| Summary: | Thesis (MScMedSc)--Stellenbosch University, 2012. === Bibliography === ENGLISH ABSTRACT: Background: In the era of antiretroviral therapy (ART), the risk of acquired immune deficiency
syndrome (AIDS) related deaths has decreased and people living with Human
Immunodeficiency Virus (HIV) now have prolonged life spans. However, an increasing trend of
non-AIDS associated deaths has been reported despite adequate control of viral loads. HIV
infection is established as a chronic inflammatory condition which is associated with an
increased risk for thrombosis. Thus HIV infected patients are at a higher risk of developing
cardiovascular disease (CVD) and other inflammatory-associated complications.
Inflammation is linked with thrombosis and promotes the formation of thrombin, which plays an
important role in platelet activation. Furthermore, activated platelets have been shown to play a
key role during infection and the inflammatory process, particularly by mediating interactions
between cells of innate immunity. Soluble markers of platelet activation have been shown to be
increased in HIV-infection. However, these have not been well documented by flow cytometry.
P-selectin CD62P is stored in the alpha granules of platelets and is expressed on the surface
only upon platelet activation. This facilitates interaction with other blood cells and the
endothelium. Activated platelets may play a role in HIV-induced atherosclerosis through the
expression and release of mediators that induce endothelial activation and support the adhesion
of leukocytes to the inflamed vessel wall. Fibrinogen is a precursor of the blood coagulatory
protein fibrin and the degradation of fibrin to D-dimer is a measure of the formation and the
subsequent dissolution of blood clots. In HIV infected patients, chronic inflammation induces the
up-regulated expression of tissue factor (TF) on monocytes which triggers the activation of the
clotting cascade and increases the level of D-dimers.
Methods: This pilot study consisted of ART naïve patients and all platelet flow analyses were
carried out on whole blood. In this study, a total of 57 adult South Africans were recruited from a
clinic in the Western Cape. These included 32 HIV positive patients and 25 HIV negative
individuals. The levels of platelet activation and platelet function were investigated using a novel
platelet cytometry assay. The method was optimized to ensure minimal platelet activation: no
centrifugation or red blood cell (RBC) lysis steps were performed. The platelet-specific markers
CD41a and CD42b were used to ensure gating on platelets only. CD62P expression was used
to evaluate platelet activation and these levels were correlated with Fibrinogen, hsCRP, Ddimer,
CD4 counts and viral load. Furthermore, platelet function was evaluated by investigating
the response of platelets to endogenous agonists which included adenosine diphosphate(ADP)
and arachidonic acid (AA) at varying concentrations. Results:This study demonstrated higher baseline levels of CD62P expression in treatment
naïve HIV positive patients as compared to uninfected controls (mean %CD62P 71.74 ± 2.18 vs
control 54.52 ± 2.42; p=<0.0001). In addition it was shown that %CD62P expression correlated
directly with platelet counts (r=0.374, p=0.042). Platelet counts showed an inverse correlation
with viral loads (give values) Fibrinogen levels correlated with the absolute WCC (r=0.659,
p=0.0021); absolute neutrophil count (r=0.619, p=0.0105); absolute monocyte count (0.562,
p=0.0235) and hsCRP (r=0.688 p=0.0011). In addition, fibrinogen showed a strong negative
correlation with CD4 counts (r=-0.594, p=0.0014) and therefore, may be a valuable marker of
both disease progression and risk of thrombosis in treatment naïve HIV positive patients.
HsCRP levels correlated with the absolute neutrophil counts (r=0.392, p=0.0005). The HIV
Group showed an overall hyper-response to ADP at a concentration 0.025 μM as compared to
uninfected controls (62.34 ± 9.7 vs control 36.90 ± 5.7, p=0.0433). Conclusions: In this study we describe a novel Flow Cytometry technique that may be used to
evaluate the levels of platelet activation and platelet function in HIV infected patients. In addition
we report a cost-effective panel in the form of fibrinogen, WCC and platelets that may be
valuable in predicting the progression of HIV infection to AIDS or other inflammatory- associated
complications in treatment naïve HIV infected patients. Platelet counts showed an inverse
correlation with viral loads and a direct correlation with the level of activated platelets. These
findings taken together suggest the potential prognostic value of platelet activation and platelet
counts in the context of asymptomatic HIV infected patients. Our findings suggest WCC and
Fibrinogen may be used to evaluate the inflammatory profile of individual HIV infected patients.
This may have a direct impact on HIV patient management prior to initiation of antiretroviral
therapy and valuable in monitoring responses to treatment. Further, we present a novel flow
cytometry based platelet functional assay and suggest the use of ADP at a concentration of
0.025 μM to evaluate platelet function optimally in HIV infected patients. The utilization of the
novel Flow Cytometry technique as described in this study would add significant value in the
assessment of thrombotic risk and disease progression in HIV infected patients and may
additionally prove to be of value in other chronic inflammatory conditions. === AFRIKAANSE OPSOMMING: Voorkennis: In die era van antiretrovirale terapie (ART), het die risiko van vigs-verwante
sterftes verminder en mense wat nou met volle naam (MIV) leef, het ‘n verlengde lewensduur.
Nogtans, word 'n toenemende neiging van nie-vigs geassosieer sterftes berig wat hoofsaaklik
toegeskryf word aan trombotiese toestande. MIV-infeksie word as 'n chroniese inflammatoriese
toestand beskou met ʼn verhoogde trombose risiko geassosieer word. Dus, MIV-besmette
pasiënte het 'n hoër risiko om kardiovaskulêre siekte (CVD) te ontwikkel ongeag of hulle ARV
naïef is of op behandeling is nie.
Inflammasie word geassosieer met trombose en bevorder die vorming van trombien, wat 'n
belangrike rol in plaatjie aktivering speel. Verder, word daar bewys dat geaktiveerde
bloedplaatjies 'n belangrike rol speel tydens infeksie en die inflammatoriese proses.Hulle
bemiddel interaksies tussen die selle van ingebore immuniteit. Daar word bewys dat oplosbare
merkers van plaatjie aktivering verhoog is in MIV-infeksie, maar die bewyse is nie so goed
gedokumenteer deur vloeisitometrie nie. P-selectin (CD62P) word gestoor in die alfa korrels van
plaatjies en word uitgedruk op die oppervlak slegs wanneer plaatjies geaktivering word;
daardeur fasilitering dit die interaksie met ander bloedselle en die endoteel. Geaktiveerde
plaatjies kan ook 'n rol in MIV-geïnduseerde aterosklerose speel deur middel van die uitdrukking
en vrylating van bemiddelaars wat endoteel aktivering induseer asook die adhesie van
leukosiete aan die ontsteekte vat wand ondersteun.. Fibrinogeen, 'n voorloper van die bloed
koagulatories proteïen fibrin en die degradasie van fibrin na D-dimeer is' n maatstaf van die
vorming en die daaropvolgende ontbinding van bloedklonte. Kroniese inflammasie in MIVbesmette
pasiënte, induseer die op-gereguleerde uitdrukking van weefsel faktor (TF) op
monosiete wat die aktivering van die stolling kaskade inisieer en die D-dimere vlakke verhoog.
Metodes: Hierdie loodsstudie bestaan uit ART naïewe pasiënte en al die plaatjie vloei ontleding
was op vol bloed uitgevoer. In hierdie studie, 'n totaal van 57 volwasse Suid-Afrikaners was van'
n kliniek in die Wes-Kaap gewerf. Dit sluit 32 MIV-positiewe pasiënte en 25 MIV negatiewe individue in. Die vlakke van plaatjie aktivering en plaatjie funksie was ge ondersoek deur middel
van 'n nuwe plaatjie sitometrie toets. Die metode was geoptimaliseer om minimale plaatjie
aktivering te verseker: dus geen sentrifugering of volle naam (RBS) liseer stappe was gebruik
nie. Die plaatjie-spesifieke merkers, CD41a en CD42b was gebruik om te verseker dat slegs
bloedplaatjes gekies word. Die uitdrukking van CD62P was gebruik vir die evaluering van
plaatjie aktivering en hierdie vlakke was gekorreleer met fibrinogeen, hsCRP, D-dimeer, CD4-
tellings en virale lading. Verder, was plaatjie funksie geëvalueer deur die reaksie van plaatjies
aan endogene agoniste wat ADP en AA by wisselende konsentrasies insluit te ondersoek.
Results: Hierdie studie het getoon hoër basislyn vlakke van CD62P uitdrukking in behandeling
naïewe MIV-positiewe pasiënte in vergelyking met onbesmette beheermaatreëls (beteken%
CD62P 71,74 ± 2,18 vs beheer 54,52 ± 2,42, p <0.0001). Daar is ook getoon dat% CD62P
uitdrukking direk gekorreleer met plaatjie tellings (r = 0,374, p = 0,042). Plaatjie tellings het 'n
omgekeerde korrelasie met virale ladings (gee waardes) fibrinogeen vlakke korreleer met die
absolute WCC (r = 0,659, p = 0,0021), absolute neutrofiel telling (r = 0,619, p = 0,0105);
absolute monosiet telling (0,562, p = 0,0235) en hsCRP (r = 0,688 p = 0,0011). Daarbenewens,
fibrinogeen het 'n sterk negatiewe korrelasie met 'n CD4-tellings (r = -0,594, p = 0,0014) en
daarom kan 'n waardevolle merker van beide die siekte en die risiko van trombose in
behandeling naïewe MIV-positiewe pasiënte. HsCRP vlakke gekorreleer met die absolute
neutrofiel tellings (r = 0,392, p = 0,0005). Die MIV-groep het 'n algehele hiper-reaksie op die
ADP by 'n konsentrasie 0,025 μM in vergelyking met onbesmette beheermaatreëls (62,34 ± 9,7
vs beheer 36,90 ± 5.7, p = 0,0433).
Gevolgtrekkings: In hierdie studie beskryf ons 'n roman vloeisitometrie tegniek wat gebruik kan
word om die vlakke van Plaatjie aktivering en plaatjie funksie in die MIV-besmette pasiënte te
evalueer. Verder het ons 'n verslag van 'n koste-effektiewe paneel in die vorm van fibrinogeen,
WCC en plaatjies wat waardevol kan wees in die voorspelling van die vordering van MIVinfeksie
tot VIGS of ander inflammatoriese-verwante komplikasies in die behandeling naïewe
MIV-besmette pasiënte. Plaatjie tellings het 'n omgekeerde korrelasie met die virale laste en 'n
direkte verband met die vlak van geaktiveerde bloedplaatjies. Hierdie bevindinge saam, dui op
die moontlike prognostiese waarde van Plaatjie aktivering en die plaatjie tel in die konteks van
die asimptomatiese MIV-geïnfekteerde pasiënte. Ons bevindinge dui daarop WCC en fibrinogeen kan gebruik word om die inflammatoriese profiel van individuele MIV-geïnfekteerde
pasiënte te evalueer. Dit kan 'n direkte impak op MIV pasiënt vooraf aan die inisiasie van
antiretrovirale terapie en waardevolle in die monitering van die reaksie op behandeling. Verder
bied ons 'n roman vloeisitometrie gebaseer plaatjie funksionele toets en dui op die gebruik van
die ADP teen 'n konsentrasie van 0,025 μM plaatjie funksie optimaal te evalueer in MIVgeïnfekteerde
pasiënte. Die benutting van die roman vloeisitometrie tegniek soos beskryf in
hierdie studie sal 'n beduidende waarde toevoeg in die beoordeling van die die trombotiese
risiko en die siekte in MIV-geïnfekteerde pasiënte en kan addisioneel bewys van waarde te
wees in 'n ander chroniese inflammatoriese toestande. === National Reserach Foundation
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