released by human gastric mucous cells in primary culture. PGE2 stimulated the release of HMG, as evidenced by measurement of total carbohydrate and protein content, in a concentration-dependent manner. At the maximally tested concentration of 10(-5) mol/l, the increase amounted to 53% and 85%, over controls, for carbohydrate and protein, respectively. The stimulated release was accompanied by alterations of HMG glycosylation. As detected by lectin-ELISA, there was a relative decrease in N-acetyl glucosamine and an increase in mannose and galactose content. The sialic acid content increased in parallel to the total carbohydrate content. These results suggest that PGE2 plays a regulatory role in the synthesis and secretion of HMG by human gastric mucous cells.

Response of germfree rat colonic mucous cells to peroral endotoxin application.

During microbial colonization, mucin-releasing goblet cells of germ-free (GF) rats proliferate and upregulate their mucin synthesis, thus improving the intestinal mucus barrier. The present study determined the significance of bacterial membrane constituents for this development. A single dose of lipopolysaccharide (LPS) (35 micrograms/100 g body weight) and lipid A (3.5 micrograms/100 g body weight, respectively), was perorally administered to GF AS/Ztm rats. One, 3 and 5 days later, sections of the proximal and distal colon served for characterization of mucin-secreting goblet cells, released mucins were isolated in parallel. Maximal goblet cell diameters were evidenced at day 3. LPS generated a maximal goblet cell hyperplasia one day after challenge, lipid A stimulated the goblet cell proliferation continuously up to day 5. Three days after challenge with one of the stimuli, either, intracellular mucins had shifted significantly to neutral constituents. In addition, mucins, adherent to the colon mucosa and submerged to the luminal content, respectively, then were augmented. At day 5, adherent mucins were similar to the controls, while luminal, soluble constituents had further increased. Histometrical and biochemical methods evidenced a transient, inflammatory response of mucin-secreting cells, followed by an upregulated release of immature mucins.

Effects of perorally applied endotoxin on colonic mucins of germfree rats.

BACKGROUND: The intestinal epithelium, with the potential to restrict luminal noxae from the host, secretes a mucous layer with various protective functions. Microbial colonization of germfree (GF) rats stimulates this mucin-secreting tissue. The present study determined the effect of bacterial lipopolysaccharides (LPS) on this process. METHODS: One, 3, and 5 days after peroral application of 35 micrograms LPS/100 g body weight (from Escherichia coli O55:B5), LPS concentrations were monitored in ingesta, intestinal tissue, and liver. Mucin high molecular weight glycoproteins (HMG), released in response to LPS, were isolated and separated into mucins, i) attached to the colonic epithelium (EM) and ii) mixed to the luminal content (LM), respectively. Subsequently, the binding capacity of both mucin fractions for various lectins and for type-1 pili expressing E. coli was determined. RESULTS: Ingesta and tissue had maximal LPS concentrations on days 3 (jejunum) and 5 (colon). Maximal EM secretion was found on day 3, release of LM further increased to day 5. Both mucin fractions had altered glycosylation patterns: augmentation of beta-galactose, alpha-N-acetyl galactosamine, and mannose coincided with a decrease in alpha-fucose. Compared with the controls, attachment of E. coli to EM increased slightly on day 1 only; the binding capacity of LM increased continuously up to day 5. CONCLUSION: Results suggest that mucins, released in response to LPS, in addition to the epithelial protection, support the gut microbial clearance system.

Response of the germfree rat colonic mucosa to dietary endotoxins.

Suggestive to induce an immunoreactive response in the colon mucosa of germfree rats 3 diets, autoclaved or gamma-irradiated, respectively, were administered to groups of 30 days old GF Ztm:SPRD rats. Diets and rats were studied in parallel after a feeding period of 20 days. The microbiological controls confirmed the sterility of diets and animals. The aqueous suspensions of one diet displayed obviously dead yeast and Gram positive and negative bacteria, which consistently were evident in colonic cast preparations of animals fed with this diet. These findings apparently were not related to the pathohistological alterations, observed in hematoxilin-eosin stained colon sections. However, dietary endotoxin concentrations between 0.6 and 10 micrograms LPS/g corresponded with the endotoxin concentrations in feces (0.3 to 3.1 micrograms LPS/g wet weight) and in colonic tissue (0.01 to 0.6 microgram LPS/g wet weight). These endotoxins obviously mediated a dose-dependent immunoreactive response of the colonic mucosa: in parallel to the dietary endotoxin content, the cellular infiltrations ranged from single mononuclear cells to severe, cell mediated, mucosal alteration. GF rats, used as an experimental animal model for inflammatory disorders of the intestine obviously necessitate diets with low endotoxin concentrations.

Response of rat colonic mucosa to a high fiber diet.

Secreting lubricating mucins, colonic crypt goblet cells, contribute to the intestinal protection against mechanical challenge. After feeding germ-free (GF) and specific pathogen-free (SPF) AS/Ztm rats for 6 weeks, the proliferative response of colonic goblet to a commercial bulky diet (37.1% fiber) was compared to that of a standard diet. (4.4% fiber). An increased uptake of the high fiber diet by GF rats significantly augmented the capacity for mucin secretion as indicated by the amount and length of crypts, crypt cells and mature goblet cells. The response of SPF rats was limited to a crypt elongation, generated by more crypt cells. In both study groups, the goblet cell replication activity was similar to their controls. The increase in the mucin-secreting capacity, induced by a constant mechanical challenge, highly suggests an improved intestinal protection.

Prostaglandin E2 alters terminal glycosylation of high molecular weight glycoproteins, released by pig gastric mucous cells in vitro.

The gastric mucus layer consists of high molecular weight glycoproteins (HMG). E-Type prostaglandins (PGs) stimulate total HMG release from isolated gastric mucous cells. We determined the effects of PGE2 on HMG glycosylation. Pig gastric mucous cells were cultured for 20 h with 1 mumol/l PGE2. Released HMG were isolated by gel chromatography and periodic acid-Schiff (PAS)-positive sugars and protein-bound [14C]GlcNAc were determined. Monosaccharides terminally linked to HMG oligosaccharide chains were monitored by lectin enzyme linked immunosorbent assay (ELISA): N-acetylglucosamine (GlcNAc) with Datura stramonium agglutinin, N-acetylgalactosamine (GalNAc) with soy bean agglutinin, fucose (Fuc) with Ulex europaeus I agglutinin and sialic acids (Sial) with Sambucus nigra agglutinin. PGE2 stimulated total HMG release, indicated by an increase of PAS-positive sugars to 170% and [14C]GlcNAc to 220% of controls. Terminal GlcNAc increased to 128%, GalNAc to 133%, Fuc to 165% and Sial to 182%. In addition to stimulation of total HMG release, PGE2 caused alterations of HMG glycosylation, which may modulate HMG viscosity and microbiological barrier function.

Mechanical challenge causes alterations of rat colonic mucosa and released mucins. Alterations of mucosa and mucins.

The effect of intraluminal challenge on rat colonic mucin producing cells and the amount and composition of released mucins was investigated. Germfree rats (GF) were maintained on a commercial high fiber (HF) diet (37% of undigestable fiber, Altromin 1640 p), in order to increase volume, dry weight and abrasive effect of the ingesta. GF control rats were fed a standard (ST) laboratory diet with 4.5% fiber (Altromin 1314 f). In the HF diet group, histological sections of the proximal and distal colon revealed a significantly increased number of mucin secreting goblet cells and an elevated goblet cell replication activity, as determined by 5'-bromo-deoxyuridine incorporation. The total amount of colonic mucins, isolated by gel filtration, was increased versus the control group. According to the results of ion exchange chromatography, carbohydrate and amino acid analysis, mucins from rats, given the HF diet, had an elevated content of acidic mucin constituents with alterations in the carbohydrate and amino acid composition. In a parallel study with specified pathogen free rats (SPF), the additional influence of the microflora on mucin secreting cells and isolated mucins was determined. An increased number of mucin secreting cells predominantly was observed in rats given the standard diet. Due to bacterial degradation, significantly less mucin was isolated from both dietary groups. The increase of acidic mucin constituents was less pronounced than in GF rat mucin, coinciding with losses of terminally linked monosaccharides. Alterations of the core protein, accompanying the presence of the microflora, were not detected.

Changes in colonic mucins of germfree rats in response to the introduction of a "normal" rat microbial flora. Rat colonic mucin.

In order to determine the influence of bacterial colonization on amount and composition of colonic mucins, germfree male AS/Ztm rats were colonized with a rat specific intestinal flora for different times (2, 7, 14, 21, 28, 35, 120 days). The amount of colonic mucins was determined by gel filtration on Sepharose CL-4B; the relative amount of acidic mucins was calculated after ion exchange chromatography. In addition, cecal weight and dry matter of feces were monitored. While germfree and SPF rats revealed similar amounts of colonic mucins (7.0 vs. 7.2 mg mucin/300 g body weight), the initial phase of association was characterized by considerably decreasing values. After four weeks of association, the total amount of colonic mucins had almost equalized in the two groups. The amount of acidic mucins, having decreased during the first three weeks of colonization, rendered values comparable to the SPF mucins after four months of adaptation. Cecomegaly in germfree rats disappeared within the first two days, while solidification of the intestinal content occurred within four months. Mucin losses during initial phase of association are attributed 1. to the disappearance of the cecal mucin pool, and 2. to the mucin degrading activity of some bacterial strains known to be present in the intestinal flora. Further development is conducted by a stimulation of mucin secretion, described to follow the colonization. The initially increased secretion of neutral mucins is attributed to a pronounced release of immature mucin glycoproteins, while the shift to more acidic mucins is considered to result from stimulated secretion as well as from a selective bacterial degradation of neutral mucin components.

Acidification of the colonic mucins following polyvalent colonization of the germ-free rat.

Colonic mucins of germ-free (GF) and conventional rats (CV) were compared. After isolation by gel filtration on Sepharose CL-4B and purification by density gradient centrifugation, the content of isolated colonic mucins was estimated by determination of PAS positive carbohydrates. Purified mucins were subjected to carbohydrate and amino acid analysis and separated into mucin subclasses by ion exchange chromatography. While the total amount of colonic mucins was not statistically different in GF and CV animals, analysis of carbohydrate composition demonstrated an increased amount of sialic acid in CV rat mucin. This was in accordance with results of ion exchange chromatography, revealing a significant higher amount of negative charged mucin subclasses in CV mucin, compared to the germ-free counterpart. The results of amino acid analysis were similar in both groups. The compositional differences in carbohydrate moieties are attributed to modulations by the intestinal flora. A selective bacterial degradation of the neutral mucin subclasses and modifications in the mucin composition due to a stimulated synthesis are discussed.

Pre-epithelial mucus layer in the colon of conventional and germ-free rats.

The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10 microns-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40 +/- 24 microns at 120 days of age and 44 +/- 22 microns at 350 days of age in conventional rats, and 25 +/- 17 microns (120 days) and 22 +/- 10 microns (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free; 185 +/- 73 microns, conventional: 350 +/- 115 microns). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.