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