Prevalence of serum antibodies to Helicobacter pylori VacA and CagA and gastric diseases in Chile

The objective of this study was to evaluate the prevalence of antibodies to Helicobacter pylori CagA and VacA proteins and correlate this prevalence with gastric diseases in colonised Chileans. The study was performed in 418 adults colonised with H. pylori: 316 with gastroduodenal pathology (152 duodenal ulcer, 14 gastric cancer and 150 gastritis patients) and 102 asymptomatic subjects. Serum IgG antibodies to H. pylori were determined by enzyme immunoassay (EIA). Antibodies to VacA and CagA proteins were detected by Western blotting. In a subgroup of the patients, the vacuolating activity was determined by HeLa cell assay and the CagA product was confirmed by PCR assay. IgG antibodies to both VacA and CagA proteins of H. pylori were found in 270 (85%) of 316 colonised gastric patients and in 72 (71%) of 102 asymptomatic subjects. Colonisation with virulent strains was significantly higher among duodenal ulcer and gastric cancer patients than in gastritis patients or asymptomatic subjects. Infections with VacA+/ CagA+ H. pylori strains is common in Chile but, in contrast to some Asian countries, this phenotype was more prevalent in isolates from patients with more severe gastric pathologies.     

Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA.

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.     

幽门螺杆菌的CagA和VacA的致病机制及其临床意义

幽门螺杆菌(Helicobacterpylori,H.pylori)是消化道的主要致病因素之一,随着对H.pylori认识的深入,人们发现H.pylori的两个标志性毒素--细胞毒素相关蛋白(cytotoxin-associatedprotein,cagA)和空泡毒素(vacuolatingcytotoxin,VacA)不仅关系紧密,而且在致病过程中扮演了重要角色.本文主要阐述CagA和VacA的致病机制和它们的临床意义.     

Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.     

Comparison of Four Serological Tests To Determine the CagA or VacA Status of Helicobacter pylori Strains

We compared four tests for antibodies to CagA or VacA, HelicoBlot 2.0, RIDA Blot Helicobacter, CHIRON RIBA H. pylori SIA, and an enzyme-linked immunosorbent assay using recombinant CagA. Immunoblot assays were accurate for determining Helicobacter pylori status but poor for determining CagA or VacA status (accuracy, 66 to 80% for CagA status and 34 to 67% for VacA status). None can be recommended for determining CagA or VacA status.     

CagA subtyping in Helicobacter pylori isolates from gastric cancer patients in an ethnic Kashmiri population

The association between gastric cancer and Helicobacter pylori has been well established. Among H. pylori virulence genes the most important determinant is the cytotoxin associated antigen gene (cagA) which is characterized by the presence of repeated EPIYA motifs at the C terminus of the protein. From the alignment and number of these EPIYA motifs, two major types of CagA protein have been identified.AimsThe aim of this study was to classify the CagA into eastern or western type and to determine the number and type of motifs present.MethodsThe CagA subtyping was done by PCR and multiplex PCR for eastern/western classification and determination of EPIYA motifs respectively.ResultsAll the isolates studied were of the western type, with 70% of the isolates having more than one EPIYA-C motifs. No statistically significant association was found between the presence of CagA and more than one EPIYA-C motifs with the clinical outcome (differentiation status of the tumour).     

Helicobacter pylori and gastric cancer: factors that modulate disease risk

Helicobacter pylori is a gastric pathogen that colonizes approximately 50% of the world's population. Infection with H. pylori causes chronic inflammation and significantly increases the risk of developing duodenal and gastric ulcer disease and gastric cancer. Infection with H. pylori is the strongest known risk factor for gastric cancer, which is the second leading cause of cancer-related deaths worldwide. Once H. pylori colonizes the gastric environment, it persists for the lifetime of the host, suggesting that the host immune response is ineffective in clearing this bacterium. In this review, we discuss the host immune response and examine other host factors that increase the pathogenic potential of this bacterium, including host polymorphisms, alterations to the apical-junctional complex, and the effects of environmental factors. In addition to host effects and responses, H. pylori strains are genetically diverse. We discuss the main virulence determinants in H. pylori strains and the correlation between these and the diverse clinical outcomes following H. pylori infection. Since H. pylori inhibits the gastric epithelium of half of the world, it is crucial that we continue to gain understanding of host and microbial factors that increase the risk of developing more severe clinical outcomes.     

Helicobacter pylori VacA toxin promotes bacterial intracellular survival in gastric epithelial cells

Helicobacter pylori colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that H. pylori can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of H. pylori invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of H. pylori's intracellular niche. We show that in AGS cells infected with H. pylori, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the H. pylori-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the H. pylori-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection.     

Helicobacter pylori infection and the risk of gastric carcinoma

BACKGROUND. Infection with Helicobacter pylori has been linked with chronic atrophic gastritis, an inflammatory precursor of gastric adenocarcinoma. In a nested case-control study, we explored whether H. pylori infection increases the risk of gastric carcinoma. METHODS. From a cohort of 128,992 persons followed since the mid-1960s at a health maintenance organization, 186 patients with gastric carcinoma were selected as case patients and were matched according to age, sex, and race with 186 control subjects without gastric carcinoma. Stored serum samples collected during the 1960s were tested for IgG antibodies to H. pylori by enzyme-linked immunosorbent assay. Data on cigarette use, blood group, ulcer disease, and gastric surgery were obtained from questionnaires administered at enrollment. Tissue sections and pathology reports were reviewed to confirm the histologic results. RESULTS. The mean time between serum collection and the diagnosis of gastric carcinoma was 14.2 years. Of the 109 patients with confirmed gastric adenocarcinoma (excluding tumors of the gastroesophageal junction), 84 percent had been infected previously with H. pylori, as compared with 61 percent of the matched control subjects (odds ratio, 3.6; 95 percent confidence interval, 1.8 to 7.3). Tumors of the gastroesophageal junction were not linked to H. pylori infection, nor were tumors in the gastric cardia. H. pylori was a particularly strong risk factor for stomach cancer in women (odds ratio, 18) and blacks (odds ratio, 9). A history of gastric surgery was independently associated with the development of cancer (odds ratio, 17; P = 0.03), but a history of peptic ulcer disease was negatively associated with subsequent gastric carcinoma (odds ratio, 0.2; P = 0.02). Neither blood group nor smoking history affected risk. CONCLUSIONS. Infection with H. pylori is associated with an increased risk of gastric adenocarcinoma and may be a cofactor in the pathogenesis of this malignant condition.     

Reliability of Helicobacter pylori and CagA serological assays

Background serological assays for Helicobacter pylori are commonly used without knowledge of reliability. This information is needed to define the ability of serological tests to determine either new cases of infection or loss of infection in longitudinal studies. We evaluated the reproducibility and the interrelationships of serological test results for H. pylori and cytotoxin-associated gene product A (CagA) enzyme-linked immunoassays within a subset of participants in a population-based study. Stored samples from 1,229 participants in the third U.S. National Health and Nutrition Examination Survey were replicate serologically tested for H. pylori and CagA. Overall disagreement was 3.4% between duplicate tests for H. pylori (or 2.3% if equivocal results were disregarded). Six percent of samples positive on the first test had an immune serum ratio at least 30% lower on repeat testing. The odds ratio for H. pylori seropositivity on retesting was 2.8 (95% confidence interval [CI] = 1.8 to 4.5) when CagA serology was positive versus when it was negative. CagA antibody was found among 47.8% of H. pylori-equivocal and 7.0% of H. pylori-negative samples. CagA-positive yet H. pylori-negative samples were more likely to occur among Mexican Americans (odds ratio, 5.2; 95% CI = 2.4 to 11.4) and non-Hispanic blacks (odds ratio, 5.5; 95% CI = 2.3 to 13.0) than among non-Hispanic whites. Relying on repeated H. pylori serological tests over time to determine infection rates may result in misinterpretation due to limits in test reproducibility. CagA testing may have a role in verifying infection.     

Helicobacter pylori infection and chronic gastritis in gastric cancer.

AIMS: To investigate the prevalence of Helicobacter pylori associated chronic gastritis in patients with gastric cancer. METHODS: Serum IgG antibodies for H pylori were determined in 54 consecutive patients with gastric carcinoma. The prevalence of H pylori in gastric mucosa was also examined histologically (modified Giemsa) in 32 patients from whom adequate biopsy specimens of the antrum and corpus were available. Thirty five patients with gastrointestinal tumours outside the stomach and 48 with non-gastrointestinal malignancies served as controls. RESULTS: Of the 54 patients, 38 (70%) had H pylori antibodies (IgG) in their serum (three additional patients had H pylori antibodies IgA, class specific but not IgG specific). This prevalence was significantly higher (p less than 0.05) than that (49%) in the 35 controls. No differences in prevalence of H pylori antibodies were found between gastric cancer cases of intestinal (IGCA) or diffuse (DGCA) type, both these types showing H pylori antibodies (IgG) in 71% of the patients. In the subgroup of 32 subjects, five patients had normal gastric mucosa and four showed corpus limited atrophy ("pernicious anaemia type" atrophy of type A). All of these nine patients had no evidence of current or previous H pylori infection in serum (no IgG antibodies) or in tissue sections (negative Giemsa staining). The remaining 23 patients had antral or pangastritis, and all had evidence of current or previous H pylori infection. CONCLUSIONS: H pylori associated chronic gastritis was the associated disease in 75% of the patients with gastric cancer occurring equally often in both IGCA and DGCA groups. About 25% of cases seem to have a normal stomach or severe corpus limited atrophy, neither of which showed evidence of concomitant H pylori infection.     

Reliability of Helicobacter pylori and CagA Serological Assays

Background serological assays for Helicobacter pylori are commonly used without knowledge of reliability. This information is needed to define the ability of serological tests to determine either new cases of infection or loss of infection in longitudinal studies. We evaluated the reproducibility and the interrelationships of serological test results for H. pylori and cytotoxin-associated gene product A (CagA) enzyme-linked immunoassays within a subset of participants in a population-based study. Stored samples from 1,229 participants in the third U.S. National Health and Nutrition Examination Survey were replicate serologically tested for H. pylori and CagA. Overall disagreement was 3.4% between duplicate tests for H. pylori (or 2.3% if equivocal results were disregarded). Six percent of samples positive on the first test had an immune serum ratio at least 30% lower on repeat testing. The odds ratio for H. pylori seropositivity on retesting was 2.8 (95% confidence interval [CI] = 1.8 to 4.5) when CagA serology was positive versus when it was negative. CagA antibody was found among 47.8% of H. pylori-equivocal and 7.0% of H. pylori-negative samples. CagA-positive yet H. pylori-negative samples were more likely to occur among Mexican Americans (odds ratio, 5.2; 95% CI = 2.4 to 11.4) and non-Hispanic blacks (odds ratio, 5.5; 95% CI = 2.3 to 13.0) than among non-Hispanic whites. Relying on repeated H. pylori serological tests over time to determine infection rates may result in misinterpretation due to limits in test reproducibility. CagA testing may have a role in verifying infection.     

The inflammatory response in CD1 mice shortly after infection with a CagA+/VacA+ Helicobacter pylori strain

To investigate the early events of Helicobacter pylori infection in a mouse model, CD1 mice were infected with a type I (CagA+/VacA+) H. pylori strain. Up to 4 weeks after infection the majority of gastric tissue biopsies were positive in culture. Immunohistochemical analysis showed that inflammatory changes started to occur after 3 weeks. Four weeks after infection a significant increase in T cells was observed in the cardia/corpus region of the stomachs of infected mice. These T cells were CD4+ and CD8+, and they were located in an area with increased expression of MHC class II antigens. In 50% of the infected mice also an increased number of mast cells was seen. Furthermore, aggregates of B and T cells were present in the submucosa. Characterization of cytokines by immunohistochemistry showed an increase in IL-5-secreting cells in the inflamed area of the infected stomach. No difference was observed between interferon-gamma (IFN-gamma)-, IL-4- and IL-10-secreting cells in control and infected mice. These results suggest that no polarized T-helper cell response was present at this early phase of infection. Infection with H. pylori also induced a serum response and especially IgG was increased after 4 weeks of infection. However, no particular increase in IgG1, IgG2a or IgG3 isotype was observed. Part of the serum antibodies was directed against lipopolysaccharide (LPS), but no evidence for anti-Lewis antibodies or antibodies against epitopes on the gastric mucosa was found.     

Helicobacter pylori infection and the development of gastric cancer

BACKGROUND: Although many studies have found an association between Helicobacter pylori infection and the development of gastric cancer, many aspects of this relation remain uncertain. METHODS: We prospectively studied 1526 Japanese patients who had duodenal ulcers, gastric ulcers, gastric hyperplasia, or nonulcer dyspepsia at the time of enrollment; 1246 had H. pylori infection and 280 did not. The mean follow-up was 7.8 years (range, 1.0 to 10.6). Patients underwent endoscopy with biopsy at enrollment and then between one and three years after enrollment. H. pylori infection was assessed by histologic examination, serologic testing, and rapid urease tests and was defined by a positive result on any of these tests. RESULTS: Gastric cancers developed in 36 (2.9 percent) of the infected and none of the uninfected patients. There were 23 intestinal-type and 13 diffuse-type cancers. Among the patients with H. pylori infection, those with severe gastric atrophy, corpus-predominant gastritis, and intestinal metaplasia were at significantly higher risk for gastric cancer. We detected gastric cancers in 21 (4.7 percent) of the 445 patients with nonulcer dyspepsia, 10 (3.4 percent) of the 297 with gastric ulcers, 5 (2.2 percent) of the 229 with gastric hyperplastic polyps, and none of the 275 with duodenal ulcers. CONCLUSIONS: Gastric cancer develops in persons infected with H. pylori but not in uninfected persons. Those with histologic findings of severe gastric atrophy, corpus-predominant gastritis, or intestinal metaplasia are at increased risk. Persons with H. pylori infection and nonulcer dyspepsia, gastric ulcers, or gastric hyperplastic polyps are also at risk, but those with duodenal ulcers are not.     

Tyrosine phosphorylation of CagA from Chinese Helicobacter pylori isolates in AGS gastric epithelial cells

Helicobacter pylori strains possessing the cag pathogenicity island (PaI) are associated with the development of gastroduodenal diseases, including gastric cancer. cag PaI products induce the secretion of interleukin-8 (IL-8) from epithelial cells and facilitate the translocation of CagA into the cell cytosol. In East Asia, where the incidence of gastric cancer is high, most strains possess the cag PaI. To date, however, no cag PaI phenotypic data have been provided for strains isolated in mainland China. Here we used 31 Chinese strains to determine the genotypic and phenotypic status of the cag PaI. All strains possessed cagA and cagE, and we observed a variation in the length of cagA variable regions. Nucleotide sequencing of the cagA variable region revealed that CagA was of two types, a short "Western" form with two tyrosine phosphorylation sites and a longer "East Asian" form with three tyrosine phosphorylation sites. Coculture of strains with AGS epithelial cells showed that strains could induce IL-8 secretion from the cells and that CagA with three phosphorylation sites became more phosphorylated than that with two and could induce significantly (P < 0.001) more cells to elongate. We hypothesize that the preponderance of the more active East Asian form of cagA may underlie the high rate of gastric cancer in China.     

High frequency of CagA+ Helicobacter pylori infection in high-grade gastric MALT B-cell lymphomas

A high incidence of Helicobacter pylori infection has been found in patients with gastric MALT (mucosa-associated lymphoid tissue) B-cell lymphoma. Recent studies have indicated that the aggressive strains of the bacterium containing the CagA gene may have direct effects on tumourigenesis. To investigate the involvement of CagA+ strains in MALT lymphomagenesis, a sensitive polymerase chain reaction (PCR)-based detection assay for the gene was developed. DNA extracts from paraffin sections of 123 H. pylori-related gastric biopsies from Italy were analysed, including 56 cases of chronic gastritis, 37 low-grade, and 30 high-grade MALT lymphomas: 30·3 per cent (17/56) of the gastritis cases, 37·8 per cent (14/37) of the low-grade, and 76·7 per cent (23/30) of the high-grade MALT lymphomas were found to contain the CagA gene. The frequency of CagA+ strain infection was signfiicantly higher (P<0·05) in high-grade than in low-grade MALT lymphoma or gastritis. These results suggest that high-grade gastric MALT lymphoma transformation may be more likely to occur following infection by CagA+ strains of H. pylori. © 1998 John Wiley & Sons, Ltd.