Helicobacter pylori and its interaction with chief and parietal cells.

Helicobacter pylori has been shown, along with other Helicobacter species to produce effector molecules that induce substantial physiological changes on acid and pepsin secretion. The effects are clinically evident, and long-term achlorhydria may be a risk factor for gastric cancer. Further identification and characterization of the factors may lead to additional understanding of gastric physiology.     

幽门螺杆菌感染与胃黏膜上皮细胞DNA甲基化的研究进展

在癌症发生表观遗传学研究中,DNA甲基化是研究最多也最深入的一种机制,异常甲基化可导致胃黏膜上皮细胞癌变.幽门螺杆菌(Helicobacter pylori,Hpylori)是胃癌的第Ⅰ类致癌原,在“慢性浅表性胃炎-慢性萎缩性胃炎-肠上皮化生-不典型增生-胃癌”的癌变模式中可能起先导作用.胃癌相关基因的异常甲基化和H.pylori的致癌机制均是目前研究的热点,本研究综述H.pylori感染与胃黏膜上皮细胞DNA异常甲基化的关系,阐述H.pylori感染介导慢性炎症诱发胃黏膜上皮细胞DNA甲基化的机制.     

Brefeldin A enhances Helicobacter pylori vacuolating cytotoxin-induced vacuolation of epithelial cells

Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.     

Proliferative activity of gastric epithelial cells in Helicobacter pylori infected children

Helicobacter pylori infection has been associated with gastric carcinogenesis. Gastric epithelial cells proliferative rate is accelerated in H. pylori infected adult patients. Our study was performed to evaluate proliferative cell activity in gastric epithelium in the course of H. pylori infection in the early stage of its natural history. Gastric antral biopsy specimens were obtained from thirteen H. pylori positive and seven negative children. To assess replication rates we used nucleolar organiser regions staining with colloidal silver nitrate technique (AgNOR). The number of AgNORs per nucleus, area of single AgNOR, and the quotient of these two parameters (AgNOR content) were analysed. The mean area of AgNOR was lower in H. pylori positive than in negative children. Conversely, both the mean number of AgNOR per nucleus and AgNOR content were higher in infected than non infected subjects. These results show accelerated proliferation of gastric antral epithelial cells in the course of H. pylori infection in children. Such alteration of cell replication occurring in an initial phase of natural history of long lasting infection provides an explanation for the association between acquisition of H. pylori infection in the first years of life and the development of gastric cancer.     

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.     

Effects of Helicobacter pylori on biological characteristics of gastric epithelial cells

Infection with Helicobacter pylori (H. pylori) strains is linked to an increased risk of inflammation and gastric cancer. To investigate the effects of H. pylori on biological characteristics of gastric epithelial cells SGC-7901, derived from human adenocarcinoma, morphological appearances of both the pathogen and these cells, as well as features of attachment and internalization were observed by using transmission electron microscopy (TEM). We also investigated cell junctions and invasion by TEM and Transwell Invasion Assay. Cell proliferation and apoptosis were assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and flow cytometry. Three types of H. pylori were observed around, attaching to, or invading tumor cells. Cellular damage was characterized by vacuolar degeneration, dilated endoplasmic reticulum (ER), and reduction of organelles. Cell junctions and cell microvilli reduced or disappeared. H. pylori inhibited cell proliferation, whereas it had no effect on apoptosis. It also promoted gastric carcinoma cell invasion. H. pylori damages cell construction, destroys cell junctions, inhibits cell proliferation, promotes cell invasive ability, and, therefore, might accelerate the malignant progress and metastasis of gastric cancer.     

Persistence factors of Helicobacter pylori

Isolates of H. pylori from patients with chronic gastritis and peptic ulcer, were found to be capable of inactivating lysozyme and intercide (the bactericidal component of human leukocytic interferon). The expression and penetration capacity of their antilysozyme activity (ALA) and antiintercide activity was determined. The wide spread of ALA among H. pylori clinical isolates associated with inflammatory changes in bioptic specimens, confirmed the leading role of this microorganism in the pathogenesis of the gastric mucosal lesions. The retrospective analysis of clinical cases made it possible to recommend the use of the ALA sign as one of the criteria for choosing the scheme of eradication therapy even at the stage of the initial diagnostics of H. pylori infection.     

Helicobacter pylori interacts with heparin and heparin-dependent growth factors

Abstract The pathogenic bacterium Helicobacter pylori , which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 × 10⁻¹² M), and also heparan sulfate and heparin with higher affinity ( K _d 9 × 10⁻⁹ M) than FGFs bind to heparin (10⁻⁸–10⁻⁹ M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori , which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanism. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.     

Helicobacter pylori: molecular evolution of a bacterial quasi-species

Helicobacter pylori persists chronically within individuals and as they spread the mutating bacteria migrate with them. The continuous selection and microevolution generates a population of closely related but different bacteria that behave like a quasi-species. Within this heterogeneity, H. pylori strains fall into distinct types, into the virulent (type I) and less virulent (type II) strains, based on the presence of a pathogenicity island (cag) that encodes a specialized secretion machinery. We propose that during chronic infection a dynamic equilibrium between bacteria expressing a disparate degree of virulence is established, and that diverse forms prevail at different times.     

Structure and interaction of VacA of Helicobacter pylori with a lipid membrane.

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.     

External membrane vesicles from Helicobacter pylori induce apoptosis in gastric epithelial cells

The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.     

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

Molecular and cellular activities of Helicobacter pylori pathogenic factors.

Stomach infection with pathogenic strains of Helicobacter pylori causes in some patients severe gastroduodenal diseases. These bacteria produce various virulence factors and, here, we review the recent acquisition on the biochemical mode of action of three major factors. We discuss the role of urease both as buffer of the stomach pH and as source of ammonia. The vacuolating toxin alters the endocytic pathway of non-polarized cells, inducing the release of acid hydrolases, the depression of extracellular ligand degradation and of antigen processing and, in the presence of ammonia, swelling of late-prelysosomal compartments. In polarized epithelial monolayers, vacuolating toxin induces an increase of the paracellular permeability, independent of vacuolation. The neutrophil activating protein induces the production of oxygen radicals in human neutrophils and could contribute to the damage of the stomach mucosa. The activities of these factors are discussed in terms of the need of the bacterium of increasing the supply of nutrients from the stomach lumen and from the mucosa.     

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.     

Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells

BACKGROUND: Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection. MATERIALS AND METHODS: Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction. RESULTS: GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively). CONCLUSIONS: This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.     

Cardiovascular risk factors in subjects with Helicobacter pylori infection

Background. It has been proposed that Helicobacter pylori infection is related to cardiovascular disease, although this has not been fully investigated. The aim of this study was to investigate whether H. pylori in‐fection is associated with cardiovascular risk factors. Subjects and Methods. One thousand six hundred and fifty people undergoing annual medical checks at Shimane Institute of Health Science between September 1998 and August 1999 were enrolled. Gender, age, body mass index, habitual smoking and drinking, systolic and diastolic blood pressure, serum level of total cholesterol, triglyceride, high‐density lipoprotein cholesterol (HDLC), blood glucose, leukocyte count and hemoglobin were compared between H. pylori seropositive and seronegative cases. Results. In H. pylori seropositive individuals, HDLC was significantly lower than that in seronegative individuals. After adjustment for possible confounding factors (gender, age, BMI, smoking and drinking habits), mean HDLC in H. pylori‐seropositive and seronegative individuals were 56.1 and 58.2 mg/dl, respectively (p < .005). The percentage of the elderly (over 50 years old) individuals with HDLC < 35 mg/dl in H. pylori seropositive and seronegative groups were 7.4% and 4.7%, respectively (p < .001). In addition, the lower HDLC level was accompanied by an increased leukocyte count. Conclusion. Long‐term infection with H. pylori may have an important role in decreasing the serum HDLC concentration.