Alteration of BBB functions in the infected hCMEC/D3 cells.
(A) Altered permeability of a monolayer of infected hCMEC/D3 cells. The endothelial cells were cocultured with irradiated MT-2 or C81-66 lymphocytes for 15 days. Then hCMEC/D3 cells were seeded on Transwell filters and permeability to FITC-dextran 70 kDa was assessed after differentiation of the monolayer. (B) Effect of endothelial cells infection on CEM lymphocyte migration. Infected endothelial cells were seeded onto filters. The migration was estimated at 24 hours of culture by fluorescence assay after labeling of lymphocytes (from the CEM, Jurkat, MT-2 of C81-66 cell-lines) with a fluorescent marker. The migration rate is expressed as ratio (%) of fluorescence intensity in the lower compartment versus total fluorescence. (C) Analysis of the expression level of ZO-1, Occludin, and viral p24 of a monolayer of hCMEC/D3 cells cocultured with irradiated MT-2 or C81-66 for 15 days. β-tubulin was used for normalization. (D) Inhibiting MLC phosphorylation has no effect on the permeability for FITC dextran 70 kDa across a monolayer of infected endothelial cells. hCMEC/D3 cells were seeded on filters. After differentiation, cultures were either left untreated of treated for 48 hours with ML-1, a specific inhibitor for MLCK activity. Permeability was then estimated as described in Materials and Methods.