Because adherence between endothelial cells and H pylori, a noninvasive bacterium, cannot occur within gastric mucosa, it is very important to estimate activation of endothelial cells by the stimulation of some substance secreted or released from H pylori. In the present study, we examined whether H pylori water extract (HPE) induces the upregulation of three important adhesion molecules, ICAM-1, VCAM-1 and E-selectin, on cultured human umbilical vein endothelial cells (HUVEC). We also analyzed the nature of the substances which mediate such upregulation.
HPE were prepared from a type strain (NCTC11637) and clinical strains (H13, H16, 930 and 933) of H pylori that were isolated from gastric antral biopsies of patients with gastric ulcers . These strains were tested for the presence of the cagA and vacA genes by a reverse transcription-polymerase chain reaction using specific primer pairs (Table 1). The organism was grown on blood agar plates as previously described . The growth medium consisted of Bacto brain-heart infusion, with 0.5% Bacto (Difco, USA) yeast extract, 2.0% Bacto agar and 7% fresh horse blood. Plates were inoculated and the bacteria were grown for 12 passages in a CO2 incubator with 12% CO2 and 100% humidity for 48 h. Cells were harvested with sterile cotton swabs and suspended in distilled water using 1.0 mL per plate (109 to 1010 bacteria). The cell suspension was kept at room temperature for 20 min before centrifugation at 12,000 rpm (17,000 g) for 15 min.
TABLE 1 Lipopolysaccharide concentration in water extracts of Helicobacter pylori, and cagA and vacA positivity of strains
|Strain||cag A||vac A||Lipopolysaccharide (EU/mL)|