Helicobacter pylori and endothelial adhesion molecules: RESULTS

LPS concentrations in the five HPEs ranged from 0.65 EU/mL to 4.16 EU/mL. Final LPS concentrations in medium containing 10% HPE ranged from 0.065 EU/mL to 0.416 EU/mL. All strains were positive for both cagA and vacA genes. Unstimulated HUVEC constitutively expressed ICAM-1 but little VCAM-1 and E-selectin (Figure 1). Coincubation with 20 U/mL of IL-1p (positive control) for 6 h significantly (P<0.05 versus unstimulated) upregulated surface expression of ICAM-1, VCAM-1 and E-selectin on HUVEC.

Ten per cent HPE from NCTC11637 significantly upregu-lated ICAM-1 (P<0.05 versus unstimulated), VCAM-1 (P<0.05 versus unstimulated) and E-selectin (P<0.05 versus unstimulated). HPE from H13, H16, 930 and 933 did not induce any significant increase in expression of ICAM-1, VCAM-1 and E-selectin. Because the LPS concentrations in medium containing 10% HPE were less than 0.74 EU/mL, the inducing ability of 0.74 EU/mL of E coli LPS, which has more biological activities than H pylori LPS we examined. The results showed that 0.74 EU/mL of E coli LPS did not upregulate ICAM-1, VCAM-1 and E-selectin.

HPE from NCTC11637 was divided into 61 fractions (fraction 20 to 80) by gel filtration. As shown in Figure 2, fractions 58 to 65 (representing molecules of approximately 7 kDa) exhibited the peak of the inducing activity for the expression of ICAM-1, VCAM-1 and E-selectin.

The authors focused on fraction 62, which corresponded to the peak activity. As shown in Figure 3, the inducing activity of fraction 62 for ICAM-1 expression was not affected by heating (56°C for 30 min or 100°C for 10 min) or coincubation with 250 mg/mL of trypsin.

Surface expression of intercellular adhesion molecule (ICAM)-1

Figure 1) Surface expression of intercellular adhesion molecule (ICAM)-1 (A), vascular cell adhesion molecule (VCAM)-1 (B), and E-selectin (C) on human umbilical vein endothelial cells (HUVEC) stimulated by 20 U/mL of interleukin beta (IL-1P), 10% Helicobacter pylori water extracts (from NCTC11637, H13, H16, 930, or 933), or 0.74 EU/mL of lipopolysaccharide (LPS). ICAM-1, VCAM-1, and E-selectin on HUVEC were assessed by enzyme-linked immunosorbent assay 6 h after stimulation. Data represent the mean ± SE of three experiments in triplicate. *P<0.05 compared with unstimulated HUVEC. OD Optical density

Surface expression

Figure 2) Surface expression of intercellular adhesion molecule (ICAM)-1 (A), vascular cell adhesion molecule (VCAM)-1 (B), and E-selectin (C) on human umbilical vein endothelial cells (HUVEC) stimulated by 10% of gel-filtrated fractions of Helicobacter pylori water extract from nCtC11637. ICAM-1, VCAM-1 and E-selectin on HUVEC were assessed by enzyme-linked immunosorbent assay 6 h after stimulation. Data represent the mean ± SE of three experiments in duplicate. OD Optical density

Effects of heat and trypsin treatment on the activity

Figure 3) Effects of heat and trypsin treatment on the activity of fraction 62 for upregulation of intercellular adhesion molecule-1 on human umbilical vein endothelial cells (HUVEC). Treatment details are given in the text. Data represent the mean ± SE of three experiments in duplicate. *P<0.05 compared with unstimulated HUVEC. IL-1P Interleukin beta; OD Optical density

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