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

Cotton, Mark Fredric (2004-12)

Thesis (PhD)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: A simple and rapid scatter-based flow cytometric assay was developed to detect apoptosis in CD4+ and CD8+ T cells from a mixed population of cells. The assay was suitable for children. Apoptotic PBMCs were confirmed by morphologic assessment in clinical samples ex vivo and after overnight culture. The scatter-based assay was validated in a number of ways. Firstly, PBMCs were irradiated with 500 rads and cultured overnight to induce apoptosis. Thereafter, PBMCs were labeled with a CD4 MAb. CD4+ cells were sorted into apoptotic and viable populations by scatter characteristics (diminished forward and increased side scatter). Morphology was assessed by fluorescence microscopy. The majority of cells with apoptotic scatter characteristics had apoptotic morphology (chromatin condensation) (80.6%). Ninety-two percent of cells from the viable region had normal morphology. CD4+ T cell apoptosis measured by scatter was then correlated with the TdT assay for DNA fragmentation. Lastly, CD4+ T cell apoptosis by scatter and annexin V uptake were also shown to correlate. In the latter experiments, PBMC morphology and cell death by trypan blue uptake were studied simultaneously and confirmed the two flow cytometric assays. Apoptosis of CD4+ and CD8+ T cells has been shown in PBMCs from HIV infected adults analyzed after overnight culture. Since cell death may be an artifact of in vitro 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 infected children and adults. Patients were studied in Denver, CO, USA. PBMCs from 21 children, 4 adolescents and 9 adults and seronegative age-matched controls were stained for CD4 and CD8 surface markers. Apoptotic cells were detected in a newly characterized flow cytometric assay by diminished forward and increased side scatter. For the scatter assay, PBMCs had been labeled initially by an indirect method involving an intermediary incubation in the presence of biotinylated MAbs at 37°C for 30 minutes prior to incubating with streptavidin-FITC at 4°C for 20 minutes. Thereafter, the intermediary incubation step was removed and PBMCs were incubated with PE-conjugated CD4+ and CD8+ MAbs. Both CD4+ and CD8+ T cell apoptosis appeared enhanced in the indirect method. The significant differences were abolished after subtraction of data from simultaneously studied time-matched controls. CD4+ and CD8+ T cell apoptosis were significantly higher in HIV-infected study subjects than in simultaneously studied seronegative controls. PBMCs were assayed immediately ex vivo and after overnight culture after stimulation by an anti-TCR MAb as well as spontaneously. There was a direct correlation between CD4+ and CD8+ T cell apoptosis and CD4+ T cell depletion. A significant correlation was also shown between apoptosis immediately ex vivo and after overnight culture. I then studied apoptosis in a South African population comprising 18 symptomatic children and 4 seroreverters. CD4+ and CD8+ T cell apoptosis were significantly higher in symptomatic HIV-1-infected children than in seroreverters and seronegative controls. CD4+ T cell apoptosis correlated with depletion of CD4+ T cell percentage in symptomatic HIV-1-infected children. I also noted elevated CD4+ T cell apoptosis in patients recovering from intercurrent disease in comparison to those who were either acutely ill or relatively asymptomatic outpatient attendees. Lastly, I compared CD4+ and CD8+ T cell apoptosis in cohorts from Denver, CO and Tygerberg Children’s Hospital, South Africa. I selected only patients with moderate or severe HIV infection from both centers. South African patients were significantly younger, more malnourished, had higher gamma globulin levels and were less likely to receive ART. CD8+ T cell apoptosis was higher in North American patients suggesting a possible impairment in CD8+ activity in the South African study subjects.

AFRIKAANSE OPSOMMING: ‘n Eenvoudige en vinnige vloei sitometriese toets is ontwikkel om apoptose aan te toon vanuit ‘n gemengde populasie selle. Dit moes geskik wees vir kinders van wie net klein volumes bloed getrek kan word. Die teenwoordigheid van apoptotiese perifere bloed mononuklere selle (PBMS) was vasgestel deur morfologiese beoordeling in kliniese monsters ex vivo en na oornag kultuur. Die ondersoek is gebasseer op die verstrooings patroon van bestraalde PBMS wat apoptose induseer. PBMS is gemerk met a CD4 MAb. CD4+ selle is gesorteer in apoptotiese en lewensvatbare populasies deur verstrooings karakteristieke. Morfologie is beoordeel deur fluoreserende mikroskopie. Die meerderheid van selle met apoptotiese verstrooings karakteristieke (verminderde voorwaartse en verhoogde sywaartse verstrooings patroon) het apoptotiese karakteristieke gehad (80.6%). Twee-en-negentig persent van selle van die lewensvatbare area het normale morfologie gehad. Verstrooings patroon is ook gekorreleer met die TdT meting vir DNA fragmentasie in kliniese monsters van MIV-geinfekteerde kinders. Daarna is Annexin V gekorreleer met verstrooings patroon, apoptotiese morfologie en trypan blou opname in selle wat blootgestel is na verskillende konsentrasies van beauvericin. Apoptose van CD4+ en CD8+ T-selle is bewys in PBMS van MIV-geinfekteerde volwassenes na oornag kultuur. Omdat sel dood ‘n artefak van in vitro kultuur kan wees, en omdat daar min inligting is oor apoptose in paediatriese MIV siekte, het ek onderneem om ‘n deursnee analiese te doen in PBMS wat onmiddelik ex vivo geanaliseer is vanaf MlV-geinfekteerde kinders en volwassenes. Die pasiente is bestudeer in Denver, Colorado, VS A. PBMS van 22 kinders, 4 adolessente en 9 volwassenes en seronegatiewe ouderdoms-gepasde kontroles is gekleur vir CD4+ en CD8+ oppervlaksmerkers. Apoptotiese selle is vloeisitometries aangedui deur verandering in verstrooings patroon. Vir die doeleindes van die verstrooings assay is die PBMS aanvanklik deur ‘n indirekte metode gemerk, wat ‘n intermediere inkubasie in die teenwoordigheid van biogetinileerde MAbs by 37°C vir 30 minute voor dit geinkubeer is met streptavidin- FITC by 4°C vir 20 minute behels. Daarna is die intermediere inkubasie stap verwyder en PBMC is geinkubeer met PE - gekonjugeerde CD4+ and CD8+ MAbs. Beide die CD4+ en CD8+ T-sel apoptose het verhoog voorgekom met die indirekte metode. Die betekenisvolle verskille het verdwyn na data van gelyktydige tyd - gepaarde kontroles afgetrek is. CD4+ en CD8+ T-sel apoptose was betekenisvol hoër in MIV-geinfekteerde studie gevalle as in gelyktydig bestudeerde seronegatiewe kontroles. PBMS assays is gedoen onmiddelik ex vivo en na oornag inkubasie na stimulasie deur ‘n anti-TCR MAb, sowel as spontaan. Daar was ‘n direkte korrelasie tussen CD4+ en CD8+ T sel apoptosis en CD4+ T sel vermindering. ‘n Beduidende korrelasie is ook getoon tussen apoptose onmiddelik ex vivo en na oornag kultuur. Daaropvolgend het ek apoptose in ‘n Suid Afrikaanse populasie van 18 simptomatiese kinders en 4 serologies terukerende gevalle bestudeer. CD4+ en CD8+ T sel apoptose was aansienlik hoër in siptomatiese MIV - 1-geinfekteerde kinders as in die serologies terukerende gevalle en seronegatiewe kontroles. CD4+ T sel apoptose het gekorrelleer met vermindering van CD4+ T sel persentasie. Ek het ook opgemerk dat daar ‘n tendens bestaan het tot verhoogde CD4+ T sel apoptose in pasiente wat besig was om te herstel van bykomende siektes. Ek het CD4+ en CD8+ T sel apoptose in kohorte van Denver, Colorado en Tygerberg, Suid Afrika vergelyk. Suid Afrikaanse pasiente was jonger en meer wangevoed as hul Noord Amerikaanse ewekniee. Suid Afrikaanse kinders het ook meer gevorderde siekte gehad. Wanneer pasiente gepas is vir die graad van ernstigheid van siekte en slegs die minder ernstige (B) en ernstige siekte (C) vergelyk is, was CD8+ T sel apoptose beduidend hoër in Noord Amerikaanse pasiente. Hierdie waarneming ondersteun die hipotese dat CD 8+ T sel aktiwiteit moontlik onderdruk mag wees in simptomatiese Suid Afrikaanse MIV-1-geinfekteerde kinders.

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