A number of other studies support the hypothesis that endogenous Nef is involved in bystander effects. The infected cells continued to produce viral particles, while the surrounding uninfected CD4 + T lymphocytes died ( 11, 47). In one study, infected cells were protected from virus-induced cell death through major histocompatibility complex down-regulation induced by intracellularly expressed Nef protein ( 11). Several studies support the argument for the bystander effect and implicate Nef. The “bystander effect” directly implicates viral proteins or indirectly implicates virally stimulated cellular factors as mediators of apoptosis. reported that apoptosis was observed in CD4 + T lymphocytes in infected individuals and is observed predominantly in uninfected or bystander cells, with a distinct lack of cell killing in the productively infected cells themselves ( 18). They suggested that a careful examination of the relevant data with a minimum number of assumptions leads to the conclusion that direct killing of infected cells did not adequately explain the consequences of HIV-1 infection.Īn alternate hypothesis to explain T lymphocyte depletion is that it is a result of programmed cell killing or apoptosis. ( 3) suggested that this model for pathogenesis made too many assumptions. It has been proposed that the depletion of T lymphocytes is directly linked to the viral load and the process of T-cell depletion is induced by viral infectivity ( 9, 64). A direct consequence of decline of cell-mediated immunity is an elevation in the viral load in plasma and circulating, peripheral blood T lymphocytes, as the HIV-infected individual progresses from the asymptomatic stage of infection to advanced AIDS. Elimination of CD4 + T lymphocytes leads to the occurrence of a wide range of opportunistic infections, as well as proliferation of virus-transformed and malignant cells. The most consistent pathogenic feature of human immunodeficiency virus type 1 (HIV-1) infection is the gradual depletion of CD4 + T lymphocytes, a phenomenon that renders the host susceptible to opportunistic infections arising from the dysregulation and dysfunction of the immune system ( 13, 27, 35, 36). This study suggests that extracellular Nef protein could contribute to the decline of CD4 counts prior to and during the onset of AIDS in patients with human immunodeficiency virus type 1 infections. Moreover, a CXCR4-deficient cell line, MDA-MB-468, which was resistant to Nef-induced apoptosis, became sensitive upon transfection with a CXCR4-expressing vector. CXCR4 antibodies, as well as the endogenous ligand SDF-1α, were effective in blocking Nef-induced apoptosis, while CCR5 and CD4 antibodies were ineffective. The cellular receptor responsible for Nef-induced apoptosis was identified through antibody- and ligand-blocking experiments as a receptor commonly involved in viral entry. Similarly, cells treated with Nef/anti-Nef antibody complexes were protected from Nef-induced apoptosis. Cells treated with Nef/protein kinase inhibitor complexes were protected from Nef-induced apoptosis, suggesting possible roles for protein kinases in the apoptosis pathway. DNA laddering and activated caspase 3 were also evident. We observed dose- and time-dependent inductions of apoptosis. CD4 +-T-cell cultures exposed to soluble Nef were analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and hallmarks of apoptosis including cytoplasmic shrinkage, nuclear fragmentation, DNA laddering, and caspase activation. The effects of soluble Nef protein on CD4 + T cells were examined.
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