Kyi T. Tham

Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori

BACKGROUND & AIMS vacA encodes the vacuolating cytotoxin of Helicobacter pylori and exhibits marked variation in signal sequence and midgene coding regions. The implications for gastroduodenal pathology are unknown. The aim of this study was to define the association of vacA genotype with gastric inflammation and injury, in vitro cytotoxin activity, and peptic ulceration. METHODS Sixty-one consecutive dyspeptic patients underwent endoscopy and gastric biopsy. The biopsy specimens were processed for H. pylori culture, and 52 specimens were also processed for histology. H. pylori vacA was typed by polymerase chain reaction and colony hybridization. Cytotoxin activity was assessed by a HeLa cell vacuolation assay. RESULTS vacA signal sequence type s1a strains were associated with greater antral mucosal neutrophil and lymphocyte infiltration than s1b or s2 strains (P < 0.05). vacA midregion type m1 strains were associated with greater gastric epithelial damage than m2 strains (P < 0.05). Both midregion and signal sequence were associated with cytotoxin activity in vitro. Duodenal ulcer disease occurred in 89% of 18 patients with s1a strains vs. 29% of 14 with s1b strains (P < 0.01), 20% of 10 with s2 strains (P < 0.001), and 16% of 19 uninfected patients (P < 0.001). CONCLUSIONS H. pylori strains of vacA signal sequence type s1a are associated with enhanced gastric inflammation and duodenal ulceration. vacA s2 strains are associated with less inflammation and lower ulcer prevalence.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Helicobacter pylori alters gastric epithelial cell cycle events and gastrin secretion in Mongolian gerbils

BACKGROUND & AIMS Human colonization with Helicobacter pylori increases the risk for distal gastric adenocarcinoma, possibly by altering gastric epithelial cell cycle events and/or gastrin secretion. This study aimed to determine whether H. pylori virulence-related characteristics affect apoptosis, proliferation, and gastrin levels in a rodent model of gastric adenocarcinoma. METHODS Mongolian gerbils were challenged with H. pylori wild-type or isogenic cagA(-) and vacA(-) mutants, and apoptotic and proliferating cells were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and proliferating cell nuclear antigen immunohistochemistry, respectively. Serum gastrin levels were determined by radioimmunoassay. RESULTS Gastric epithelial cell turnover was no different after infection with the wild-type, cagA(-), or vacA(-) strains. H. pylori infection significantly increased antral apoptosis 2-4 weeks after challenge, before apoptotic indices decreased to baseline. In contrast, antral proliferation rates were significantly higher 16-20 weeks after inoculation, but then decreased by 40 weeks. Antral proliferation was significantly related to serum gastrin levels, whereas antral apoptosis was inversely related to acute inflammation and lymphoid follicles. CONCLUSIONS In H. pylori-infected gerbils, enhanced antral apoptosis is an early and transient cell cycle event. Epithelial cell proliferation peaks later and is significantly related to increased gastrin levels, suggesting that epithelial cell growth in H. pylori-colonized mucosa may be mediated by gastrin-dependent mechanisms.

Quantitative detection of Helicobacter pylori gene expression in vivo and relationship to gastric pathology.

ABSTRACT The iceA locus of Helicobacter pyloriincludes one of two mutually exclusive gene families, iceA1and iceA2. Colonization with iceA1 strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression oficeA1 but not iceA2 mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of iceA transcription in vivo may contribute to disease pathogenesis. RNA from 41 H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1, iceA2, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluorescein-labeled probe. iceAgenotypes were determined by PCR and sequence analysis. All 41 samples contained detectable H. pylori 16S rRNA, with similar levels in iceA1- (n = 10) andiceA2 (n = 31)-colonized patients (P = 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with iceA1 or iceA2strains, respectively, had detectable iceA RNA. Acute inflammatory scores were significantly higher in iceA1RNA-positive patients than in iceA1 RNA-negative,iceA2 RNA-positive, or iceA2 RNA-negative subjects (P ≤ 0.05 for each). Within theiceA2 RNA-positive group, H. pylori strains with a single 35-amino-acid cassette were associated with significantly higher mucosal iceA2 transcript levels (P= 0.014 versus strains with two cassettes). These results indicate that the levels of transcription of H. pylori iceA1 andiceA2 and of 16S rRNA are independent and that particulariceA2 gene structures are associated with enhanced transcription. The finding that iceA1 transcription levels are significantly associated with the intensity of neutrophilic infiltration suggests that heterogeneity in inflammatory scores among persons colonized with H. pylori iceA1 strains reflects levels of iceA1 transcription in vivo.

Local and systemic immune and inflammatory responses to Helicobacter pylori strains.

ABSTRACT Colonization with Helicobacter pylori eventuates in varied clinical outcomes, which relate to both bacterial and host factors. Here we examine the relationships between cagA status, serum and gastric juice antibody responses, and gastric inflammation in dyspeptic patients. Serum, gastric juice, and gastric biopsy specimens were obtained from 89 patients undergoing endoscopy. H. pylori colonization and cagA status were determined by histology, culture, and PCR methods, and acute inflammation and chronic inflammation in the gastric mucosa were scored by a single pathologist. Serum and gastric juice antibodies to H. pylori whole-cell and CagA antigens were determined by enzyme-linked immunosorbent assay. Relationships between variables were sequentially analyzed using univariate and multivariate statistical methods. Of the 89 subjects, 62 were colonized by H. pylori. By univariate analyses, levels of serum immunoglobulin G (IgG) and IgA and gastric juice IgA antibodies against whole-cell and CagA antigens each were significantly higher in the H. pylori-positive group than in the H. pylori-negative group (P < 0.001). H. pylori and CagA seropositivities were both significantly associated with enhanced inflammation in gastric antrum and body (P < 0.02). The presence of gastric juice antibodies to H. pylori antigens was associated with more severe gastric inflammation. However, in multivariate analyses, only the presence of serum antibodies against CagA and, to a lesser extent, whole-cell antigens remained significantly associated with acute and chronic inflammation in antrum and body (P < 0.05). Thus, serum antibody response to CagA correlates with severity of gastric inflammation. Furthermore, given the relationships demonstrated by multivariate analysis, determination of gastric juice antibodies may provide a better representation of serum, rather than secretory, immune response.