Takafumi Ando

Since the associations between Helicobacter pylori genotype and disease differ in Asia and the West, we investigated the correlation between HP0638, encoding an outer membrane protein, and potential markers of virulence ( cagA , vacA , and iceA ). For 109 strains from nine countries, the status of cagA , vacA , and iceA was determined by PCR and/or a line probe assay. We also studied 18 strains from 8 patients (parents and 6 daughters) from a Dutch family and paired strains collected on average 8 years apart from 11 patients. When the HP0638 signal sequences were amplified by PCR and DNA sequence determinations were performed, 89 (96%) of 93 cagA -positive strains had HP0638 in frame, versus none (0%) of 16 cagA -negative strains ( P < 0.001). Among strains in which HP0638 was in frame, a six-CT dinucleotide repeat pattern was dominant in Western countries (23 of 33 strains [70%]), while a pattern of three CT repeats with another CT after four T’s (3 + 1-CT-repeat pattern) was dominant in East Asia (31 of 46 strains [67%]); however, specific CT repeat patterns did not correlate with clinical outcome. HP0638 phylogenetic trees also showed geographic characters. The HP0638 frame status and CT dinucleotide repeat patterns were identical for 9 of 11 pairs of strains obtained on average 8 years apart from individuals and the 15 strains obtained from the mother and all six daughters. Thus, HP0638 frame status and cagA status are strongly correlated. The CT dinucleotide repeat pattern in the putative HP0638 signal sequence has geographic characters and appears stable in particular patients and families over a period of years. Analysis of HP0638 CT polymorphisms may serve as a new typing system to discriminate H . pylori isolates for epidemiological purposes.

Associations of Helicobacter pylori genotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-three cagA⁺ vacAs1/m1 H. pylori isolates (United States, n = 24; Japan, n = 39) and eight cagA-negative vacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/or cag island mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and Japanese cagA⁺ vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frame oipA open reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus, H. pylori cagA⁺ vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by the cag island in vitro. Assessment of apoptosis in vitro identified a group of H. pylori isolates that induce more severe gastric inflammation and atrophy.

Helicobacter pyloripersists in the human stomach despite eliciting both cellular and humoral immune responses and inducing proinflammatory cytokines. To determine whether local humoral and cytokine responses are related to each other and to histologic responses, we studied 66 Japanese patients who underwent gastroscopy. Using specific enzyme-linked immunosorbent assays, we examined gastric antral mucosal-organ biopsy culture supernatants to assess interleukin-6 (IL-6) and interleukin-8 (IL-8) levels and antibody responses toH. pyloriwhole-cell antigens CagA, HspA, and HspB. Of the patients studied, 11 wereH. pylorinegative and 55 wereH. pyloripositive; by PCR, all strains werecagA⁺. As expected, compared toH. pylori-negative patients,H. pylori-positive patients had significantly higher humoral responses to allH. pyloriantigens and had higher IL-8 (47.8 ± 3.5 versus 10.1 ± 4.3 ng/mg of biopsy protein;P< 0.001) and IL-6 levels (2.8 ± 0.3 versus 0.26 ± 0.2 ng/mg of protein;P< 0.001). Among theH. pylori-positive patients, supernatant anti-CagA immunoglobulin G (IgG) levels were significantly associated withH. pyloridensity (P< 0.005) and neutrophil infiltration (P< 0.005) scores. Anti-CagA immunoglobulin A levels were correlated with intestinal metaplasia (P< 0.05). Mononuclear cell infiltration scores were significantly associated with supernatant IL-6 levels (P< 0.005) and with IgG responses to whole-cell antigens (P< 0.05). Supernatant IL-8 levels were significantly associated with anti-CagA IgG (r= 0.75,P< 0.001). Anti-CagA responses correlated with neutrophil infiltration, intestinal metaplasia,H. pyloridensity, and IL-8 levels, suggesting that the absolute levels of these antibodies may be markers for gastric inflammation and premalignant changes in individual hosts.