Stability of HIV-1 RNA in blood during specimen handling and storage prior to amplification by NASBA-QT.

The influence of different storage temperatures and anticoagulation conditions on the HIV-1 RNA load as measured by NASBA-QT was examined. Blood specimens from 14 HIV-1 infected individuals were processed within 2 h after collection. The HIV-1 RNA load remained stable for at least 6 months when samples were frozen directly at -70 degrees C in lysis buffer. This lysis buffer fully inactivated the virus. When whole EDTA blood was stored, the HIV-1 RNA load was stable for 72 h at 25 degrees C, but it declined within 24 h at 4 degrees C. The HIV-1 RNA load in whole heparinized blood declined significantly after 24 h at both 4 and 25 degrees C. It was slightly lower (average of 0.18 log ml-1) than in whole EDTA blood. At 4 degrees C, the HIV-RNA load in serum and EDTA-plasma stored with lysis buffer did not decline up to 14 days. At + 30 degrees C, however, the load declined significantly after 2 days. Of clinical significance, the mean load in EDTA plasma was 0.5 log ml-1 higher than in serum. This difference was patient dependent (range 0.1-0.7 log ml-1). We thus recommend, for quantifying HIV-1 RNA by NASBA, to use preferably EDTA blood which is kept at room temperature until plasma separation. When using heparinized blood, the plasma should be stored frozen within 8 h.

The stimulation of human peripheral blood lymphocytes by oral bacteria: macrophage and T-cell dependence.

Human peripheral blood mononuclear lymphocytes from individuals with moderate periodontitis were separated into purified subpopulations of T lymphocytes and B lymphocytes by rosetting with sheep red blood cells (E). All three lymphocyte subpopulations were compared for proliferative responses to cell walls from seven oral bacteria, phytohemagglutinin (PHA), pokeweed mitogen (PWM), lipopolysaccharide (LPS), and streptolysin-O (SLO). Mononuclear cells and a re-combined subpopulation consisting of four parts purified T lymphocytes and one part B lymphocytes responded significantly to all of the stimulants. Purified T lymphocytes by themselves responded significantly to PHA and PWM, but were unresponsive to oral bacteria and SLO; however, T lymphocytes cultured with 2% autologous macrophages responded significantly to all seven oral bacterial cell walls and to SLO, which indicates that T-cell responses to oral bacteria are macrophage-dependent. T-cell-depleted non-E-rosette-forming B cells by themselves were poorly responsive to all of the tested stimulants; however, the responses of these cells to oral bacteria, PWM, LPS, and SLO increased significantly in the presence of 10% mitomycin-C-treated T cells, demonstrating that B cell proliferation to these stimulants is T-cell-dependent.

The stimulation of human peripheral blood lymphocytes by solubilized dental plaque: macrophage and T-cell dependence.

This study compared the ability of human peripheral blood lymphocyte subpopulations from individuals with moderate periodontitis to proliferate in response to stimulation with solubilized dental plaque, phytohemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative (PPD). Unseparated mononuclear lymphocytes and recombined purified T and B lymphocytes (4/1) responded significantly to all of the stimulants. DNA synthesis in response to solubilized dental plaque was maximal after 6 to 7 days of culture; the optimum dose was usually 10 to 20 micrograms/ml. T lymphocyte subpopulations purified by rosetting with sheep red blood cells and density gradient centrifugation responded well to PHA and PWM, but were unresponsive to solubilized dental plaque and PPD unless supplemented with 2% autologous macrophages, demonstrating that T cell responses to solubilized dental plaque and to PPD are macrophage-dependent. T cell-depleted enriched B lymphocyte subpopulations were poorly responsive to all of the tested stimulants; however, the responses of these cells to solubilized dental plaque and to the known T cell-dependent B cell mitogens PWM and PPD were increased significantly by the presence of 10% mitomycin C-treated T cells, demonstrating that B cell proliferation to solubilized dental plaque is T cell-dependent. Thus, cellular interactions between macrophages, T lymphocytes and B lymphocytes are required to obtain optimal proliferative lymphocyte responses to solubilized dental plaque. Since both T and B lymphocytes respond to dental plaque stimulants, they both have the potential to mediate periodontal inflammation and tissue destruction whenever dental plaque stimulants gain entrance into the periodontal tissues.