Detection of rabies virus antigen or antibody using flow cytometry.

BACKGROUND: Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer-automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT). METHODS: Pasteur virus (PV)-infected mouse neuroblastoma (MNA) cells were stained with rabies virus antinucleocapsid antibody, fluorescein isothiocyanate (FITC) conjugate, and the percentage of infected cells at 24, 48, and 72 h postinfection (PI) was determined using flow cytometry. Serum samples containing known antibody titres estimated by RFFIT in terms of IU/ml were used to neutralize 50 FFD50 dose of PV. The percentage of MNA cells infected by the un-neutralized virus was estimated by flow cytometry. Using the value of the percentage of cells infected in the presence of known negative serum as 100%, the infection inhibition caused by antibodies at each dilution of positive reference serum was calculated and a regression equation generated for the prediction of rabies virus antibody titres in test sera samples as equivalent units per ml (EU/ml). RESULTS: There was a significant increase in the percentage of infected cells between 24 and 48 h PI from 26.45 to 75.28%. The percentage of cells having high side scatter was also highest at 72 h PI (11.11%). Antibody titres predicted by flow cytometry and those estimated by RFFIT as IU/ml showed a correlation coefficient of 0.74. CONCLUSIONS: Thus, flow cytometry could be used to detect rabies virus antigen in infected cells and to predict serum antibody titres from a single dilution of serum tested with the potential advantages of automation, rapidity, and lack of subjectivity. It has the potential to replace the time-tested RFFIT in rabies serology in the years to come.

Dietary flavonoids modulate PCB-induced oxidative stress, CYP1A1 induction, and AhR-DNA binding activity in vascular endothelial cells.

Polychlorinated biphenyls (PCBs), especially the more coplanar PCBs, have been shown to induce oxidative stress, various transcription factors, and subsequent inflammatory processes critical to atherosclerosis in vascular endothelial cells. Dietary flavonoids such as catechins and quercetin possess antioxidant and anti-inflammatory properties. To test the hypothesis that flavonoids can modify PCB-mediated endothelial cytotoxicity, endothelial cells were treated with epigallocatechin-3-gallate (EGCG; 5 to 50 muM) or quercetin (10 to 100 muM) with or without PCB 77 (3,3',4,4'-tetrachlorobiphenyl, 3.4 muM) for 6 h. EGCG and quercetin strongly, and in a concentration-dependent manner, inhibited oxidative stress induced by PCB 77 as measured by DCF fluorescence. The role of cytochrome P450 1A1 (CYP1A1) in the PCB-induced toxicity was investigated. EGCG at 50 muM and quercetin at 100 muM concentrations markedly inhibited CYP1A1 mRNA levels and enzyme activity. Furthermore, EGCG and quercetin downregulated the PCB 77-mediated increase in aryl hydrocarbon receptor (AhR)-DNA binding activity. These data suggest that protective effects of EGCG and quercetin are initiated upstream from CYP1A1 and that these flavonoids may be of value for inhibiting the toxic effects of PCBs on vascular endothelial cells.