Effects of Vibrio vulnificus cytolysin on the numbers of mitochondria and the expression of CD62L in murine liver CD4+T cells / 中华微生物学和免疫学杂志

Objective To understand how Vibrio vulnificus hemolysin (VvhA) affects the viability of murine liver CD4+ T cells as well as its effects on the numbers of mitochondria and the expression of CD62L. Methods The primary murine liver monocytes (MNs) were isolated from C57BL/ 6 mice and then treated with recombinant VvhA (rVvhA) for 6 hours in vitro. The viability of murine liver CD4+T cells and the expression of CD62L were measured by staining with anti-mouse CD4, CD8, CD44, CD62L and cell via-bility fluorescent dye or fluorescent antibody. Moreover, the cells were simply incubated with MitoTracker or JC-1 probes to label mitochondria and mitochondrial membrane potential, which were further analyzed by using flow cytometry analysis. Results With the increase in the doses of rVvhA, the viability of murine liv-er CD4+T cells was decreased from 81. 5% to 15. 8% . The expression of CD62L on the surface of murine liver CD4+T cells was dramatically decreased. Both the murine liver na?ve and effector CD4+ T cells were sensitive to the cytotoxicity of rVvhA. Moreover, treating murine liver CD4+ T cells with rVvhA resulted in significantly decreased numbers of mitochondria and lower mitochondrial membrane potential. Conclusion The cytotoxicity of rVvhA to murine liver CD4+T cells might be achieved through inhibiting the expression of CD62L, decreasing the numbers of mitochondria and lowering mitochondrial membrane potential.

Role of rVvhA in inducing THP-1 cells damage / 中华微生物学和免疫学杂志

Objective To investigate the role of recombinant Vibrio vulnificus cytolysin (rVvhA) in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K +and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2+induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

Study on VVC recombinant protein cytolytic activity mechanism of an extracellular cytolytic toxin of Vibrio vulnificus / 中华微生物学和免疫学杂志

Objective To investigate the cytolytic activity of extracellular cytolytic toxin rVVC of Vibrio vulnificus on the apoptosis of human ECV304 cells, and to analyze the activities of Caspase-3,-8 and -9. Methods The cytotoxic effect of refolded rVVC on the growth and apoptosis of ECV304 cells was identified by MTT, Hochest33342/PI fluorescent staining, flow cytometry and DNA agarose electrophoresis analysis, respectively. The activities of Caspase-3, -8 and -9 was measured using a colorimetric method. Results The viability of human ECV304 cells exposed to rVVC was inhibited by rVVC after 24 h. 2.0 HU/ml rVVC groups had a better cytotoxic effect to human ECV304 than that of 0.5 HU /ml rVVC groups. The apoptosis of human ECV304 cells in 2.0 HU/ml rVVC+40 μmol/L Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ml of rVVC groups. After 0.5 h treatment with 2.0 HU/ml of rVVC, the Caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 h (versus control groups, P<0.01). The activity of Caspase-8 and -9 remained unchanging. Conclusion The rVVC has cytotoxic effect on human ECV304 and the cytolysin is probably correlated with Caspase-3.

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

The Therapeutic Effect of Anti - inflammatory Agents on the Lethal Effect of Toxemia Evoked by Vibrio vulnificus Cytolysin / 대한피부과학회지

BACKGROUND: Many pathophysiological derangements associated with Vibrio vulnificus sepsis result from the release of toxins and enzymes into the circulation. Its effects are mediated via complex interaction of many endogenous inflammatory mediators such as tumor necrosis factor (TNF)-a, bradykinin, histamine, and nitric oxide. OBJECTIVE: The purpose of this study is to evaluate the effects of antinflammatory agents (antiTNF-a, antihistamine, steroid, antibradykinin, and nitric oxide synthase inhibitor) on the lethal effect of toxemia evoked by Vibrio vulnificus cytolysin. METHODS: The study consisted of 4 groups of mice. Control mice received a bolus intravenous infusion of cytolysin or anti-inflammatory modalities. Drug administration was designed in accordance with the time of cytokine release(TNF-a, IL-6,8) and scored for survival rate at 72 hours after last infusion. Group I mice(early treatment group) received intraperitoneal infusions of each anti-inflammatory modalities at 1 hour after cytolysin infusion. Group II mice(delayed treatment group) received each anti-inflammatory agent treatment at 2, 4, 12 hours after the cytolysin infusion. Group III(early combined treatment group) received intraperitoneal infusions of combined anti-inflamrnatory agents at 1 hour after cytolysin infusion. Group IV(delayed combined treatment. group) received combined anti-inflammatory agents at 2, 4, 12 hours after cytolysin infusion. Autopsies were performed in dead mice after cytolysin infusion for gross and microscopic studies. RESULTS: In the control group, all mice infused with cytolysin died and all mice treated with antiinflammatory agents survived. Survival rate of group I showed 75% in aprotinin, 88% in prednisolone, 75% in N-methyl-L-arginine, 88% in pentoxifylline, 100% in hydroxyzine HC1. Group II showed 40%, 40%, 60 %, 60%, 80% in order of the agents, respectively, Mean survival rate of each agents showed 85 % in group I and 56% in group II. Results of treatment revealed 100% survival in group III and 80% in group IV. In evaluation of effectiveness of therapeutic modalities, all died in the no therapeutic group and a 76.9% survaal rate in the therapeutic group was noted. The gross and microscopic finding showed similar findings to sepsis. CONCLUSION: These results suggest that inhibitors of endogenous inflammatory mediators may improve the survival rate in th treatment of septic shock caused by Vibrio vulnificus and ot,her grarn negative bacilli. Also this study support the proposal that early treatment in V, vulnificus septicemia is essential for reduced mortality.