The relevance of apoptosis in the pathogenesis of human immunodeficiency virus-1 disease

[摘要] ENGLISH ABSTRACT:A simple and rapid scatter-based flow cytometric assay was developed to detectapoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay wassuitable for children.Apoptotic PBMCs were confirmed by morphologic assessment in clinical samplesex vivo and after overnight culture. The scatter-based assay was validated in a number ofways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induceapoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sortedinto apoptotic and viable populations by scatter characteristics (diminished forward andincreased side scatter). Morphology was assessed by fluorescence microscopy. Themajority of cells with apoptotic scatter characteristics had apoptotic morphology(chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region hadnormal morphology. CD4+ T cell apoptosis measured by scatter was then correlated withthe TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter andannexin V uptake were also shown to correlate. In the latter experiments, PBMCmorphology and cell death by trypan blue uptake were studied simultaneously andconfirmed the two flow cytometric assays.Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infectedadults analyzed after overnight culture. Since cell death may be an artifact of invitro culture, and because there is little information on apoptosis in paediatric HIV disease,I undertook a cross-sectional analysis in PBMCs analyzed immediately ex vivo from HIV infectedchildren and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stainedfor CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterizedflow cytometric assay by diminished forward and increased side scatter.For the scatter assay, PBMCs had been labeled initially by an indirect methodinvolving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, theintermediary incubation step was removed and PBMCs were incubated with PE-conjugatedCD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appearedenhanced in the indirect method. The significant differences were abolished aftersubtraction of data from simultaneously studied time-matched controls.CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected studysubjects than in simultaneously studied seronegative controls. PBMCs were assayedimmediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb aswell as spontaneously. There was a direct correlation between CD4+ and CD8+ T cellapoptosis and CD4+ T cell depletion. A significant correlation was also shown betweenapoptosis immediately ex vivo and after overnight culture.I then studied apoptosis in a South African population comprising 18symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis weresignificantly higher in symptomatic HIV-1-infected children than in seroreverters andseronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cellpercentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cellapoptosis in patients recovering from intercurrent disease in comparison to those whowere either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, COand Tygerberg Children's Hospital, South Africa. I selected only patients with moderateor severe HIV infection from both centers. South African patients were significantlyyounger, more malnourished, had higher gamma globulin levels and were less likely toreceive ART. CD8+ T cell apoptosis was higher in North American patients suggesting apossible impairment in CD8+ activity in the South African study subjects.

[发布机构] Stellenbosch University