Helicobacter pylori infection up-regulates gland mucous cell-type mucins in gastric pyloric mucosa

Background. Two types of mucous cell are present in gastric mucosa: surface mucous cells (SMCs) and gland mucous cells (GMCs), which consist of cardiac gland cells, mucous neck cells, and pyloric gland cells. We have previously reported that the patterns of glycosylation of SMC mucins are reversibly altered by Helicobacter pylori infection. In this study, we evaluated the effects of H. pylori infection on the expression of GMC mucins in pyloric gland cells. Methods. Gastric biopsy specimens from the antrums of 30 H. pylori‐infected patients before and after eradication of H. pylori and 10 normal uninfected volunteers were examined by immunostaining for MUC6 (a core protein of GMC mucins), α1,4‐N‐acetyl‐glucosaminyl transferase (α4GnT) (the glycosyltransferase which forms GlcNAcα1‐4Galβ‐R), and GlcNAcα1‐4Galβ‐R (a GMC mucin‐specific glycan). Results. MUC6, α4GnT, and HIK1083‐reactive glycan were expressed in the cytoplasm, supranuclear region, and secretory granules in pyloric gland cells, respectively. The immunoreactivity of MUC6 and α4GnT, but not of GlcNAcα1‐4Galβ‐R, in the pyloric gland increased in H. pylori‐associated gastritis, and after the eradication of H. pylori, the increased expression of MUC6 and α4GnT in the gastric mucosa of H. pylori‐infected patients decreased to almost normal levels. This up‐regulation was correlated with the degree of inflammation. Conclusions. In addition to the synthesis of GMC mucins increasing reversibly, their metabolism or release may also increase reversibly in H. pylori‐associated gastritis. The up‐regulation of the expression of gastric GMC mucins may be involved in defense against H. pylori infection in the gastric surface mucous gel layer and on the gastric mucosa.     

Helicobacter pylori infection increases cell kinetics in human gastric epithelial cells without adhering to proliferating cells

We investigated the effect of H. pylori infection on cell proliferation of gastric mucosa using immunostaining for H. pylori or Ki67. H. pylori cells attached to surface mucous cells covering luminal surface and the upper part of gastric foveolae, and up-regulated the proliferative activity of gastric epithelial cells without adhering to the proliferating epithelial cells.     

pH-dependent binding of Helicobacter pylori to pig gastric mucins

A microtiter-based assay was developed to study the binding of Helicobacter pylori to pig gastric mucins purified by density-gradient centrifugation in CsCl/4 M guanidinium chloride. Binding of H. pylori was observed over the 'mucin' band as well as with 'low-density' components in the gradients, and binding to the latter was more pronounced when incubations were performed at 37 degrees C as compared to 20 degrees C. At a lower pH, binding of H. pylori (strain SVA 40) to the 'high-density' mucins from pig antrum was increased but binding to the 'low-density' ones was decreased. Binding of the P466 strain (Le(b)-specific) was mainly associated with the 'mucin' band, whereas the MO19 strain reacted preferentially with the 'low-density' components. In summary, H. pylori may bind to gastric mucins and the binding is influenced by temperature, pH and the repertoire of bacterial adhesins.     

Four modes of adhesion are used during Helicobacter pylori binding to human mucins in the oral and gastric niches

Background:  Helicobacter pylori causes peptic ulcer disease and gastric cancer, and the oral cavity is likely to serve as a reservoir for this pathogen. We investigated the binding of H. pylori to the mucins covering the mucosal surfaces in the niches along the oral to gastric infection route and during gastric disease and modeled the outcome of these interactions.
Materials and Methods:  A panel of seven H. pylori strains with defined binding properties was used to identify binding to human mucins from saliva, gastric juice, cardia, corpus, and antrum of healthy stomachs and of stomachs affected by gastritis at pH 7.4 and 3.0 using a microtiter-based method.
Results:  H. pylori binding to mucins differed substantially with the anatomic site, mucin type, pH, gastritis status, and H. pylori strain all having effect on binding. Mucins from saliva and gastric juice displayed the most diverse binding patterns, involving four modes of H. pylori adhesion and the MUC5B, MUC7, and MUC5AC mucins as well as the salivary agglutinin. Binding occurred via the blood-group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), a charge/low pH-dependent mechanism, and a novel saliva-binding adhesin. In the healthy gastric mucus layer only BabA and acid/charge affect binding to the mucins, whereas in gastritis, the BabA/Le^b-dependent binding to MUC5AC remained, and SabA and low pH binding increased.
Conclusions:  The four H. pylori adhesion modes binding to mucins are likely to play different roles during colonization of the oral to gastric niches and during long-term infection.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

Inhibition of Helicobacter pylori haemogglutination activity by human salivary mucins

Abstract Thirty isolates of Helicobacter pylori from gastric biopsies agglutinated human erthyrocyte suspensions. Crude mucin preparation derived from saliva of 20 different donors were examined for their ability to inhibit haemagglutination. All mucin preparations exhibited strong inhibitory activity. Removal of sialic residues from mucin preparations by treatment with neuraminidase resulted in a substantial reduction of their inhibitory activity. The mucin prepations had no bactericidal or aggregation activity for H. pylori . These results are discussed in the context of the role of mucins in colonization of the gastric mucosa by H. pylori     

Strain-dependent proliferation in response to human gastric mucin and adhesion properties of Helicobacter pylori are not affected by co-isolated Lactobacillus sp

Background: Helicobacter pylori colonize the mucus layer that covers the gastric epithelium and can cause gastritis, ulcers, and gastric cancer. Recently, Lactobacillus sp. have also been found to reside in this niche permanently. This study compares adhesive properties and proliferation of co‐isolated lactobacilli and H. pylori in the presence of mucins and investigates possibilities for lactobacilli‐mediated inhibition of H. pylori. Materials and methods: Binding and proliferation of four H. pylori and four Lactobacillus strains, simultaneously isolated after residing in the stomachs of four patients for >4 years, to human gastric mucins were investigated using microtiter‐based methods. Results: The H. pylori strains co‐isolated with lactobacilli exhibited the same mucin binding properties as demonstrated for H. pylori strains previously. In contrast, no binding to mucins was detected with the Lactobacillus strains. Proliferation of mucin‐binding H. pylori strains was stimulated by the presence of mucins, whereas proliferation of non‐binding H. pylori and Lactobacillus strains was unaffected. Associative cultures of co‐isolated H. pylori and Lactobacillus strains showed no inhibition of H. pylori proliferation because of the presence of whole bacteria or supernatant of lactobacilli. Conclusions: The presence of lactobacilli in the stomach did not select for different mucin binding properties of H. pylori, and Lactobacillus sp. did neither compete for binding sites nor inhibit the growth of co‐isolated H. pylori. The effects of human gastric mucins on H. pylori proliferation vary between strains, and the host–bacteria interaction in the mucus niche thus depends on both the H. pylori strain and the microenvironment provided by the host mucins.     

The MUC5AC glycoprotein is the primary receptor for Helicobacter pylori in the human stomach

Background and objectives. Helicobacter pylori shows a characteristic tropism for the mucus‐producing gastric epithelium. In infected patients, H. pylori colocalizes in situ with the gastric secretory mucin MUC5AC. The carbohydrate blood‐group antigen Lewis B (LeB) was deemed responsible for the adherence of H. pylori to the gastric surface epithelium. We sought to determine if MUC5AC is the carrier of LeB, and thus if MUC5AC is the underlying gene product functioning as the main receptor for H. pylori in the stomach. Methods. We studied three types of human tissue producing MUC5AC: Barrett's esophagus (BE), normal gastric tissue, and gastric metaplasia of the duodenum (GMD). Tissue sections were immuno‐fluorescently stained for MUC5AC or LeB, and subsequently incubated with one of three strains of Texas red‐labeled H. pylori, one of which was unable to bind to LeB. We determined the colocalization of MUC5AC or LeB with adherent H. pylori. Results. The binding patterns for the two LeB‐binding strains to all tissues were similar, whereas the strain unable to bind to LeB did not bind to any of the tissues. In normal gastric tissue, the LeB‐binding strains always bound to MUC5AC‐ and LeB‐positive epithelial cells. In four nonsecretor patients, colocalization of the LeB‐binding strains was found to MUC5AC‐positive gastric epithelial cells. In BE, the LeB‐binding H. pylori strains colocalized very specifically to MUC5AC‐positive cells. MUC5AC‐producing cells in GMD contained LeB. Yet, LeB‐binding H. pylori not only colocalized to MUC5AC or LeB present in GMD, but also bound to the LeB‐positive brush border of normal duodenal epithelium. Conclusions. Mucin MUC5AC is the most important carrier of the LeB carbohydrate structure in normal gastric tissue and forms the major receptor for H. pylori.     

Clinicopathological analysis of early-stage gastric cancers detected after successful eradication of Helicobacter pylori

Background and Aims: The results of a randomized controlled study and meta‐analysis study have recently proved that Helicobacter pylori eradication has a preventive effect against the development of metachronous and primary gastric cancer. However, gastric cancer is sometimes detected after successful eradication. There is a lack of study about gastric cancers in eradicated patients. To clarify the characteristics of gastric cancers detected after H. pylori eradication, we analyzed the clinicopathological features of these cancers. Methods: The subjects were 18 early‐stage gastric cancer specimens resected from 17 patients who had received successful eradication of H. pylori from February 1995 to March 2009. The control group consisted of 36 specimens from noneradicated patients with persistent H. pylori infection who were matched with the subjects in age, sex, and depth of invasion. Clinicopathological features and mucin phenotypes of gastric cancer were clinically and immunohistologically evaluated. Results: The average diameter of gastric cancer was smaller and Ki‐67 index was lower in the eradication group. The morphological distribution of depression types was significantly lower in the control group. Immunohistochemical phenotyping revealed that 72.2% of the lesions in the eradicated group were complete gastric type or gastric predominant mixed type, whereas the percentages of gastric type and intestinal type in the control group were similar. Conclusion: Our findings indicate that the clinicopathological characteristics of gastric cancers detected after H. pylori eradication are different from those of gastric cancers in patients with persistent H. pylori infection. H. pylori eradication may suppress intestinalization during the development of gastric cancer.     

Additive effect of pronase on the efficacy of eradication therapy against Helicobacter pylori

Background. Helicobacter pylori (H. pylori) colonizes not only the surface of the surface mucous cells but also the surface mucous gel layer (SMGL). Thus, we examined the possible value of pronase, a mucolytic agent, as a potential eradication therapy. Materials and Methods. One hundred and thirty‐five patients were randomly assigned to two treatment groups. Sixty‐eight patients received 30 mg of lansoprazole once daily, 500 mg of amoxicillin and 250 mg of metronidazole thrice daily for 2 weeks (LAM group), while the other 67 patients received the same dosage of those agents plus 18,000 tyrosine units of pronase thrice daily for 2 weeks (LAMP group). Eradication was assessed 4–6 weeks after treatment by immunohistochemical tests and cultures. We also determined the in vitro activity of pronase against H. pylori, and evaluated the synergistic effects between pronase and the other three drugs. To investigate the effect of pronase on the structure of the SMGL, surgically removed stomachs obtained from patients who had taken pronase were examined histopathologically. Results. The cure rates for H. pylori infection in the LAMP group were significantly higher than those in the LAM group (intention to treat analysis: 94.0 vs. 76.5%, p = .0041). Pronase exhibited no antibacterial activity against H. pylori., and no in vitro synergistic effects were observed. In the patients who took pronase before surgery, the SMGL was thinner than in the patients who did not take pronase, and the structure of the SMGL was markedly disrupted. Conclusions. Pronase has an additive effect in curing H. pylori infection. Pronase has no apparent in vitro activity against H. pylori, but may improve the local delivery of antibiotics by virtue of its removal and disruption of the SMGL.     

Bacterial factors that mediate colonization of the stomach and virulence of Helicobacter pylori

Helicobacter pylori is a Gram-negative microaerophilic organism that colonizes the gastric mucosa of humans. Helicobacter pylori is one of the most common infections in humans and results in the development of gastritis in all infected individuals, although the majority of people are asymptomatic. A subset of infected people develop serious disease including duodenal ulceration and gastric cancer. Helicobacter pylori exhibits many striking characteristics. It lives in the hostile environment of the stomach and displays a very strict host and tissue tropism. Despite a vigorous immune response, infection persists for the lifetime of the host unless eradicated with antimicrobials. Why H. pylori is so pathogenic in some individuals and not in others is unknown but is thought to be due to a variety of host, environmental and bacterial factors. In this review, some of the bacterial factors that mediate colonization of the gastric mucosa and play a role in the pathogenesis of this organism have been considered.     

Antiadhesive property of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin

The emergence of antibiotic-resistant Helicobacter pylori is of concern in the treatment of H. pylori-associated gastroduodenal diseases. As the organism was reported to bind gastric mucin, we used porcine gastric mucin as substrate to assess the antiadhesive property of polysaccharides derived from Spirulina (PS), a commercially available microalga, against the binding of H. pylori to gastric mucin. Results show that polysaccharides prevented H. pylori from binding to gastric mucin optimally at pH 2.0, without affecting the viability of either bacteria or gastric epithelial cells, thus favouring its antiadhesive action in a gastric environment. Using ligand overlay analysis, polysaccharide was demonstrated to bind H. pylori alkyl hydroperoxide reductase (AhpC) and urease, which have shown here to possess mucin-binding activity. An in vivo study demonstrated that bacteria load was reduced by >90% in BALB/c mice treated with either Spirulina or polysaccharides. It is thus suggested that polysaccharides may function as a potential antiadhesive agent against H. pylori colonization of gastric mucin.     

Protein changes in gastric epithelial cells RGM-1 in response to Helicobacter pylori infection

Helicobacter pylori-induced inflammation significantly increases the risk of gastric cancer. To investigate the role of H. pylori infection in gastric epithelial cell carcinogenesis, flow cytometry was used to analyze the apoptosis of gastric epithelial cells infected by H. pylori. Next, LTQ MS mass spectrometry (MS) was applied to identify protein changes in gastric epithelial cells infected with H. pylori, and then bioinformatics was adopted to analyze the cellular localization and biological function of differential proteins. LTQ MS/MS successfully identified identified 22 differential proteins successfully, including 20 host-cell proteins and two H. pylori bacterial proteins. Also, human proteins were located in all areas of cells and involved in various cell biological functions. The oncogene proteins p53, p16, and C-erbB-2 proteins in H. pylori-infected RGM-1 cells were remarkably increased from the analysis by Western blot analysis. H. pylori infection of gastric epithelial cells leads to changes in various protein components in the cell, and enhances the expression of oncogene proteins, thereby increasing the possibility of possibility of carcinogenesis of H. pylori infection.     

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells

Background. Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. Methods. Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. Results. DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5–75.8), n = 18 and 21% (11.9–29.8), n = 65, respectively, p < .001]. Intermediate levels were found in reactive gastritis [55.5% (41.3–71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3–60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3–51.0), p = .007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = –.92 and –.88 for normal mucosa and H. pylori gastritis, respectively). Conclusions. Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.