The measurement of apoptosis in HIV-1 infection

Yu, J. (2006-03)

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2006.


Acquired immunodeficiency syndrome (AIDS) was first reported in 5 homosexual men in Unite States of America in 1981 as a series of opportunistic infections which occasionally occurred in adults. Subsequently, it has been achieved that human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS and this aetiological agent has spread all over the world. The virus primarily attacks CD4+ T cells and gradually leads to progressive depletion of CD4 T lymphocytes from peripheral blood and lymphoid organs. Since CD4+ T cells are vital immune cells in induction and regulation of both cell-mediated and humoral immune responses, depletion of these cells ultimately results in a profound immunodeficiency characterized by susceptibility to variety of opportunistic infection. Apoptosis have been commonly proposed as the mechanism of CD4 depletion because elevated levels of apoptosis were observed in HIV-1 infected individuals (Ameisen et al., 1991; Groux et al., 1992 & Oyaizu et al., 1993). Nevertheless, there was evidence showing that HIV-1 infected cells died not from apoptosis (Bolton et al., 2002) and another study reported that inhibition of apoptosis resulted in high viral production (Antoni et al., 1995). These controversial views indicated that the mechanism of CD4 depletion and the immuno-pathogenesis of apoptosis should be considered. As a pilot sub-study, eight HIV-1 infected subjects were enrolled to determine the methods in measuring apoptosis. Three different cell separations: (1) whole blood cells, (2) buffy coat cells and (3) isolated PBMCs were prepared to determine whether different cell preparations result in different measurements of apoptosis. In addition, FITC-labelled Annexin V, an early marker of apoptosis, and flow-cytometer based scatter methods based on characteristics of apoptotic cells were used to investigate the difference in analytical methods in determining the levels of apoptosis. Firstly, it was found that whole blood samples yielded more precise measurements in measuring apoptosis, followed by Buffy coat and then PBMC samples. Secondly, this sub-study also indicated that the scatter method as well as fluorenscent labelled Annexin V could be useful markers for apoptosis. Secondly, different surface markers of apoptosis were used to investigate apoptosis in HIV-1 infected adults. Fifty-eight HIV-1 infected adults were involved in this sub-study. They were classified into three categories based on CDC CD4 category classification (CDC, 1993). According to the data, the level of apoptotic CD4+ T cells measured by the scatter method was high in CD4 category 1, decreased in category 2 and finally increased again in category 3. This tendency was in parallel with CD95 (Fas) expression on CD4+ T cells. The curve formed a “V” shape according to the three CD4 categories. Together with the gradually increased plasma viral load, these data reflect an activated immune response at early stage of infection and under controlled viraemia. This possibly represents the immune response trying to eliminate infected cells as a means of survival. The high level of apoptosis in category 3 could indicate a disordered immune system accounting for the rapid loss of CD4+ T cells and progression to AIDS. A novel finding of this study was the presence of two CD4+ populations in 10 HIV-1 infected subjects, which were CD4dim and CD4bright. These 10 subjects had relatively high CD4 count and low viral replication. Statistical analysis showed they had significantly higher levels of apoptosis in CD4 and CD8 T lymphocytes, measured by the scatter method, than those subjects presenting single CD4 population. In addition, when comparing the two CD4 subpopulations, it was found that CD4dim cells had significant higher level of apoptosis and CD95 expression than the CD4bright cells. Finally, the virological and immunological effects of antiretroviral therapy (ART) were investigated in two cohorts of HIV-1 infected children. Fourteen HIV-1 infected children were involved in investigation of 12-month long-term effect, while another five children were involved in a short-term 1-month follow-up study. In addition, a different assay of detecting apoptosis: terminal deoxynucleotidyltransferase deoxyuridine triphosphates nick end labeling (TUNEL) was conducted to measure the level of apoptotic PBMCs. According to the findings from 12-month and 1-month sub-studies, it appeared that ART could be effective in suppression of viral replication at an early stage. However, the immunological effect, such as CD4 reconstitution, could only be seen as a long-term effect, since immune recovery would take a long time. In addition, different regimens containing protease inhibitors (PIs) might be more effective in inhibiting apoptosis than non-nucleoside reverse transcriptase inhibitors (NNRTIs).

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