Background: Immunological events due to infection by the human immunodeficiency virus (HIV) perturb mitochondrial function which augments virus-induced metabolic imbalances. Organic acids, established biomarkers of mitochondrial dysfunction have not yet been studied as indicators of HIV-induced changes in this organelle. In this study, mass spectrometry (MS) was used to determine the organic acid profile and flow cytometry the corresponding immune changes in biofluids of clinically stable patients, with the aim of identifying HIV-influenced molecules which could potentially be developed into diagnostic and/or prognostic markers. Methodology and Results: Gas chromatography mass spectrometry (GC-MS) was used to determine HIV-induced mitochondrial dysfunction by means of organic acid profiling of sera, peripheral blood mononuclear cells (PBMCs) and urine. The Metabolomics Ion-based Data Extraction Algorithm (MET-IDEA) proved more suitable for data analysis than other software packages. The biofluids analyzed differed in the type of metabolites identified but provided related biological information. An overlap in the metabolic profiles of HIV seronegative (HIV-) and seropositive (HIV+) groups was observed. When cases in the advanced stage of the disease were included an improved separation between the groups was observed. Metabolites altered as a result of HIV infection were representative of disrupted mitochondrial metabolism, changes in lipid, sugar, energy and neurometabolism as well as oxidative stress. Metabolite detection was found to be influenced by viral load. Corresponding immune parameters were measured by detecting oxidative stress, apoptosis and cytokine changes. As expected, the HIV+ individuals experience constant oxidative stress. Significantly higher amounts of reactive oxygen species (ROS, p =0.004) were detected in infected sera. Apoptosis in the HIV+ cells was significantly higher than that occurring in the HIV- cells (p< 0.0001). When gating T cells, a greater percentage apoptosis was measured in the CD8 positive cell population (p=0.0269). Since the CD4 cells of the patient group were not depleted these cells were able to produce the soluble factor needed for apoptosis to occur in CD8 cells. In vitro stimulation of the infected PBMCs with viral peptides led to an increase in the percentage T cells which produced intracellular interferon gamma (IFN-γ). The T helper type 1 (Th1), Th2 and Th17 cytokine profile in aliquots of HIV and HIV+ sera measured using Cytometric Bead Array (CBA) technology and analyzed using multivariate statistics, correctly classified over 70 % of the cases as HIV- or HIV+. Interleukin (IL)-6 and IL-10 were found to be the key immune markers altered during HIV infection. Analyzing cytokines in this manner follows a cytokinomics approach. Conclusion: Organic acids detected agree with the oxidative, apoptotic and cytokine responses. The impact of HIV on the metabolic signature and immune system is detectable in the early asymptomatic phase of infection by using MS, flow cytometry and spectroscopy. The observed changes share a biochemical relationship and are supportive of the link between the metabolic and immune systems. The data was collected using different forms of spectroscopy and spectrometry and these approaches may therefore have a future in the management of HIV infection and the acquired immunodeficiency syndrome (AIDS). / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/25142 |
| Date | 30 May 2012 |
| Creators | Williams, Aurelia Alvina |
| Contributors | Prof D Meyer, Prof C Reinecke, s28584661@tuks.co.za |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
| Rights | © 2012 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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