4 research outputs found

    <i>R. montanensis</i> enhanced VE-cadherin tyrosine phosphorylation.

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    <p>Human cerebral microvascular endothelial cells were mock-infected (control) or infected with <i>R. montanensis</i> at a MOI of 10. At 24, 48, or 72 hr post-infection, cells were harvested for Western immunoblot or immunoprecipitated with anti-VE-cadherin antibody using a magnetic bead system. <b>3A</b>). The Western immunoblot for ╬▒-tubulin served as a control to verify equal loading and transfer. There was no significant difference in VE-cadherin expression detected by Western immunoblot. <b>3B and 3C</b>). Loading of VE-cadherin was detected by anti-VE-cadherin antibody. The normalized relative densities from IP phosphorylation assay showed a 2.16- and 4.48-fold increase in phosphorylation of VE-cadherin (P-VE-cadherin) at 48 hr and 72 hr post-infection, respectively, compared to control cells (* p<0.05). These data are representative of three independent experiments.</p

    Immunofluorescence studies show rickettsiae (red) located in human cerebral microvascular endothelial cells at 24, 48 and 72 hr after infection.

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    <p>Dual immunofluorescence staining of SFG rickettsiae (red) and VE-cadherin (green) using dual wave lengths filter system reveals that, compared to normal controls, <i>R. montanensis</i> infection (10 MOI) resulted in degradation of the density of VE-cadherin, suggesting disruption in the continuity of VE-cadherin at neighbouring areas at 72 hr post-infection.</p

    AFM measurements of the de-adhesion forces between VE-cadherin and endothelial cells.

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    <p><b>4A</b>). Typical force-extension curves obtained between cells and VE-cadherin coated AFM tips. The dashed lines indicate zero force. The experiments were carried out in uninfected and infected cells at different time points post-<i>R. montanensis</i> infection (48 hr and 72 hr). <b>4B</b>). Work of de-adhesion between VE-cadherin beads and endothelial cells at different time points post-infection. <i>R. montanensis</i>-infected cells required a significantly lower level of average work to break the interaction compared with uninfected cells. The addition of a monoclonal antibody against VE-cadherin significantly blocked the VE-cadherin-endothelial cell interaction. Ab: anti-VE-cadherin antibody.</p

    <i>R. montanensis</i> infection enhanced human cerebral microvascular endothelial cell permeability.

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    <p>Endothelial cells were seeded on type I rat-tail collagen-coated polycarbonate transwell filters and infected with <i>R. montanensis</i> at an MOI of 10 in triplicate, or mock infected. After 24, 48, and 72 hr, FITC-dextran was added to the upper chamber medium, and the presence of FITC dextran in the lower chamber was quantified after 3 hr. The results are expressed as the fold-increase in monolayer permeability over basal permeability levels (* p<0.05).</p