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HIV-induced apoptosis
Besides the above-mentioned cytokine phenotypes an array of other immunological changes is induced following HIV infection. Firstly, whole virus as well as viral proteins causes hyperactivation of the immune system (Ross, 2001) inducing a chronic state of inflammation within the host (Martin and Emery 2009). Failure by the immune system to control infection ultimately results in immunodeficiency measurable by a decrease in CD4+ T helper cells. The depletion of these cells is speculated to occur primarily through apoptosis which is an active, energy-requiring process that forms part of the normal development and maturation cycle of cells. Apoptosis is well-regulated and executed either through the extrinsic (death receptor pathway) or intrinsic pathways (mitochondrial pathway, Shedlock et al., 2008; Boya et al., 2004). For an illustration of the differences between these pathways see Figure 2.8. Briefly, extrinsic signals cause an upregulation in the expression of the tumor necrosis factor (TNF) receptor, the expression of the type 1 transmembrane protein, Fas and Fas ligand (Fas L) respectively, other “death receptors” and their ligands (Badley et al., 2003). The binding of molecules (TNF-α, IFN-γ) to their receptors triggers intracellular signalling, caspase activation and apoptosis. In contrast, during the intrinsic pathway deathsignals act directly on mitochondria prior to the caspases being activated (Boya et al., 2004).
The intrinsic pathway is the most common cell death pathway following intracellular infections. It is usually activated before the extrinsic pathway and is associated with mitochondrial damage (Genini et al., 2001), elevated ROS production and an increase in oxidative stress (Bayir and Kagan 2008). The intrinsic pathway is usually activated when cells are stressed (Lecoeur et al., 2008) and involves the translocation of pro-apoptotic proteins to mitochondria. These proteins cause a change in the organelles’ membrane potential causing the release of intermembrane space proteins (such as cytochrome c), apoptosis inducing factors and a range of metabolic intermediates (Lemasters et al., 1998).
Externalization of phosphatidylserine then follows signalling early apoptosis. Late apoptosis is characterized by DNA fragmentation and degradation. When the cell is damaged necrosis becomes evident and the cell dies.
Detection of apoptosis
The various changes that cells undergo following HIV-induced apoptosis is measured through flow cytometry (elaborated on in Section 2.10.3) and include for example a loss in plasma membrane integrity, a decrease in mitochondrial membrane potential as well as morphological changes (Lecoeur et al., 2008). One of the primary changes involves the shrinking of cells. Using flow cytometry this is observed by a decrease in forward scatter (FSC) which relates to the size of cells (Vermes et al., 2000). Other changes are usually detected through the addition of fluorescent labels. In the context of HIV infection apoptosis may be beneficial or detrimental to both the host and pathogen respectively (Goldberg and Stricker 1999). For example, an increase in cell death through apoptosis may limit viral replication since the host reservoir becomes depleted. In contrast cell death may facilitate viral spread when damaged cells release their intracellular contents. The reverse also holds true i.e. apoptosis may be inhibited so that the host cells are not depleted and productive infection is maintained (Selliah and Finkel 2001).
During HIV infection not all the CD4+ cells are infected with virus. The numbers of apoptotic cells are usually more than the percentage of infected cells. This is largely due to the apoptosis of uninfected bystander cells (Figure 2.9, Selliah and Finkel 2001). In an attempt to better understand HIV-specific apoptosis and the mechanisms leading to this form of cell death in uninfected and HIV-infected T lymphocytes in vitro, Herbein et al (1998) isolated peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs) from a healthy individual and subjected these cells to in vitro HIV infection.
CHAPTER 1
INTRODUCTION
1. Introduction
CHAPTER 2
LITERATURE REVIEW
2. HIV/AIDS and its effect on the immune and metabolic systems
2.1 History of HIV/AIDS
2.2 Classification
2.3 Virion Structure
2.4 HIV-1 Genome
2.5 HIV-1 Life Cycle
2.6 Clinical Course of HIV-1 infection
2.7 The Immune System and HIV
2.8 Host Metabolism
2.9 Rationale, Research Questions/Objectives and Hypothesis
2.10 Current tools for measuring HIV infection, HIV-induced changes and disease progression
CHAPTER 3
EXPERIMENTAL DESIGN & PRACTICAL CONSIDERATIONS
3. Design and Practical Considerations
3.1 Ethics Approval
3.2 Selection of Biochemical/Metabolic Pathway for MS analysis
3.3 Selection of Immune Parameters
3.4 Biofluid Selection
3.5 Analysis Techniques
3.6 Sample Selection
3.7 Statistical Methods
CHAPTER 4
METABONOMICS PROFILE OF HIV INFECTED BIOFLUID
4. Summary
4.1 Introduction
4.2 Materials and Methods
4.3 Results and Discussion
4.4 Conclusion
CHAPTER 5
IMMUNOLOGICAL PROFILE OF HIV INFECTED INDIVIDUALS
5. Summary
5.1 Introduction
5.2 Materials and Methods
5.3 Results and Discussion
5.4 Conclusion
CHAPTER 6
CONCLUDING CHAPTER
6. OVERVIEW
6.1 Metabonomics Profile of HIV infected Individuals
6.2 Immune Profile of HIV-infected individuals
6.3 Linking metabolic and Immune changes
6.4 Answers to Questions raised
6.5 Significance of the Project
6.6 Limitations of this study
6.7 Novel Aspects
6.8 Recommendations and Future Considerations
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