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ABSTRACTPersistent Helicobacter pyloriinfection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylorifacilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pyloriMV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pyloristrains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pyloriMV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pyloriMV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity.

ABSTRACTThe present report describes a novel method for genotyping the virulence-associated vacAintermediate (i) region of Helicobacter pyloriin archive material. vacAi-region genotypes as determined by the novel method were completely concordant with those of sequence analysis and with those of functional vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective characterization of the vacAi region was obtained in 191 of 192 (99.5%) frozen and in 186 of 194 (95.9%) formalin-fixed paraffin-embedded gastric biopsy specimens, respectively. The genotyping method was next used to address the relationship between the vacAgenotypes and the cagAstatus. The vacAi1 genotype was associated with vacAs1 (where s indicates signal region), vacAm1 (where m indicates middle region), and cagA-positive genotypes (P< 0.0001), while the vacAi2 genotype was closely related with vacAs2, vacAm2, and cagA-negative genotypes (P< 0.0001). The relationship between H. pylori vacAi-region genotypes and gastric disease development was subsequently evaluated in the Portuguese population. Patients infected with vacAi1 strains showed an increased risk for gastric atrophy and for gastric carcinoma, with odds ratios of 8.0 (95% confidence interval [CI], 2.3 to 27) and of 22 (95% CI, 7.9 to 63), respectively. Taken together, the results show that this novel H. pylori vacAi-region genotyping method can be applied directly to archive material, providing a fast evaluation of strain virulence determinants without the need of culture. The results further emphasize that the characterization of the vacAi region may be useful to identify patients at higher risk of gastric carcinoma development.

ABSTRACTHelicobacter pyloricauses peptic ulceration and gastric adenocarcinoma; the latter is common in Iran but not in Iraq. We hypothesized that more virulent H. pyloristrains may be found in Iran than in Iraq and so compared established and newly described virulence factors in strains from these countries. We studied 59 unselected dyspeptic patients from Iran and 49 from Iraq. cagAwas found in similar proportions of strains from both countries (76% in Iran versus 71% in Iraq) and was significantly associated with peptic ulcer disease in Iraq (P= 0.01) but not in Iran. cagAalleles encoding four or more tyrosine phosphorylation motifs were found in 12% of the Iranian strains but none of the Iraqi strains (P= 0.02). There were no significant differences in the vacAsignal-, middle-, or intermediate-region types between Iranian and Iraqi strains. Among the strains from Iran, vacAgenotypes showed no specific peptic ulcer associations, but among the strains from Iraq, vacAi1 strains were associated with gastric ulcer (P= 0.02), mimicking their previously demonstrated association with gastric cancer in Iran. dupAwas found in similar proportions of Iranian and Iraqi strains (38% and 32%, respectively) and was associated with peptic ulceration in Iraqi patients (P= 0.01) but not Iranian patients. H. pyloristrains from Iraq and Iran possess virulence factors similar to those in Western countries. The presence of cagAwith more phosphorylation motifs in Iranian strains may contribute to the higher incidence of gastric cancer. However, the association between strain virulence markers and disease in Iraq but not Iran suggests that other host and environmental factors may be more important in the disease-prone Iranian population.

ABSTRACTHelicobacter pyloristrains possessing the cagpathogenicity island (PaI) are associated with the development of gastroduodenal diseases, including gastric cancer. cagPaI 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 cagPaI. To date, however, no cagPaI 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 cagPaI. All strains possessed cagAand cagE, and we observed a variation in the length of cagAvariable regions. Nucleotide sequencing of the cagAvariable 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 cagAmay underlie the high rate of gastric cancer in China.

ABSTRACTWe show here that Helicobacter pyloribroth culture supernatants disrupt the actin cytoskeleton of epithelial cell lines, leading to cell rounding and apoptosis through anoikis. We demonstrate that there are marked quantitative differences between strains and that there are different cell line sensitivities. By constructing VacA null isogenic mutants, we show that the effect is not due to the vacuolating cytotoxin.