Briefly, AGS cells were treated with a mixture of lipofectamine and plasmid DNA in a ratio of 1 : 3 for 4 hours. Thereafter, the serum-free medium was aspirated and cells were grown in Ham’s

F12 medium with 10% fetal bovine serum for 24 hours. The coverslips were then washed thrice with 1X phosphate buffer saline and fixed in 4% paraformaldehyde and probed with anti-rabbit HP0986 antibody. This was followed by 1 hour incubation with peroxidase-conjugated goat anti-rabbit IgG. Slides were washed and mounted with Vectashield mounting medium containing DAPI Sirolimus (Invitrogen). Expression vector pEGFPN-1 without any insert was used as negative control. Two tailed student t-test was used to demonstrate the level of secretion of IL-8 in treated cells when compared with untreated cells. Further, Mann–Whitney’s U test was carried out to compare antibody responses. All the data were expressed

as mean ± SEM. p values of less than .05 were read as statistically significant. To investigate the in vitro expression of HP0986 in H. pylori clinical isolates and to confirm it as a virulence marker linked to disease outcome, we checked the epidemiologic consistency of HP0986 across the Malaysian patient population selected by us (see in methods). Firstly, we performed PCR-based detection of HP0986 using the genomic DNA isolated from all (n = 110) H. pylori isolates and with the help of gene specific primers as described earlier [14]. Among the 110 clinical isolates, HP0986 gene p38 MAPK signaling pathway was found in 31% (n = 34) of the samples. To investigate if PCR-based detection of HP0986 also entails expression of HP0986, a quantitative real time PCR was

performed on the above 34 isolates so as to analyze the in vitro expression of the gene. Galeterone A single primer set, complementary to a highly conserved region, which specifically amplifies HP0986 was used to perform the in vitro expression analysis. This precluded the possibility of any primer mismatches. The mRNA expression of HP0986 was recorded as cycle threshold relative to the strain P12 of H. pylori. (Fig. 1). Our results revealed that the presence of HP0986 genotypes corroborated with the in vitro expression of HP0986. The prevalence of HP0986 in the clinical samples varied among three ethnic groups and it was highest among the Indian origin patients (88%) followed by Chinese (10%) and Malay subjects (2%). This demonstrated that there was a differential prevalence of HP0986 in H. pylori clinical isolates corresponding to different ethnic groups in Malaysia. We analyzed the expression of HP0986 mRNA in gastric biopsy specimens and the analysis of relative HP0986 transcript levels was performed by quantitative RT PCR. On a pilot scale, relative mRNA was measured only in 10 gastric biopsy specimens. These 10 biopsy specimens were different from the 110 clinical isolates used for in vitro expression analysis.