This thesis embodies much of my work done over the past 25 years. The impetus for these studies was the need to provide the best tissue typing available for organ transplantation and to overcome the problems of defining HLA antigens in different ethnic groups. These goals were achieved by extensive international collaboration and participation in the International Histocompatibility Workshops. The discovery that the HLA antigens are associated with many diseases led to an epidemic of investigations in which over 500 diseases have been studied. In retrospect, it is not surprising that auto-immune diseases such as diabetes and rheumatoid arthritis showed such marked associations with HLA antigens. The studies in Part II of this thesis were aimed at finding out if the HLA associations reported in Caucasian populations were also present in the Black and Indian populations. These research interests led to my being invited by the National Science Council of the Republic of China in Taiwan to be a Visiting Professor at the National Taiwan University in Taipei for the 1989 academic year. I investigated the association between HLA and naso-pharyngeal carcinoma in Chinese during that year. I wish to express my appreciation to Dr Peter Brain who inspired the early investigations and continued to encourage and support my research. I am grateful to all my co-authors and the many colleagues, clinicians and laboratory staff who have contributed to the various research programmes. Studies of the relationship of the HLA system to cancer, diabetes, arthritis and other diseases have been supported in part by grants from the National Cancer Association and the Medical Research Council of South Africa. / Thesis (D.Sc.)-University of Natal, Durban, 1992.

A study of the causes of liver enlargement amongst black patients at King Edward VIII Hospital, Durban, South Africa has revealed that congestive cardiac failure (36.7%), amoebic liver abscess (7.1%), hepatocellular carcinoma (5.8%) and cirrhosis (5.4%) are the most common causes in this population. Liver biopsy was needed to determine the cause in 28.7% of patients studied. The diagnostic yield of percutaneous liver biopsy was increased by obtaining 2 or 3 consecutive specimens for histological examination by redirecting the biopsy needle through a single entry site. This benefit was achieved without an increase in morbidity or mortality. Fatalities and complications associated with liver biopsy were more frequent at this hospital than in hospitals in Europe, The United Kingdom and North America. The complication rates after percutaneous or peritoneoscopic biopsy were 2.0% and 2.3% respectively. A total of 6 deaths was recorded. The morbidity and mortality rates were not increased when more than one specimen was taken during percutaneous biopsy. In the majority of patients in whom biopsy was carried out, after-care was either non-existent or inadequate. The "Tru-Cut" needle was used for all percutaneous liver biopsies at King Edward VIII Hospital. Two techniques, including the method recommended by the manufacturer, have been found to be incorrect; the needle must be used correctly if an adequate biopsy specimen is to be obtained for histological examination and if serious complications are to be avoided. Hepatic tuberculosis was diagnosed in 9% of patients with unexplained hepatomegaly who were subjected to liver biopsy. This disease did not yield any consistent clinical findings. In addition, liver function tests were of little diagnostic value and results of hepatic imaging techniques were often normal. Accordingly, a high index of suspicion is needed and liver biopsy is essential in patients with unexplained hepatomegaly or hepatospienomegaly, or pyrexia of unknown origin since biopsy provides the only means of diagnosing hepatic tuberculosis. The accuracy of both ultrasonography and scintigraphy in distinguishing between normal and diseased livers was low (68% and 74% respectively). These techniques performed better at detecting focal than diffuse liver disease; the sensitivity of ultrasonography and scintigraphy in focal and diffuse disease were 88% and 92%, and 27% and 54% respectively. The specificity of both procedures was high for both types of liver disease (range 91-96%). Overlap between the ultrasonographic features of amoebic liver abscess, hepatocellular carcinoma and metastatic carcinoma resulted in a correct final diagnosis being made in only 81% of patients with amoebic liver abscess, 29% with hepatocellular carcinoma and 43% of patients with metastatic carcinoma who had an ultrasound scan. Neither technique was capable of determining the cause of diffuse liver disease. Therefore, when diffuse parenchymal liver disease is suspected, liver biopsy is needed to determine the presence and nature of the disease. In addition, liver biopsy or aspiration is usually required to determine the cause of focal disease in selected patients in whom space-occupying lesions are detected on hepatic imaging studies. / Thesis (M.D.)-University of Natal, Durban, 1990.

No abstract available. / Thesis (Ph.D.)-University of Natal, Durban, 1973.

In South Africa, co-infection with HIV and intestinal parasites is a major challenge in disadvantaged communities who live in densely populated under-serviced urban informal settlements. This pilot cross sectional study evaluates the immunological effects of co-infection with Ascaris lumbricoides and Trichuris trichura on the immune response to HIV. The work was a substudy of a prospective double blind, placebo-controlled investigation to test whether regular deworming changes the immune profile of HIV positive individuals with concurrent helminth infection. The substudy has a cross sectional design and presents pilot data that defines immune profiles of HIV-1 positive individuals with and without gastrointestinal helminth (Ascaris lumbricoides and Trichuris trichura) infection. The hypothesis was that concurrent helminth infection adversely affects immune responses against HIV. It was conducted in an area of high helminth endemnicity and limited infrastructural resources. Individuals with known HIV infection were recruited from an HIV Support Group and HIV negative individuals residing in the same area (for demographic matching) were used for comparison. The substudy was to provide pilot data for future larger scale and possible interventional studies. The current work is limited by the cross sectional design, moderate sample size and practical challenges. The profile of lymphocyte phenotypes, viral loads, eosinophils, activation markers, expression of the nuclear proliferation antigen-Ki67 and activation regulator antigen CTLA-4 were analysed using flow cytometry in HIV positive and negative subgroups with or without helminth infection. The type-1, type-2 and inflammatory cytokines were analysed using multiplex cytokine array technology. These were correlated with immune responses to HIV. Non parametric statistics were used to describe differences in the variables between the subgroups. A major finding of the study was the result of the supplementary use of the serological marker, Ascaris lumbricoides-specific IgE in addition to the presence (or absence) of helminth eggs in stools to classify intestinal helminth infection status. Two significant outcomes of this measure were the enhancement of diagnosis of current or recent helminth infection and, more importantly, the distinction of different phenotypes of individuals who displayed different immunological responses to co-infection with HIV and helminths. The different helminth infection phenotypes are defined by stool egg positivity (egg⁺) or negativity (egg⁻) with either high or low Ascaris-specific IgE (IgEhi or IgElo) respectively. The four subgroups, egg⁺IgEhi, egg⁺IgElo, egg⁻IgEhi and egg⁻IgElo showed different interactions with regards to immune response to HIV. It should be noted that no Trichuris specific IgE tests are commercially available but that there is significant antigenic cross-reactivity with Ascaris antigen. The presence of helminth stool eggs and high Ascaris IgE (egg⁺IgEhi) was associated with the following characteristics: reduction in numbers of all lymphocyte populations, frequent eosinophilia, highly activated immune profiles, antigen specific proliferative hyporesponsiveness, impaired type 1 cytokine responses in unstimulated and antigen stimulated cells and increased TNFα levels. In HIV infected individuals, the egg⁺IgEhi helminth infection status was associated with lower but not significant CD4⁺ counts and higher viral loads. A strong negative correlation was observed between viral loads, CD4⁺ and CD8⁺ cells in this subgroup. Subgroups with high IgE (egg⁺IgEhi and egg⁻IgEhi) had elevated Th2 markers with lower CD4⁺ counts and higher viral loads in the HIV⁺ group. The inverse correlation between viral load and CD4⁺ counts found in all the HIV⁺ participants was strongest in these two subgroups. Individuals with parasite eggs in stool and low Ascaris IgE (egg⁺/IgElo) presented a modified Th2 profile. This subgroup had high absolute numbers of all lymphocyte subsets in both HIV⁻ and HIV⁺ groups with higher CD4⁺ counts in the HIV⁻ and lower viral load in the HIV⁺ groups as well as higher interferon gamma, lower IL-4 and higher IL-10. In conclusion, the results suggest that helminth infections may be associated with deleterious effects on the immune responses to HIV in certain groups of susceptible individuals. The underlying reasons for the different stool egg/Ascaris IgE combinations in settings with high exposure to helminthes is currently not clear but genetic predisposition and environmental factors could play a role. Future studies of helminth- HIV co-infection have to ensure adequate definition of helminth infection status by the use of both stool examination and measurement of helminth-specific IgE as the infection phenotype is associated with differential effects on HIV associated immune responses. This may also apply to co-infection with other pathogens, including tuberculosis. The long-term effect of helminth co-infection in HIV positive people was not assessed in this study but requires further studies. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2009.

The manifestation of HIV-1 infection is different in children and adults. Most of the children who acquire HIV perinatally progress to disease within the first two years of life, while adults can remain asymptomatic for up to ten years. However, a small minority group of children can control the virus for years in the absence of antiretroviral therapy. We characterized CD8+ T cell responses critical for the containment of HIV infection in a cohort of infants HIV infected from birth using IFN- γ ELISPOT, multicolour flow cytometry and viral sequencing of the Gag protein. We investigated whether the age at the time of infection, specificity and functionality of the generated responses, genetic make up and the maternal immune responses to HIV, influenced disease progression in the child. We found that the majority of in-utero infected infants mounted CD8+ T cell responses from the first days of life. In contrast to chronically infected children or adults, the specificity of the initial response in acutely infected infants was directed towards Env and Rev proteins and CD4+ T cell responses were minimal during the first 6 months of life. Slow progression to disease was associated with possession of one of the protective HLA-B alleles by either the mother or the child (P=0.007) and targeting of Gag epitopes presented by the protective HLA-B alleles. Mothers who expressed protective alleles but whose children did not possess these alleles, transmitted less fit viruses that benefited their children. Furthermore, slow progressor children had more polyfunctional CD8+ T cell responses in early infection when compared to rapid progressors (P=0.05). The ability of infants to induce CD8+ T cell responses early in life is encouraging for vaccine interventions. The differences in the specificity of the initial responses between adults and children, insufficient priming of these responses as a result of minimal CD4+ T cell help during infancy and possession of non-protective HLA alleles shared between mother and child, may explain the rapid disease progression generally noted in most infants. However, slow progression to disease in the minority group of children may be attributed to functional capacity of the CD8+ T cells generated by the child, mediation by protective HLA alleles, acquisition of low fitness viruses from the mother or de novo attenuation of the virus by the child’s own immune responses. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

The test for antineutrophil cytoplasmic antibodies (ANCA) was introduced into the author's laboratory in 1987. An improved indirect immunofluorescent method was developed, using a system which allows 16 instead of one serum sample to be screened on each microscope slide. The known disease associations of ANCA that have been explored include systemic vasculitis, renal limited vasculitis, chronic inflammatory bowel disease and HIV disease. In general the findings are similar to those which are emerging from other centres and confirm the value not only of the positivity but also the relevance of the intracellular disposition of the neutrophil cytoplasmic fluorescence in diagnosis. In this study 85% of patients with Wegener's granulomatosis were found to have C-ANCA. C, P and X-ANCA staining patterns were found in 57% of patients with ulcerative colitis. Forty one per cent of patients with symptomatic HIV have ANCA. Certain histological features such as neutrophil and vascular damage in invasive amoebiasis, and the established lytic effect of amoebae on neutrophils prompted the investigation of the possibility that ANCA may be generated in this disease. Seventy eight amoebiasis sera were screened and 98,70/0 gave a positive ANCA test with a pattern of fluorescence resembling that found in Wegener's granulomatosis. An ELISA test for specificity confirmed that, as in Wegener's granulomatosis, this amoebiasis-associated ANCA had proteinase 3 specificity. Of practical clinical importance is the fact that both HIV and amoebiasis are associated with a high level of ANCA positivity. These findings will need to be considered when ANCA tests are used in clinical decision making in an area where HIV disease and amoebiasis are endemic. A large number of normal volunteer blood donors have been tested and the false positivity rate of 0,5% confirms the specificity of the test. / Thesis (M.Med.)-University of Natal, Durban, 1993.

HIV infection has risen to pandemic proportions. Interleukin-10 (IL-10), a potent antiinflammatory cytokine has been shown to enhance the establishment and persistence of chronic viral infections through inactivation of effector antiviral immune responses and it may also directly influence HIV-1 replication in cells of diverse lineages. IL-10 promoter polymorphisms have been shown to affect HIV-1 susceptibility and pathogenesis. However, the underlying mechanisms are poorly understood. We investigated the relationship between IL-10 promoter variants, plasma IL-10 levels, and markers of disease outcome in chronically HIV-1-infected individuals. To investigate the mechanistic role of IL-10 and its genetic variants on HIV pathogenesis, we studied markers of activation on B cells, CD4+ and CD8+ T cells, and assessed effects on CD4+ T cell proliferation with and without blockade of the IL- 10 pathway. We used Taqman genotyping assays to genotype three IL-10 promoter single nucleotide polymorphisms (SNPs) in our study cohort. Baseline plasma IL-10 levels were measured using Luminex technology for 112 individuals. Viral load, CD4+ T cell counts and cytotoxic T lymphocyte (CTL) immune responses were measured at baseline. The rate of CD4+ T cell decrease was calculated in 300 individuals with a median follow-up of 25 months. CD38, CD95, Ki67, IgG and PD-1, markers of activation or exhaustion were measured on B cells, and CD38, CD95, Ki67, HLA-DR and PD-1 were measured on CD4+ and CD8+ T cells in a subset of 63 individuals. CD4+ T cell proliferation was measured using Carboxyfluorescein succinimidyl ester (CFSE) assays, following IL-10 receptor blockade in a subset of 31 individuals. The IL-10 -1082G, -592A and -3575 variants were observed at frequencies of 0.3, 0.34 and 0.23 respectively, in our study cohort. Plasma IL-10 levels were significantly higher in the - 1082GG group than in the combined AA/AG group (p=0.0006). There was a significant association between the 592AA genotype and a greater breadth of CTL responses compared to the CC and CA (p= 0.002 and 0.004 respectively). The -592AA genotype associated significantly with an attenuated loss of CD4 cells (p= 0.0496), with -592AA having the least change in CD4 cells per year. The median expression of HLA-DR, a marker of T cell activation was significantly higher in the-1082AA group for CD8 cells (p= 0.047), and the - 592AA group for CD4 T cells (p= 0.01). The median expression of IgG on the surface of B cells was significantly higher in the -1082GG genotype and the -592CC genotype (p=0.0183 and 0.0659 respectively). Overall, IL-10 variants correlated with IL-10 expression and CD4 decline during chronic HIV-1 infection. IL-10 promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels, which in-turn, may affect the breadth of CTL responses. Furthermore, the increased expression of HLA-DR and PD-1 on CD8+ and CD4+ T cells, indicates that lower IL-10 levels are associated with increased immune activation and immune exhaustion. The increased expression of IgG on B cells, suggests that in a setting of lower IL-10, there is possibly a bias towards a Th2 immune response. These data suggest a significant role for IL-10 genetic variants and IL-10 in HIV pathogenesis. Further studies to determine whether and how the IL-10 pathway may be manipulated for therapeutic or vaccine strategies for HIV are warranted. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

HIV-1 specific CD4+ T cell activity in clade C infected subjects has not been studied. CD4+ T cells play a vital role in controlling infectious diseases and there is a need to augment our knowledge of HIV immunology to aid vaccine design. We therefore embarked on a study to characterize HIV-1 specific CD4+ T cell activity in both adults and infants; assess the relationship between CD4+ and CD8+ immune responses; and the relationship between CD4+ T cell activity and markers of disease progression (viral loads and CD4 counts). Our study revealed that the magnitude of CD8+ T cell responses correlated significantly with CD4+ T cell responses, but that the percentage of CD8+ T cells directed against HIV-1 was always greater than that of CD4+ T cells. Gag was the frequently targeted HIV-1 protein by CD4+ T cells and had the highest density of epitopes targeted by CD4+ T cells. Patients with either a dominant CD4 or CD8 T cell response against Gag had significantly lower viral loads than patients in whom non-Gag proteins were the main target (p< 0.0001 for CD4 activity and p= 0.007 for CD8 responses). Single IFN- producing CD4+ T cells were present in significantly higher numbers than cells producing both IFN- and IL-2 simultaneously (p=0.009). Gag also dominated the CD4+ T cell response in acutely infected infants with IFN- production detected more frequently than IL-2 or TNF- . Longitudinal analysis of infants receiving early ARV treatment and then ceasing after 12 months revealed that early treatment conferred no protection against increasing viremia and disease progression. CD4+ T cell responses were detected sporadically in untreated infants indicating a dysfunctional immune response in the face of constant exposure to high levels of viremia. Taken together, the data reveal that a vaccine inducing Gag specific CD4+ T cell responses has the potential to confer some degree of protection, but other immunological parameters need to be investigated especially in infants. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

This study was conducted to investigate the role of natural killer cell surface receptors, KIRs and their cognate HLA ligands in preventing HIV-1 acquisition and disease progression in HIV-1 exposed infants. Using DBS stored for 8 years from 21 pregnant South African women we evaluated 3 methods of gDNA extraction with and without whole genome amplification (WGA) to characterize immune-related genes: IL-10, KIR and HLA class I. However, IL-10 SNP typing was only for testing the quality of gDNA. QIAamp DNA mini kit yielded the highest gDNA quality (p<0.05; Wilcoxon Signed Rank Test) with sufficient yield for subsequent analyses. In contrast, WGA was not reliable for SSP-PCR analysis of KIR2DL1, KIR2DS1, KIR2DL5, and KIR2DL3 or high resolution HLA genotyping using a sequence-based approach. A cohort of 370 infants; 124 HIV-1 perinatally infected, 120 exposed uninfected and 126 unexposed healthy infants was used for KIR and HLA genotyping. After adjustment for viral load and multiple comparisons, the frequency of HLA-Cw*04:01 allele was likely to be associated with susceptibility to mother-to-child acquisition of HIV-1 in exposed infected (EI) infants (p=0.05; Logistic Regression analysis). HLA-A*23:01 was likely to be associated with decreased CD4 T lymphocyte count in HIV-1 infected infants (p=0.01; ANOVA), whereas HLA-B*81 tended to be associated with higher CD4 T lymphocyte count (p=0.04, ANOVA). We speculate that HLA-Cw*04:01 interacts with KIR2DL1 and inhibit NK cell responses which predispose the infants to HIV-1 infection. KIR2DS1 and KIR2DL5 were both associated with faster HIV-1 disease progression. Identified protective HLA-class I alleles could be used to present viral epitopes to either NK cells via KIRs or CTLs and enhance immune activation which may promote resistance to HIV-1 infection. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2012.