Joseph W. Oakes

Detection of HIV-1 proviral DNA in sperm from HIV-1-infected men.

ObjectiveSexual transmission is a major mode of the spread of HIV-1, although the cellular and molecular mechanisms are poorly defined. In this study, we sought to assess the cellular reservoirs of HIV-1 proviral DNA in the semen of HIV-1-infected men. Design and methodsAn in situ polymerase chain reaction (IS-PCR), which amplifies specific genes within intact cells, was used to evaluate levels of HIV-1 provirus in seminal cells from HIV-1-infected men in various stages of clinical disease. ResultsInitial studies demonstated HIV-1 provirus in relatively low numbers (1:100 to 1:6000) of both the seminal mononuclear cells and sperm from certain HIV-1-infected men. To extend these findings, 94 seminal samples from HIV-1-infected men were evaluated. HIV-1 proviral DNA was detected in seminal cells of a significant percentage of HIV-1-infected men (45%) at all stages of clinical immunodeficiency. Both seminal mononuclear cells and sperm (35 and 33% of samples studied, respectively) harbored HIV-1 proviral sequences. HIV-1-harboring sperm are shown to stain positively for HIV-1 in the mid-pieces of these cells, with rarer staining of the sperm heads. ConclusionsHIV-1 proviral DNA can be demonstrated by IS-PCR in seminal mononuclear cells and sperm from certain HIV-1-infected men. The role played by proviral DNA in these cells in the sexual transmission of this retroviral agent will require further study.

Sodium butyrate stimulation of HIV-1 gene expression : a novel mechanism of induction independent of NF-κB

Nuclear factor-kappa B (NF-kappa B) has been shown to play a central role in stimulating human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed viral gene expression. We have previously described a cell line (TE671/RD) that fails to respond to phorbol myristate acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha) in terms of amplifying HIV-1 LTR-driven gene expression unless it is concurrently treated with sodium butyrate. It was not determined whether this lack of response stemmed from an inability of these cells to produce free NF-kappa B or from ineffectual interaction of this sequence-specific transcriptional factor with its target. We now show that these cells are in fact capable of inducing a free nuclear NF-kappa B-binding activity when stimulated with PMA but not when treated with sodium butyrate alone. Furthermore, we show that sodium butyrate alone is equally potent in stimulating HIV-1 LTR-directed gene expression in latently infected U1 and ACH-2 cells in the absence of induction of nuclear NF-kappa B, as compared with PMA, which induces NF-kappa B activation in these cells. We also show that stimulation of HIV-1 expression in U1 cells with sodium butyrate is not blocked by N-acetylcysteine, whereas that of PMA stimulation is blocked. These observations are discussed in the context of a model where chromatin structure participates in the maintenance of restricted HIV-1 viral gene expression in these cells.

High percentages of CD4-positive lymphocytes harbor the HIV-1 provirus in the blood of certain infected individuals.

ObjectivesHIV-1 infection of humans leads to states of immunosuppression. Therefore, we sought to determine precise levels of HIV-1 infection of cells in vivo, as these data may assist in the understanding of the pathogenetic processes involved in HIV infection. Design and methodsWe have developed an in situ polymerase chain reaction (IS-PCR), which allows amplification of various genetic elements within intact cells. Initial studies using this technique have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells (PBMC) of infected individuals than have been demonstrated in many previous studies using standard PCR techniques. This study describes a combined protocol in which an immunomagnetic bead separation technique is used with IS-PCR to specifically determine cellular reservoirs for HIV-1 and levels of infected cell types in the peripheral blood. ResultsCD4-positive lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected patients evaluated. The percentages of HIV-1-infected CD4-positive lymphocytes increased significantly with advancing stages of disease. These procedures also demonstrated that, with the exception of small percentages of infected peripheral blood monocytes, the CD4-positive lymphocyte is clearly the major cellular reservoir for HIV-1 in the peripheral blood. ConclusionsThese data suggest that, in certain infected individuals, high levels of CD4-positive lymphocytes may harbor the HIV-1 provirus. Thus, the levels of infected lymphocytes are consistent with possible direct effects of HIV-1 on lymphocyte depletion in vivo.

Potentiation in the intact rat of the hepatotoxicity of acetaminophen by 1,3-bis(2-Chloroethyl)-1-nitrosourea

Studies of the killing of cultured hepatocytes by acetaminophen indicate that the cells are injured by an oxidative stress that accompanies the metabolism of the toxin (J. L. Farber et al. (1988) Arch. Biochem. Biophys. 267, 640-650). The present report documents that the essential features of the killing of cultured hepatocytes by acetaminophen are reproduced in the intact animal. Male rats had no evidence of liver necrosis 24 h after administration of up to 1000 mg/kg of acetaminophen. Induction of mixed function oxidase activity by 3-methylcholanthrene increased the hepatotoxicity of acetaminophen. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the hepatotoxicity of acetaminophen in male rats induced with 3-methylcholanthrene. Whereas the pretreatment with BCNU reduced the GSH content by 40%, a comparable depletion of GSH by diethylmaleate did not potentiate the toxicity of acetaminophen. The antioxidant diphenylphenylenediamine (25 mg/kg) and the ferric iron chelator deferoxamine (1000 mg/kg) prevented the liver necrosis produced by 500 mg/kg acetaminophen in rats pretreated with BCNU. Neither protective agent prevented the fall in GSH produced by acetaminophen. It is concluded the conditions of the irreversible injury of cultured hepatocytes by acetaminophen previously reported are not necessarily different from those that obtain in the intact rat with this toxin.

A quantitative reverse transcriptase-polymerase chain reaction for HIV-l-specific RNA species

The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.

Frequency of cells positive for HIV-1 sequences assessed by in situ polymerase chain reaction.

ObjectiveHIV-1 infection of humans causes immunosuppression. The determination of precise levels of HIV-1 infection of cells in vivo may assist in the understanding of the pathogenetic processes involved in clinical infection with this virus. Studies using polymerase chain reaction have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells of infected subjects than many previous studies. MethodsWe developed a new, highly sensitive, polymerase chain reaction method that amplifies selected genetic regions within intact single cells. We used this technique before and after immunomagnetic bead separation to determine the proportion of unfractionated peripheral blood mononuclear cells and CD4+ lymphocytes carrying the HIV-1 provirus in infected subjects in different stages of disease. ResultsNone of the peripheral blood mononuclear cells from 11 HIV-1-seronegative subjects were positive for HIV-1 provirus by the in situ polymerase chain reaction method. In 56 subjects infected with HIV-1, the percentage of peripheral blood mononuclear cells with HIV-1 ranged from 0.1 to 13.5%. CD4+ lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected subjects evaluated. The percentages of HIV-1-infected CD4+ lymphocytes increased significantly with an advancing stage of disease. The proportion of peripheral blood mononuclear cells infected with HIV-1 appeared to be at least 10 times higher than previously described. ConclusionThese data suggest that in certain infected patients, high levels of infected lymphocytes may harbor the HIV-1 provirus.