| Summary: | Thesis (PhD)--Stellenbosch University, 2004. === 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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