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A recent study showed that a polymorphism ~35kb upstream of the HLA-C gene (-35 SNP) correlates with host control of HIV-1 in Caucasians, with the minor allele (C) associating with significantly lower set point viral loads than the major allele (T). A link between viral load and HLA-C is suggested by linkage of the two SNP alleles with different HLA-C alleles and by the fact that HLA-C, in contrast to HLA-A and -B, is not down-regulated by HIV-1 Nef protein. In addition, the -35C variant has been shown to associate with higher HLA-C messenger RNA expression in EBV-transformed B cell lines. We initially propose that increased surface expression of HLA-C in subjects with the protective SNP leads to increased breadth and magnitude of HLA-C restricted T cell responses, explaining the decrease in viral load in these subjects. This study initially investigates whether the -35 SNP correlates with the surface level of HLA-C using the monoclonal antibodies DT9, which recognises both HLA-C and HLA-E, and 3D12, which is specific for HLA-E. The lymphocytes from -35 CC subjects expressed a significantly higher level of surface HLA-C when compared to those from -35 TT subjects, but this difference in HLA-C expression can be attributed primarily to the very low expression of a single allelic product, HLA-Cw*07. Increased surface HLA-C should translate to functional differences between CC and TT subjects. This study confirmed that HLA-C restricted CD8 T cell responses against HIV-1 do exist, even for HLA-Cw*07, but represent a minority of total T cell responses. They were detected in all -35 SNP genotypes but there were no functional differences, making it unlikely that the protective effect of this SNP on viral load set point could be accounted for solely by HLA-C restricted T cell responses. Finally, a viral suppression assay was used to investigate the capacity of CD8 T cells to suppress HIV-1 replication in Caucasian and African subjects. We provide evidence that the -35 SNP effect on viral load is indeed T cell mediated. However, we suggest that the protective effect of the -35 SNP on viral load set point manifests as a result of linkage disequilibrium of this polymorphism with both favourable and unfavourable HLA-B and HLA-C alleles.

Thesis (M Med.(Community Health))--University of Limpopo, 2009. / The threat posed by infectious diseases is progressively growing on a global scale. With 2010 rapidly approaching, when South Africa will host the Soccer World Cup, there will be a massive influx of foreigners into the country. The purpose of the study was evaluate to the status of Gauteng tertiary academic hospitals with respect to outbreak response and, with the help of existing local and international policies and research, to develop a generic model that can be used by hospitals in developing outbreak response policies and standard operating procedures. Methods A descriptive cross-sectional survey using a semi-structured questionnaire was utilized to evaluate the preparedness of tertiary health care facilities in South Africa. The target population consisted of Clinical Directors, Senior Clinical Executives/ Medical Superintendents, Infection Control Nurses and / or Quality Assurance Managers and Infection Control Nurses. These categories of health professionals were targeted as they are normally delegated responsibility for outbreak response activities. Results Twelve tertiary academic hospitals were included in the survey and nine responded to the survey questionnaire giving a 75% response rate. Other significant findings were: • 71% of the responding hospitals had clear terms of reference for their response team. v • 43% of the responding hospitals had a functional preparedness and response strategy / plan for priority diseases. • The most frequent point of entry in the tertiary academic hospitals is the casualty / emergency unit, followed by the trauma and OPD areas • There are very few ‘protective environment wards’ and ‘airborne infection isolation rooms’ in Gauteng Province. • Only 15% of responding hospitals have infection control compatible ventilation and only 42% could manage a patient that requires quarantine in the casualty/ emergency unit area. Most hospitals did not have the capacity to quarantine large number of patients. The study has also illustrated that there is no model easily available, suitable for the South African context, that can be used by hospital management in facility specific planning for infectious disease outbreaks. Conclusions It can be concluded from the findings of this study that academic hospitals in Gauteng, as well as in other areas of South Africa, are not adequately prepared for the management of an infectious disease outbreak.

Epstein-Barr virus (EBV) has infected 95% of the adult population. Yet, EBV stays as a harmless passenger in infected B-cells of nearly every host. EBV depends on a careful balance between the immune system and the virus that becomes evident when the host is immunocompromised. In such individuals, EBV can manifest as one of many associated malignancies. In children who have undergone solid organ transplantation, EBV-driven post-transplant lymphoproliferative disease (PTLD) can cause significant morbidity and mortality. We examined EBV-infected cells in non-diseased pediatric transplant recipients with elevated viral loads in their peripheral blood. Examination of high EBV genome copy cells in high load patients with a combined fluorescent in situ hybridization and immunofluorescence procedure demonstrated that the majority of high copy cells had no discernible expression of immunoglobulin on the surface (Ig-null cells). Such cells are lacking the crucial survival signal provided by an intact BCR and should not survive in the circulation. By flow cytometry, high load patients were shown to have the highest percentage of Ig-null cells in their peripheral blood; those with low viral loads and non-detectable viral loads had lower percentages. The phenotype of Ig-null cells was shown to differ from the resting memory B2 phenotype of normal latently infected B cells, with variable expression of CD20, CD40, and HLA Class I and II. Sorting Ig-null cells from the peripheral blood of high load carriers further demonstrated that in all patients examined, a large portion of the viral load was carried in the Ig-null compartment. Virus was also detected in the Ig-null, CD20- and HLA Class I- compartment, with a variable enrichment of the viral load in these compartments from patient to patient. Ig-null cells have been reported in the tumors of other EBV-associated malignancies, including PTLD, but never in the peripheral blood or in a non-disease state. This study has public health relevance because PTLD carries significant morbidity and mortality to transplant recipients; the presence in the blood of aberrant Ig-null cells which should have followed a program of apoptosis might be a risk factor for the development of PTLD or another EBV-associated disease.

Human herpes virus8 (HHV-8) is a known carcinogenic agent. This report investigates five populations to determine if HHV-8 was associated with disease onset. Detection and levels of antibodies were measured using an enhanced immunofluorescent assay and were analyzed with the statistical program, SPSS. In the first study, we tested the hypothesis that HHV-8 was the causative agent in Langerhans cell histiocytosis (LCH). The seroprevalence of HHV-8 among 159 LCH patients was similar to the control group, indicating that HHV-8 is not the etiological agent of LCH. In the second study, we tested the hypothesis that HHV-8 reactivation occurs in solid-organ transplant (SOT) patients, following immunosuppression. We found a significant increase in HHV-8 seropositivity when comparing pre-transplant to post-transplant samples (p-less than .01). There was also an overall increase in viral antibody titers following transplantation (p=less than .001), indicating viral reactivation. In the third study, we compare the SOT results to bone-marrow transplant patients (BMT). Longitudinal serum samples from 34 BMT patients did not demonstrate a significant association with HHV-8 as compared to the control (p=.716) or the SOT populations (p=.180). In addition, HHV-8 reactivation did not occur post-transplantation. In the fourth study, we tested the hypothesis that HHV-8 is associated with increased risk of prostate cancer (PrCa). There was greater than a 2-fold association between HHV-8 seroprevalence and PrCa among African-Caribbean men from Tobago (p=.003). A similar trend was present in a PrCa cohort from the United States, p=greater than .05. In a fifth study, we tested the hypothesis that HHV-8 increased the risk of PrCa among men who carried genetic polymorphisms in the androgen (AR) and estrogen receptor (ESR1) genes. This study analyzed an expanded Tobago cohort, which demonstrated an association between HHV-8 and PrCa (OR 1.74, p=.032). An increased association was found among seropositive men carrying the high-risk AR allele (OR=2.46, p=.023) and ESR1 allele (OR=3.10, p=.004). The strongest association was found in seropositive men with both high-risk alleles (OR=5.20, p=.017). This study demonstrates the use of HHV-8 serology as a marker for an increased public health cancer detectable risk, due to viral prevalence or reactivation.

Virus-specific memory CD8+ T cells play a prominent role in protection of a host from recurring and persistent virus infection. It is known that memory CD8+ T cells undergo a series of differentiation stages to become fully matured effector cells. There are several important aspects of the current CD8+T cell memory phenotype model that need to be more thoroughly defined. In specific aim 1, it was hypothesized that CD27+CD28+ undifferentiated CD8+ memory T cells specific for non-persistent virus influenza A (FluA) would have phenotypic markers associated with more differentiated (effector) phenotypes. Results showed that in spite of the phenotypic enrichment of FluA-specific memory CD8+ T cells in the undifferentiated stage, they displayed effector markers indicative of late stage differentiated effector cells. In specific aim 2, it was further hypothesized that the most undifferentiated CD62L+ central memory CD8+T cells would have the effector function including immediate cytoplasmic production of gamma-IFN upon antigenic-stimulation. Results showed that CD62L+ CD8+ T cells are capable of immediate gamma IFN production after antigen-specific stimulation in the presence of the CD62 sheddase inhibitor, GM6001, highlighting the need to re-evaluate the defining markers of virus-specific central memory CD8+ cells and/or their functions. In specific aim 3, this dissertation tests the hypothesis that memory-effector differentiation of HIV-1-specific memory CD8+ T cells is impaired during the course of persistent HIV-1 infection. Detailed comparison of CD27 and CD57 co-expression on HIV-1-specific CD8+ T cells showed that these cells had a significantly lower proportion of the CD27CD57high effector subset. Moreover, these cells did not display progression from CD27+CD57 (immature memory), through CD27lowCD57low (transitional memory-effector) to CD27CD57high (effector subset) that was seen in well differentiated EBV-specific CD8+ T cells and was common in CMV-specific CD8+ T cells. These observations suggest that the normal course of HIV-1-specific CD8+ T cell memory-effector differentiation is impaired during the course of persistent HIV-1 infection. Elucidation of memory-effector differentiation of virus-specific CD8+ T cells has significant public health implications. Understanding the impairment of memory-effector differentiation of HIV-1-specific CD8+ T cells, for instance, will greatly facilitate a design of effective vaccine against progressive HIV-1 infection.

Approximately 25-30% of people infected with human immunodeficiency virus 1 (HIV-1) develop HIV-associated encephalitis and HIV-associated dementia. The underlying mechanisms leading to HIV encephalitis remain unclear. In an attempt to understand the molecular events that lead to encephalitis and subsequent dementia, I focused on identifying differentially expressed genes in the central nervous system (CNS) using SIV infected rhesus macaques as an experimental model system by using methods serial analysis of gene expression (SAGE), and microarray hybridization. I studied two different brain regions, caudate and globus pallidus, in non-infected, acutely infected, and mildly encephalitic animals. Since my analysis of macaque SAGE data utilized existing human nucleotide sequence databases, identification of the genes from which the SAGE tags were obtained proved to be challenging. I successfully identified the genes from which two of the tags were obtained. These were major histocompatibility complex class I (MHCI), differentially expressed during disease and neurogranin (Nrg), differentially expressed in caudate relative to globus pallidus. The differential expression of these two genes was confirmed by real-time RT-PCR and in situ hybridization techniques. I further characterized the localization of MHCI in the CNS tissue and found that whereas in non-infected tissues, endothelial cells were the major cell types expressing MHCI mRNA, during acute infection and mild encephalitis, when local virus replication was low or absent, all CNS cell types could express this mRNA. In addition, I observed upregulation of interferon-stimulated genes (ISGs), MxA, OAS2, and G1P3, both in the CNS and in the periphery that could be potential surrogate markers for SIV infection. Since encephalitis is observed only at end-stage disease, traditional thinking has been that the CNS remains relatively unaffected until later stages of infection. Our findings indicate that immune activation within the CNS might occur early in infection and persist in a chronic manner thereby causing continuous damage, which might affect the development of end-stage encephalitis and dementia. Therefore, early, potent, suppression of systemic viral replication could potentially inhibit the development of virus-mediated neuropathology later on. Such an approach would be of important public heath significance.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus known to cause respiratory tract infections primarily in children. This previously unknown pathogen remained undetected for years due to very slow replication in vitro and an inconsistent CPE. More recently, detection of hMPV by means of quantitative molecular techniques has proved to be more effective than culture methods. In this study we describe the development of a quantitative real time RT-PCR assay targeting the hMPV nucleoprotein (N) gene. This assay is compared to a real time nucleic acid sequence based amplification (NASBA) test, developed by bioMérieux, using control material from hMPV strains Can97-83 and Can98-75 representative of the two main lineages A and B, respectively. Using control material the real time RT-PCR, designed to detect all four sublineages of hMPV, can detect as low as 50 and 100 copies of viral RNA from the A and B lineages respectively. The real time NASBA assay can also detect 50 copies of viral RNA from the A strain but only detects 1000 copies of strain B viral RNA. In this study, hMPV has been detected in both immunosuppressed lung transplant recipients (2.14%) and children with respiratory symptoms (1.83%). This research is of major public health significance due to the amount of respiratory infections that are going undiagnosed or being treated with unnecessary antibiotics. It is important for our physicians to not only know that hMPV is present in our community but also to be able to detect and treat it appropriately. This study reports the first evidence of hMPV in the Pittsburgh area and demonstrates the importance of this virus as a critical player among respiratory pathogens in both immunosuppressed lung transplant recipients and children. In conclusion, we have successfully developed a real time RT-PCR assay targeting the hMPV N gene. Using this assay along with the real time NASBA assay developed by bioMérieux, we have detected hMPV infections in lung transplant recipients in a year long study. Using the real time RT-PCR assay alone hMPV has also been detected in children suspected of respiratory infection during the early winter season.

Molecular determinants of malaria pathogenesis are largely undefined. Chemokines and chemokine receptors, regulate immune responses, may thus determine malaria severity. Further, by regulating HIV pathogenesis, they may constitute a crucial link in malaria-HIV interaction. Understanding biologic mechanisms underlying malaria-HIV interaction has important public health utility in designing rational therapeutic and preventive strategies. Malaria could potentially modulate HIV-1 infection through alteration in expression of CD4 and chemokine receptors, required for cellular entry. This study has determined circulating levels and transcriptional profiles of β-chemokines (MIP-1α, MIP-1β, and RANTES) in ex vivo peripheral blood mononuclear cells (PBMCs) of children with varying degrees of malaria severity. Additional in vitro experiments assessed the effects of stimulation of PBMCs with crude hemozoin (Hz) or synthetic hemozoin (sHz) on CD4, β-chemokine and chemokine receptor (CCR5 and CXCR4) protein expression and transcript formation. Plasma MIP-1α and MIP-1β levels were significantly elevated in mild and severe malaria, while RANTES levels decreased with increasing disease severity. β-chemokine gene expression closely matched circulating β-chemokine profile, illustrating that PBMCs are a primary source for β-chemokine production during malaria. Healthy children with a history of severe malaria had lower baseline RANTES production than children with a history of mild malaria, suggesting inherent differences in RANTES production. In vitro experiments in PBMCs from healthy malaria-naïve donors showed that Hz and sHz promote a similar pattern of β-chemokine protein secretion and transcript expression. FACS analysis showed that Hz and sHz induced similar patterns of cellular surface expression of CD4, CCR5 and CXCR4 on PBMCs. Hz or sHz-exerted differential effects on CD14+ and CD3+ subsets, and this modulatory effect part to transcriptional regulation based on gene expression profiles obtained for respective antigens. Additional studies showed that HIV-1 replicates differently in monoctye-derived macrophages (MDMs) stimulated with either Hz or sHz. sHz enhanced HIV-1 replication while Hz had an inhibitory effect. Results presented here demonstrate a distinct profile of β-chemokine expression in children with severe malaria, which is promoted by P. falciparum derived hemozoin. Further, Hz modulates expression of surface antigens required for HIV-1 entry, defining a possible mechanism for HIV-malaria interaction.

HIV-1 Associated Dementia (HAD), the most severe neurological complication associated with HIV-1 infection, is commonly characterized by inflammation of the brain and neuronal degeneration, known as HIV Encephalitis (HIVE). HIVE develops in 20-30% of patients infected with HIV, which means that 9.5 million people are affected by HIVE throughout the world. While the introduction of highly active antiretroviral therapy (HAART) has decreased the incidence of severe late-stage HAD, the prevalence of its precursor HIVE is actually rising. Several HIV-1 viral proteins have been shown using in vitro models to have a role in the neurotoxic effects causing the neurodegeneration seen during HIVE. HIV-1 Viral Protein R (Vpr), a virion associated gene product which induces apoptosis in non-proliferating cells including neurons, is thought to contribute to the neuropathogenesis associated with HIVE. Previous studies have shown the presence of detectable levels of Vpr in the cerebrospinal fluid of HIV-1 infected patients. Extracellular Vpr released from HIV-1 infected macrophages has also been shown to be capable of transducing into cells not normally infected by HIV-1, causing death of these bystander cells. Additionally, Vpr has been shown in vitro to be able to induce apoptosis in human neurons. Although current research suggests that Vpr plays a significant role in neuropathogenesis, no work has been done yet in vivo to show the presence of Vpr in the brain tissue of HIVE patients. Using a panel of eight HIVE and four HIV seronegative patient brain tissue sections, I performed immunohistochemistry staining for Vpr, p24, and brain cell specific markers. Results indicate that Vpr was present in detectable amounts in both the basal ganglia and frontal cortex of all eight HIVE brain tissue samples tested. Double label immunohistochemistry was performed using antibodies specific for astrocytes macrophages and neurons. I detected the presence of Vpr in the macrophages and neurons, but not in the astrocytes, of HIVE patients. The results of this study strongly support the role of Vpr in the neuropathogenesis seen during HIVE. Further studies based on these findings could lead to the development of effective therapeutic treatments necessary to reduce, and possibly prevent, this public health epidemic.