Briefly, 200 ng of each sample DNA was mixed with denaturing buffer and spotted onto a Hybond N+ membrane (Amersham Biosciences, Buckinghamshire, Caspase inhibitor UK) using a 96-well Bio-Dot apparatus (Bio-Rad, Ivry-sur-Seine, France). DNA of the reference strain
ATCC43504 and human DNA were also transferred to the membrane as positive and negative controls, respectively. The cagA of strain ATCC43504 was amplified by PCR with the above-mentioned primer sets. The amplified fragments were purified with an Illustra GFX PCR DNA and Gel Band Purification Kit and used as probes. The probes were labeled with horseradish peroxidase, hybridized to the membranes overnight at 42°C, and finally exposed to Hyperfilm ECL using ECL Direct signaling pathway Nucleic Acid Labeling and Detection Systems (Amersham Biosciences, Buckinghamshire, UK). Histological analysis Three biopsy specimens from the antrum, corpus and upper part of the lesser curvature were used for histological examination. The biopsy specimens were fixed in 10% buffered formalin, and thinly
sliced sections were stained with hematoxylin and eosin (H&E) and Giemsa. Histological features of neutrophil infiltration, mononuclear cell infiltration, grade of atrophy and grade of intestinal metaplasia were scored into four grades in accordance with the Updated Sydney system (0: none, 1: mild, 2: moderate, 3: severe) . Statistical analysis Statistical analysis of the distribution of H. pylori genotypes was performed using Fisher’s exact test. The Mann-Whitney rank sum test was used for Wnt inhibitor assessing differences between ordered categories such as histological grade. The effects of the H. pylori genotypes on the risk for developing peptic ulcer in patients were expressed as odds ratios with 95% confidence intervals with reference to subjects with gastritis. Multiple linear regression analysis was performed to determine which factor(s) was related to the severity of Phosphoglycerate kinase histology, where age, sex, bacterial factors and clinical outcome were explanatory variables. Variables were selected by backward stepwise deletion in the logistic
regression and by the F-out and F-in stepwise method in the linear regression, where F values were both 2.0. Differences at P < 0.05 were accepted as statistically significant. Calculations were carried out using the statistical software package ”JMP IN(R) 5.1J” (SAS Institute, Cary, NC) or ”HALBAU” (Gendai Sugaku-sha, Kyoto, Japan). Nucleotide sequence data reported are available under the DDBJ accession numbers AB469377, and AB469561 to AB469657. Acknowledgements This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (20790285). This work was also supported in part by the Office of Research and Development, Medical Research Service Department of Veterans Affairs, and by a Public Health Service grant DK56338, which funds the Texas Medical Center Digestive Diseases Center.