Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction
In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus
(HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency
syndrome (AIDS) related complications now prevails in the aging HIV infected population.
Increased levels of inflammation and chronic immune activation are associated with HIV
infection. In the era of ART people living with HIV are at an increased risk of cardiovascular
disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and
upon activation platelets degranulate and secrete various inflammatory, coagulatory and
adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key
component of the coagulation pathway and serve as a link between inflammation and
thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease
and have been identified as immune cells that play a crucial role in pathogen recognition and
modulation of immune cells during infections. Several antiviral and antibacterial properties of
platelet alpha granule contents have been established. Platelet aggregometry remains the most
widely used technique to evaluate platelet function even though this technique is limited by many
pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of
platelet function in their physiological environment with minimal artefactual activation. Few
studies have however reported on standardized methods to evaluate platelet function in the
context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve
individuals remains scarce. The aim of this project was to examine the relationship between
platelet function and immune activation in patients with HIV
Materials and methods
This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation
were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected
healthy controls) using; flow cytometry and haemotology analyzers. The relationship between
these indices and markers of platelet activation, disease progression and immune activation
were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a
cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy
controls), using a novel whole blood flow cytometry based functional assay. These baseline
levels were then correlated with markers of immune activation and disease progression in HIV.
In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve)
and 38 uninfected controls was evaluated using flow cytometry. Platelet response was
measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce
reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet
function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV
naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP,
arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry.
Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow
cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected
healthy controls. Associations between PLAs, immune activation and disease progression in HIV
infected individuals were determined. The final study evaluated platelet aggregates, platelet
derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected
(ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet
aggregates and markers of immune activation and disease progression were evaluated.
Results
HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85
vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and
viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated
(r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with
platelet aggregation. HIV infected individuals showed increased levels of platelet activation
(%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV,
platelet function is retained and platelets showed increased response to submaximal
concentrations of endogenous agonists. HIV infected individuals showed increased levels of
circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36-
18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8
(r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels
of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160);
PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133);
activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6],
p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group
0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals.
Conclusion
This study supports the potential use of the MPV and PDW as readily available markers of
platelet activation and immune activation in HIV. We also showed elevated levels of activated
platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a
concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in
the evaluation of platelet function in HIV. The measurement of platelet function using flow
cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV
infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay
for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and
levels of activated platelets and aggregates which mimics the in vivo physiological environment
of MPs. To the best of our knowledge, this study is the first to report on a novel approach in
evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based
platelet assays. In addition, minimal work has been performed previously on platelet function in
the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as
defined for this study. / AFRIKAANSE OPSOMMING: Inleiding
In die era van antiretrovirale terapie (ART), het mense wat met die menslike
immuniteitsgebreksvirus (MIV) leef, het nou 'n verlengde lewensduur. 'N opkomende neiging van
nie-verworwe immuniteitsgebreksindroom (vigs) heers nou in die verouderende MIV-besmette
bevolking. Verhoogde vlakke van inflammasie en chroniese immuun aktivering word
geassosieer met MIV-infeksie en in die era van ART loop mense wat met MIV leef, 'n verhoogde
risiko van kardiovaskulêre siekte (KVS). Plaatjies speel 'n belangrike rol in beide inflammasie en
immuun aktivering en met aktivering degranulate en skei plaatjies verskeie inflammatoriese,
coagulatory en adhesie molecule af. Geaktiveerde plaatjies druk oppervlak P-selectin (CD62P)
is 'n belangrike komponent van die stollings weg en dien as 'n skakel tussen inflammasie en
trombose. Geaktiveerde plaatjies is in beide inflammasie en kardiovaskulêre siekte betrokke en
is geïdentifiseer as immuun selle wat 'n deurslaggewende rol speel in die patogeen erkenning
en modulasie van immuun selle tydens infeksies. Verskeie antivirale en antibakteriese
eienskappe van plaatjie alpha korrel inhoud is vasgestel. Plaatjie aggregometry bly die mees
gebruikte tegniek om plaatjie funksie te evalueer, alhoewel hierdie tegniek is beperk deur baie
pre-analitiese veranderlikes. Plaatjie vloeisitometrie aan die ander kant bied 'n vinnige meting
van plaatjie funksie in hul fisiologiese omgewing met 'n minimale artefactual aktivering. Min
studies het egter berig op gestandaardiseerde metodes om plaatjie funksie in die konteks van
MIV te evalueer. Plaatjie funksie is steeds onduidelik en data oor MIV besmet behandeling naïef
individue bly skaars. Die doel van hierdie projek was om die verhouding tussen die plaatjie
funksie en immuun aktivering in pasiënte met MIV te ondersoek.
Materiaal en metodes
Hierdie studie het bestaan uit vyf sub-studies. In die eerste plekis plaatjie indekse en vlakke van
plaatjie aktivering bepaal in 'n groep van 330 deelnemers (185 MIV-besmette ARV naïef en 145
onbesmette gesonde kontrole) met behulp van vloeisitometrie en hematologie ontleders. Die
verhouding tussen hierdie indekse en merkers van plaatjie aktivering, die siekte se progressive
en immuun aktivering is beoordeel. Verder is die vlakke van plaatjie aktivering en samevoeging
in 'n groep van 82 deelnemers (41 MIV-besmette (ARV naïef) individue en 41 onbesmette
gesonde kontrole) geëvalueer, met behulp van 'n nuwe vol bloed vloeisitometrie gebaseerde
funksionele toets. Hierdie basislyn vlakke is dan gekorreleer met merkers van immuun aktivering
en die progreessie van die siekte in MIV.
In 'n daaropvolgende studie, is plaatjie funksie in 'n groep wat bestaan uit 58 MIV besmet te
(ARV naïef) en 38 onbesmette beheer geëvalueer met behulp van vloeisitometrie. Plaatjie
reaksie is na stimulasie gemeet met adenosine diphophate (ADP) by konsentrasies bekend
omkeer (0.04mM) te oorreed en onomkeerbaar (0.2mm) plaatjie aggregasie. Ten einde plaatjie
funksie in MIV te evalueer, is plaatjie reaksie in 'n groep wat bestaan uit 58 MIV-besmette (ARV
naïef) en 38 onbesmette kontrole geëvalueer. Die plaatjies is geaktiveer deur gebruik te maak
van wisselende konsentrasies van ADP, is aragidoonsuur (AA) en kollageen en plaatjie funksie
gemeet met behulp van vloeisitometrie. Vlakke van sirkulerende plaatjie leukosiet gemiddeldes
is ook gemeet met behulp van vloeisitometrie in 'n groep wat bestaan uit 35 MIV-positiewe (ARV
naïef) individue en 32 onbesmette gesonde kontrole. Assosiasies tussen leukosiet gemiddeldes,
immuun aktivering en die progressive van ie siekte in MIV-besmette individue is ook bepaal. Die
finale studie het plaatjie-gemiddeldes, plaatjie afgelei mikrodeeltjies en mikrodeeltjies
geëvalueer in 'n groep wat bestaan uit 46 MIV besmet (ARV-naïewe) en 40 onbesmette
gesonde kontrole. Assosiasies tussen mikrodeeltjies, plaatjie afgelei, plaatjie gemiddeldes en
merkers van immuun aktivering en die siekte se progressie is geëvalueer.
Resultate
MIV-besmette individue het gedaalde gemiddelde plaatjie volume vlakke getoon (HIV
gemiddelde 7,91 ± 0,85 8,52 ± 1,12 teen, p <0,0001) wat direk gekorreleer het met CD4-tellings
(r = -0,2898, p = 0,0075) en virale (r = 0,2680, p = 0,0177). Plaatjie verspreiding breedte vlakke
het direk gekorreleer met (r = 0,3455, p = 0,0362) met 'n aktiewe koagulasie en omgekeerd
gekorreleer (r = -0,3666, p = 0,0463) met plaatjie aggregasie. MIV-besmette individue het
verhoogde vlakke van plaatjie aktivering getoon (% CD62P mediaan 11,33 [5,96-29,36] teen
kontrole groep 2,48 [1,56-6,04], p = 0,0001). In MIV, was plaatjie funksie behou en plaatjies het
'n verhoogde reaksie op submaksimale konsentrasies van endogene agoniste getoon. MIVbesmette
individue het verhoogde vlakke van sirkuleer plaatjie monosiet-gemiddeldes
gedemonstreer (25.26 [16,16-32,28] teen kontrole groep 14,12 [8,36-18,83], p = 0,0001) wat
direk gekorreleer het met merkers van immuun aktivering; % CD38 / 8 (r = 0,54624, p = 0,0155),
virale lading (r = 0,633, p <0,009). Verder rapporteer ons op verhoogde vlakke van sirkulerende
mikrodeeltjies (mediaan% LP 1.7 [0,95-2,83] teen kontrole groep 1,12 [0,63-1,57], p = 0,0160);
PMPs (mediaan% PMPs 26,64 [11,33-36,62] teen kontrole groep 20,02 [18,08-26,08], p =
0,0133); geaktiveer PMPs (mediaan CD62P MFI 3,81 [3,46-4,54] teen kontrole groep 3,41 [3,16-
3,6], p = 0,0037) en plaatjie gemiddeldes (Mediaan% CD62P 14,10 [5,49-39,94] teen 0.17 [0,10-
10,99], p= 0.0097) in MIV besmet asimptomatiese individue.
Gevolgtrekking
Hierdie studie ondersteun die potensiële gebruik van die MPV en PDW as waardevolle geredelik
waardevolle merkers van plaatjie aktivering en immuun aktivering in MIV. Ons het ook getoon
verhoogde vlakke van geaktiveer de plaatjies in MIV-besmette individue getoon wat hyper
reageer op endogene agoniste was in 'n konsentrasie-afhanklike wyse. Plaatjie vloeisitometrie is
'n vinnige en waardevolle tegniek in die evaluering van plaatjie funksie in MIV. Die meting van
plaatjie funksie gebruik vloei cytometry maak die evaluering van plaatjie sein paaie wat in MIVgeïnfekteerde
individue verander moontlik. Laastens het ons beskryf 'n hele bloed
vloeisitometrie gebaseer de toets vir die evaluering van sirkulerende mikrodeeltjies, plaatjie
afgelei mikrodeeltjies en vlakke van geaktiveer plaatjies en gemiddeldes wat lyk soos die in vivo
fisiologiese omgewing van MP's. Na die beste van ons kennis, is hierdie studie die eerste om te
rapporteer oor 'n nuwe benadering in die evaluering van plaatjie funksie in MIV met behulp van
'n reeks van new hele bloed vloeisitometrie gebaseer de plaatjie toetse. Daarbenewens is
minimale werk voorheen uitgevoer op die plaatjie funksie in die konteks van MIV-infeksie; en
veral in 'n groep van asimptomatiese, onbehandelde pasiënte soos vir hierdie studie. Hierdie
projek het bewyse bygevoeg tot die teorie dat plaatjies, in MIV, kan 'n skakel wees tussen die
aktiewe inflammatoriese reaksie en die toename in die aantal trombotische en kardiovaskulêre
siekte waargeneem in pasiënte wat met hierdie siekte saamleef.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/97022 |
| Date | 04 1900 |
| Creators | Nkambule, Bongani Brian |
| Contributors | Ipp, Hayley, Davison, Glenda, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Haematological Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxvi, 151 pages : illustrations (some colour) |
| Rights | Stellenbosch University |
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