Respiratory burst by dengue-virus-induced cytotoxic factor

This study investigates the induction and release of the superoxide anion (O- 2) and hydrogen peroxide (H2O2) by mouse spleen cells on stimulation with dengue type 2 virus (DV) and a DV-induced cytokine, the cytotoxic factor (mCF). Methods: Normal mice or their spleen cell cultures were inoculated with DV or mCF. At different time periods, the spleen cell supernatants were assayed for the production of O- 2 and H2O2. Results: Inoculation of DV in spleen cell cultures resulted in peak production of O- 2 and H2O2 at 48 and 72 h, respectively, while in DV-infected mouse spleen, the maximum production was on days 7 and 8, which correlated with the appearance of mCF in the milieu. Maximum O- 2 and H2O2 production occurred at 45 min and 1 h after inoculation of 5 µ g of mCF. Pretreatment of mCF with anti-mCF-antiserum inhibited O- 2 and H2O2 release indicating the specificity of the induction by mCF. The enriched subpopulations of macrophages and T cells produced O- 2 and H2O2 and not B cells. Treatment of the cells with superoxide dismutase increased H2O2 release but inhibited O- 2 release and the cytotoxicity in a dose-dependent manner. Conclusion: This showed that O- 2 is responsible for the cytotoxic activity of mCF and not H2O2. In conjunction with our earlier findings that pretreatment with NG-monomethyl-L-arginine inhibited mCF-induced production of NO and the cytotoxicity, it is concluded that the presence of both O- 2 and NO is required for the cytotoxic activity of mCF, thereby indicating a possible role of peroxynitrite

High-Performance Capillary Electrophoresis for Determining HIV-1 Tat Protein in Neurons

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders

Dengue virus-induced cytotoxin releases nitrite by spleen cells

Dengue type-2 virus (DV) infection in mice induces T cells to produce a cytokine, the cytotoxic factor (CF), which induces H2-A positive macrophages to produce another cytokine, cyotoxin (CF2), which amplifies its cytotoxic effects on target cells. The present study was undertaken to investigate the production of nitrite (NO−2) by the spleen cells of mice in vitro and in vivo following inoculation of CF2. Maximum NO−2 production occurred at 1 hour after inoculation of 100 μg CF2. Pretreatment of CF2 with anti-CF2-antisera (CF2-As) inhibited the production of NO−2. Pretreatment of the spleen cells with NG-monomethyl- l-arginine (NMA) or with arginase inhibited NO−2 production. The NO−2 production was diminished in a dose dependent manner by treatment of spleen cells with the Ca2+ channel blocking drug, nifedipine and Zn2+ as ZnSO4. The findings of the present study thus demonstrate that CF2 induces production of NO−2 in the spleen cells in a CA2+-dependent manner which may be a mechanism of target cell killing

Release of reactive oxygen intermediates by dengue virus-induced macrophage cytotoxin

Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mice to produce a cytokine, cytotoxic factor (mCF), which induces H-2A positive macrophages to produce macrophage cytotoxin (CF2). The present study was undertaken to investigate the mechanism of cytotoxicity of CF2. It was observed that CF2 induced production of superoxide anion (O−2) and hydrogen peroxide (H2O2) by the spleen cells of mice in vitro and in vivo. The maximum production of O−2 (260 ±10 n M/4 × 106 cells) was at 45 minutes while that of H2O2 was at 90 minutes after inoculation of CF2. Pretreatment of mice or spleen cells with anti-CF2-antisera inhibited O−2 and H2O2 production in a dose-dependent manner. Superoxide dismutase (SOD) inhibited O−2 production and cytotoxicity while H2O2 production was increased by increasing SOD concentration in the culture. This indicated that O−2 production is necessary for the cytotoxic activity of CF2. Pretreatment of the cells with Ca2+ channel blocking drugs, nifedipine or verapamil, inhibited CF2-induced O−2 and H2O2 production in a dose-dependent manner. We have shown earlier that the cytotoxic activity of CF2 is known to be Ca2+ dependent and CF2-induced production of nitrite and the cytotoxicity is inhibited by NG-monomethyl- L-arginine. Thus, it is suggested that O−2 and nitrite are necessary for cell killing by CF2 in a Ca2+ manner and the killing may possibly be by generation of peroxynitrite

Empirical bayes prediction intervals in a normal regression model: higher order asymptotics

We explore two proposals for finding empirical Bayes prediction intervals under a normal regression model. The coverage probabilities and expected lengths of such intervals are studied and compared via appropriate higher-order asymptotics

Empirical Bayes prediction intervals in a normal regression model: higher order asymptotics

We explore two proposals for finding empirical Bayes prediction intervals under a normal regression model. The coverage probabilities and expected lengths of such intervals are studied and compared via appropriate higher-order asymptotics.Coverage probability Equal tail Expected length

Human immunodeficiency virus type 1 Tat prevents dephosphorylation of Sp1 by TCF-4 in astrocytes

Previous examination of the effect of TCF-4 on transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in human astrocytic cells found that TCF-4 affects the HIV-1 promoter through the GC-rich domain (nt -80 to nt -68). Here, the physical interaction and a functional consequence of TCF4-Sp1 contact were characterized. It was shown that expression of TCF-4 in U-87 MG (human astrocytic) cells decreased basal and Sp1-mediated transcription of the HIV-1 promoter. Results from a GST pull-down assay, as well as combined immunoprecipitation and Western blot analysis of protein extracts from U-87 MG cells, revealed an interaction of Sp1 with TCF-4. Using in vitro protein chromatography, the region of Sp1 that contacts TCF-4 was mapped to aa 266-350. It was also found that, in cell-free extracts, TCF-4 prevented dsDNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation. Surprisingly, TCF-4 failed to decrease Sp1-mediated transcription of the HIV-1 long terminal repeat (LTR) and Sp1 phosphorylation in cells expressing HIV-1 Tat. Results from immunoprecipitation/Western blotting demonstrated that TCF-4 lost its ability to interact with Sp1, but not with Tat, in Tat-transfected cells. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1, which is affected by Tat and DNA-PK. Interactions among TCF-4, Sp1 and/or Tat may determine the level of viral gene transcription in human astrocytic cells