A better understanding of the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome requires the precise identification of dengue virus (DV) permissive target cells. To examine the relative DV permissiveness among cell subsets, we inoculated unfractionated human peripheral blood mononuclear cells with DV2-16681 in the presence or absence of pooled DV-immune human sera (PHS), and assessed infection with fluorescent dye labeled DV-specific monoclonal antibody and cell surface markers using flow cytometry. We found significantly higher levels of DV antigen staining on DVinfected than mock-infected primary monocytes (3.54 AE 3.42% vs. 0.50 AE 0.38%; P ¼ 0.001). The magnitude of infection was markedly enhanced in the presence of highly diluted PHS (10.04 AE 6.10% vs. 3.54 AE 3.42%; P ¼ 0.015). Under identical experimental conditions, primary T or B cells were not infected either with or without the addition of PHS (0.06 AE 0.04% and 0.44 AE 0.22% for T and B cells, respectively). Furthermore, depletion of CD14þ monocytes prior to DV inoculation abrogated the detection of infected cells, and the addition of monoclonal antibodies to either FcgRI (CD64) or FcgRII (CD32) led to a 50-70% reduction in antibody-dependent enhancement (ADE) of DV infection. Collectively, these results provide further support to the notion that primary monocytes and FcgRs expressed on these cells may be important in the initial steps of immune enhancement observed in some patients with natural DV infection. They also demonstrate that using modern experimental technology, DV infection, and neutralization and enhancement of DV infection can be easily assessed simultaneously in multiple cell types.

Background The blood brain barrier tightly regulates the passage of molecules into the brain and becomes leaky following obstructive cholestasis. The aim of this study was to determine if increased serum bile acids observed during cholestasis permeabilize the blood brain barrier. Methods Rats underwent bile duct ligation or deoxycholic or chenodeoxycholic acid injections and blood brain barrier permeability assessed. In vitro, the permeability of rat brain microvessel endothelial cell monolayers, the expression and phosphorylation of occludin, ZO-1 and ZO-2 as well as the activity of Rac1 was assessed after treatment with plasma from cholestatic rats, or bile acid treatment, in the presence of a Rac1 inhibitor. Results Blood brain barrier permeability was increased in vivo and in vitro following bile duct ligation or treatment with bile acids. Associated with the bile acid-stimulated increase in endothelial cell monolayer permeability was elevated Rac1 activity and increased phosphorylation of occludin. Pretreatment of endothelial cell monolayers with a Rac1 inhibitor prevented the effects of bile acid treatment on occludin phosphorylation and monolayer permeability. Conclusions These data suggest that increased circulating serum bile acids may contribute to the increased permeability of the blood brain barrier seen during obstructive cholestasis via disruption of tight junctions.

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