AIM The objective of this study is to determine factors that may contribute to hospital admissions in a cohort of medically insured South African patients in the era of HAART. METHODS This was a retrospective cohort of all HIV-positive adult and paediatric patients enrolled on a medical aid disease management programme in South Africa over a period of three years. Patient-specific demographic and clinical information were obtained from the medical aid records. Survival analysis was used to analyse time to first admission looking at admissions occurring after enrolment to the programme, during the study period of between 01 January 2006 and 31 December 2008. Only the right censored cases were included in the analyses. Descriptive analyses were conducted on the key prognostic factors. Variables that were significant in the univariate were considered in the multivariate Cox proportional hazards model. RESULTS A total of 8440 patients were included in the analysis. Half of these patients had at least one admission during the observation periods with 43.28% having had 2 or more admissions. The average admission rate was 2 admissions per patient over the 36 month observation period. Young children, adolescents and the very old (> 60 years) were significantly more likely to be admitted than the middle age groups, HR = 1.30 [95%CI 1.21 -1.40] p<0.01, 1.24 [95%CI 1.10 – 1.41] and 1.13 [95% CI 1.10 – 1.27] p<0.01 respectively. Low CD4 cell counts of < 200 cells/ µL were significantly associated with a higher likelihood of hospitalizations with hazard ratios even greater for CD4 cell counts of less than 100 cells/ µL, HR= 1.34 [95%CI 1.29 – 1.39], p<0.01. Cases were more likely to be admitted by a clinical haematologist or gynaecologist than by other specialist categories.HR =1.58 [95%CI 1.29 –1.94] and 1.17[95%CI 1.08 – 1.27] respectively with p<0.01. CONCLUSION Factors that are associated with hospital admissions in this private sector, medically insured population are a younger and older age, low CD4 cell counts and admission by a clinical haematologist and gynaecologist. These results suggest that disease management strategies should be intensified for the younger and older age groups. All HIV-positive patients should be closely monitored for CD4 deterioration so that treatment is initiated timeously. Routine haematological investigations should be recommended for all HIV-positive patients in order to pick up and treat haematological conditions before they result in a hospital admission. Evidence based guidelines, outlining the place of caesarian section deliveries in the HIV population, should be developed for use by gynaecologists specifically in the private sector. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Clinical Epidemiology / unrestricted

Objectives: The aim of the study was to describe the prevalence and nature of auditory and otological manifestations in adults with HIV/AIDS according to clinical examinations and self-reported symptoms. Auditory profiles of HIV individuals were compared to that of a matched control group. Study design: A descriptive, cross-sectional group design was utilized in the first section of the study while a comparative, control matched research design was used to compare the HIV group and matched control group. Methods: Two hundred HIV positive adult patients attending the Infectious Disease Clinic of the 1 Military Hospital were included through convenience sampling. Participants were interviewed, medical files were reviewed and clinical examinations, including otoscopy, tympanometry, pure tone audiometry and distortion product oto-acoustic emissions, were completed. A control group of 184 individuals were compiled, matched to 184 of the HIV infected participants according to age, gender, ethnicity as well as working environment. Audiological thresholds at 0.5kHz – 4kHz were compared among these groups. Results: A prevalence of self-reported tinnitus (26%), vertigo (25%) hearing loss (27.5%), otalgia (19%) and pruritis (38%) was recorded. The onset of hearing loss was reported to be mostly (82%) of a slow progressive nature. Abnormalities in tympanometry, otoscopy and oto acoustic emissions were found in respectively 41%, 55% and 44% of participants. Hearing loss greater than 25 dB (PTA) was recorded in 14% of participants compared to 39% for hearing loss greater than 15 dB (PTA). Although not statistically significant (p<.05), self reported vertigo, self reported hearing loss, OAE abnormalities, hearing loss (PTA>15dB and PTA>25dB) and occurrence of mild hearing loss occurred throughout the CDC categories which were used as a measure of disease progression. A statistically significant increase (p<.05) in sensorineural hearing loss was seen with disease progression. In the comparative section, statistically significant (p<.05) worse thresholds were found in the HIV group as opposed to the control group at all frequencies (0.5 kHz – 4 kHz). Conclusions: Auditory and otological symptoms occurred frequently in this sample, while an increase in some symptoms as well as hearing loss was seen throughout disease progression. Sensorineural hearing loss increased significantly through disease progression. Hearing loss occurred more frequently in HIV individuals as opposed to individuals in the control group, while hearing loss occur more frequently in the more advanced stages of HIV infection. / Dissertation (MCommunication Pathology)--University of Pretoria, 2011. / Speech-Language Pathology and Audiology / Unrestricted

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-κB, facilitated nuclear translocation of NF-κB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-κB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-κB translocation, which are the characteristics of pulmonary endothelial cell activation.

Since the first cases were reported over thirty years ago, great strides have been made to control disease progression in people living with HIV/AIDS. However, current estimates report that there are about 34 million individuals infected with HIV worldwide. Critical in the ongoing fight against this pandemic is the continuing development of highly active anti-retroviral therapies, ideally those with novel mechanisms of action. Currently, there are no medications approved for use that exploit the HIV-1 MA protein, despite its central role in multiple stages of the virus life cycle. This thesis sought to examine whether a highly conserved glutamate residue at position 99 in the understudied C-terminal helix of MA is required for HIV-1 replication. I characterized a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. In doing so, I found that substitution of this glutamate with either a valine (E99V) or lysine (E99K) residue disrupted Env incorporation into nascent HIV particles, and abrogated their ability to fuse with target-cell membranes. In determining that the strain of HIV could affect the magnitude of E99V-associated defects, I identified a compensatory substitution at MA residue 84 that rescued both E99V- and E99K-associated impairments. I further characterized the MA E99V and E99K mutations by truncating HIV Env and pseudotyping with heterologous envelope proteins in an attempt to overcome the Env incorporation defect. Unexpectedly, I found that facilitating fusion at the plasma membrane was not sufficient to reverse the severe impairments in virus infectivity. Using quantitative PCR, I determined that an early post-entry step is disrupted in these particles that contain the E99V or E99K MA substitutions. However, allowing entry of mutant virus particles into cells through an endosomal route conferred a partial rescue in infectivity. As the characterization of this post-entry defect was limited by established virological methods, I designed a novel technique to analyze post-fusion events in retroviral infection. Thus, I present preliminary data regarding the development of a novel PCR-based assay that monitors trafficking of the viral reverse transcription complex (RTC) in an infected cell. The data presented in this thesis indicate that a single residue in MA, E99, has a previously unsuspected and key role in multiple facets of HIV-1 MA function. The pleiotropic defects that arise from specific substitutions of this amino acid implicate a hydrophobic pocket in MA in Env incorporation and an early post-entry function of the protein. These findings suggest that this understudied region of MA could be an important target in the development of a novel antiretroviral therapy.

Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens. Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe. In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease. Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution. This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/ Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. The functions of Vpx/ Vpr are not well understood. This study presents evidence that the Vpx proteins of HIV-2/ SIVSMpromote HIV-1 infection by antagonizing an antiviral restriction in myeloid cells. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in transovercame the restriction to HIV-1 and SIV infection. Similarly, the cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx rendered macrophages permissive to MLV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. This study further demonstrates that this restriction prevents transduction of quiescent monocytes by HIV-1. Although terminally differentiated macrophages are partially permissive to HIV-1, quiescent monocytes, which are macrophage precursors, are highly refractory to lentiviral infection. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection, suggesting the resistance of quiescent monocytes to HIV-1 transduction is governed by a restriction factor. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Introduction of Vpx into monocytes by pre-infection also rendered quiescent monocytes permissive to HIV-1 infection. Infection of monocytes by HIV-1 either with or without Vpx did not have an effect on temporal expression of CD71. In addition, Vpx increased permissivity of CD71– and CD71+cells to HIV-1 infection with no apparent bias. These results confirm that Vpx directly renders undifferentiated monocytes permissive to HIV-1 transduction without inducing their differentiation. The introduction of Vpx did not significantly alter APOBEC3G complex distribution, suggesting a restriction other than APOBEC3G was responsible for the resistance of monocytes to HIV-1. Collectively our results indicate that macrophages and monocytes harbor a potent antiviral restriction that is counteracted by the Vpx protein. The relative ability of primate lentiviruses and gammaretroviruses to transduce non-dividing myeloid-cells is dependent upon their ability to neutralize this restriction.

Philosophiae Doctor - PhD / In the decade since first identified, the Acquired Immunodeficiency syndrome (AIDS) has become a serious global disease. The nature of the Human Immunodeficiency Virus (HIV) that causes AIDS, whereby a carrier may be asymptomatic yet remain infectious, has enabled its dramatic spread. The number of AIDS cases is increasing exponentially, averaging a doubling time of between 8-15 months in different countries. Of the millions of HIV carriers, it is now estimated that all will eventually go on to develop full-blown AIDS and probably die within 15 years. Unlike other infectiqus diseases, there is currently no known vaccine or cure. Further, HIV is now virtually completely dependent on volitional sexual behaviours for transmission to occur. It is therefore an entirely preventable disease. However, since the behaviours that contribute to HIV-transmission are influenced by biological, psychological, and social factors, their alteration in line with safer sexual practices has been shown to be considerably complex and difficult. Intervention strategies that have relied on imparting knowledge about the disease have achieved limited success in influencing behaviour change. Unsafe sexual practices, and the risk of HIV-infection, often continue even when knowledge regarding prevention is adequate. It has therefore become apparent that other variables intrude which may mediate between knowledge acquisition, attitude formation, and consequent sexual behaviours. There appear to be no models which adequately explain the complexities in this area, and which enable adequate intervention strategies to be developed. The present study was undertaken to redress this problem, and to explore those variables that mediate in the area. Various psychological and social factors appear to be implicated in influencing sexual attitudes and behaviours. In order to adequately test the impact of psychosocial variables that were found to have significant associations in an exploratory study, a measuring instrument was developed. The AIDS Psychosocial Scale was statistically validated using content, frequency, factor, and reliability analyses and included psychological factors of self concept, defenses of denial, repression, and rationalisation, perceived empowerment in the form of locus of control and selfefficacy, and the social factor of peer pressure susceptibility. The impact of these psychosocial variables on indices of knowledge, condom attitude, and sexual practices, and on other epidemiological variables was tested using a sample of students at the University of the Western Cape (n=308). Results indicated a number of correlational and causal links between variables, confirming the mediational role psychosocial factors have in influencing knowledge acquisition, attitude formation, and behaviour outcome. A profile of lower self concept, higher defenses, lower self-efficacy, more external locus of control, and higher peer pressure susceptibility emerged which was associated with poorer knowledge, more negative attitudes, and higher unsafe sex. Based on this study, a model of psychosocial mediation is developed and its implications for intervention strategies discussed.

Resilient Educators Support Programme (REds) for HIV and AIDS affected educators was initiated by the University of North West in 2006 following a research project in 2005 that highlighted the need for a support programme that addresses the challenges of educators affected by HIV and AIDS, as existing support structures were found to be inadequate. REds is implemented in phases, and with each phase it is modified to meet the needs of a broader audience of educators. The first phase was implemented in the Gauteng Province in 2006. Recommendations made from this phase were used to modify it for phase two, that is, implementation in Mpumalanga Province and two additional provinces. The goal of the research project was to evaluate the effectiveness of the Resilient Educators Support Programme (REds) in empowering and supporting HIV and AIDS affected educators in Mpumalanga. Programme evaluation research was adopted, using the dominant-less dominant model of combining both quantitative and qualitative research approaches, with the quantitative approach being the dominant approach. The research design for the quantitative research approach was the quasi-experimental one group pre-test post-test design whilst the collective case study design was used for the qualitative research approach. Quantitative data was collected through a group administered standardized questionnaire, the Professional Quality of Life Scale (ProQOL) and for qualitative data, a non-standardized questionnaire with drawings, semi-structured interviews, observation and field notes were used. Data was collected before and after the respondents were exposed to REds. The respondents, ten educators from Sozama High School in Middelburg, were recruited using non-probability volunteer sampling method. Additional qualitative data was collected from the five members of school management team using semi-structured interviews, after the respondents were exposed to REds. Although the quantitative empirical research findings showed no significant differences between the pre- and post-test data, it seems from the qualitative approach as if the respondents found the programme addressing their support needs as HIV and AIDS affected educators and they felt empowered with knowledge and skills that they lacked and thus making them resilient. Conclusions drawn from the qualitative research findings indicated that REds Support Programme was effective in meeting the support needs of HIV and AIDS affected educators in Mpumalanga. From a quantitative perspective, it is recommended that the reason/s for the non-significant test results from the standardized questionnaire be investigated. Copyright / Dissertation (MSW)--University of Pretoria, 2008. / Social Work and Criminology / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18186号 / 医科博第51号 / 新制||医科||4(附属図書館) / 31044 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 松岡 雅雄, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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