Includes abstract. / Includes bibliographical references. / Leishmania major is a protozoan parasite and infection in the human host causes severe cutaneous Leishmaniasis. The study aims to determine how signaling via the IL-4Rα on CD4+T cells causes susceptibility to L. major. We compared gene expression patterns early during infection in CD4+ T cells in the absence or presence of IL-4Rα signaling. Non-healer BALB/c mice with a deletion of the IL-4Rα on all cells (IL-4Rα-/-) or CD4+ T cells only (iLCKcreIL-4Rαlox/-) and their controls (wild-type (WT) C57BL/6, WT BALB/c and littermate IL-4Rαlox/-) were subcutaneously infected with L. major. As expected, the C57BL/6 “healer” mice produced a predominant TH1 response, whereas the iLCKcreIL-4Rα-/lox mice and susceptible BALB/c mice produced a TH2 response.

Background The majority of invasive fungal disease in South African hospitals is HIV-related or associated with another secondary immunodeficiency e.g. haematopoietic stem cell transplant. After excluding secondary immunodeficiency, a detailed immune work-up can lead to a diagnosis of primary immunodeficiency. Objective To detail an appropriate step-wise immunological work-up for a series of patients with invasive fungal diseases and possible underlying primary immune deficiency. Methods Detailed review of all culture- or histologically confirmed cases of invasive fungal disease (IFD) at Groote Schuur Hospital between 2007-2017. Step-wise immunological work-up of IFD patients with no secondary immunodeficiency. Clinical characteristics and step-wise immunological profiles were evaluated. Results Sixty-seven adults with IFD were identified; 72% (48/67) were HIV-related. 8/19 HIVnegative cases were either deceased (4) or lost-to-follow-up (4). Work-up of the remaining 11 cases found five with non-HIV secondary immunodeficiencies (Lupus, liver transplant, endstage renal failure and haematological malignancy). A primary immunodeficiency was suspected in six cases, but 1 case of cutaneous sporotrichosis was excluded; with five cases (4 with disseminated Cryptococcus neoformans and 1 with cerebral aspergillosis) undergoing detailed immune work-up. A case of idiopathic CD4 lymphopenia was diagnosed; but all other cases had no evidence of neutrophil or a cell-mediated immune defect; including investigations of naïve and memory T-cell subsets and cytokine responses to PHA and candida. All cases were noted to have low baseline vaccine responses and Vitamin D deficiency. Conclusion Invasive fungal disease is predominantly associated with HIV and secondary immunodeficiency in South Africa. Known primary immunodeficiencies can be identified with basic immune work-up; but no obvious functional immune defect is evident in the majority of these cases.

Includes bibliographical references.

Bibliography: leaves 174-198.

Includes bibliographical references (leaves 114-142). / Lymphocyte-specific reconstitution of IL-4Ra was recently established by intercrossing lymphocyte-specific human IL-R4a transgenic mice with mIL-4Ra-deficient mice. Human IL-4Ra may bind to mouse yc resulting in a chimeric receptor specific for human IL-4 but not mouse IL-4. This provides us with and inducible IL-4 system. The aim of this study was to investigate in vitro and in vivo characteristics of our novel hlL-4Ra Tg/mIL-4Ra+/- mouse model.

Includes bibliographical references. / African trypanosomiasis encompasses diseases caused by pathogenic trypanosomes, infecting both humans and animals alike. To determine the immunological role of IL=12 family members in Trypanosoma brucei brucei, Trypanosoma evansi and Trypanosoma congolense infections, IL-12p35¯/¯, IL-12p40¯/¯ and IL-12p35¯/¯/p40¯/¯ mice were used. While the two latter mouse strains lack all IL-12 homologues, IL-12p35¯/¯ mice still produce IL-12p80 homodimers and IL-23. In infection with T.b. brucei and T.evansi; IL-12p35¯/¯, IL-12p40¯/¯ or IL-12p35¯/¯/p40¯/¯ mice were susceptible to both these pathogens, demonstrated by increased mortality compared to wild type C57BL/6 mice. The different IL-12 deficient mouse strains showed similar mortality kinetics, suggesting that IL-12p70 but not the IL-12p80 homodimer or IL-23 plays a crucial role in survival. Similarly, parasitemia control was reduced in the absence of IL-12p70. While plasma levels of IgM and IgG2c were similar between IL-12 deficient mice and wild type mice, IF-γ production. As IFN-γR¯/¯ mice were also highly susceptible to both T.b. brucei and T. evansi, IL-12p70-dependent IFN-γ production seems to be important mechanism involved in resistance against both these pathogens.

The non-Hodgkin's lymphomas (NHL) are a heterogeneous group of malignancies characterised by the uncontrolled proliferation of lymphoid cells. The Working Formulation divides these neoplasms into low, intermediate and high-grade categories according to their natural history. Low grade NHL (LG-NHL) is clinically indolent whereas high grade NHL is more aggressive. Most LG-NHL patients respond well to chemotherapy, but are rarely cured, making death from LG-NHL virtually inevitable. In this study on LG-NHL patients, all patients received combination chemotherapy for 6 weeks, which consisted of cyclophosphamide, oncovin (vincristine) and prednisone (COP). After this treatment patients received either oral cyclophosphamide or alpha-interferon (α-IFN) as maintenance treatment for a period of two years. The ability to predict patients' treatment response at diagnosis would be extremely helpful for both the patient and the clinician. This would enable appropriate therapy to be instituted at diagnosis, whether it be cyclophosphamide or α-IFN, which might increase the subsequent survival of these patients, as this would prevent patients from receiving treatment to which they would not respond. Furthermore, patients who were unlikely to respond to either of these two treatments could be targeted for alternative treatment or form part of a clinical research trial. LG-NHL patients were assessed at diagnosis for a number of immune parameters. These included: - natural killer activity (NKA), α-IFN enhanced NKA, mitogen and antigen proliferation, lymphokine activated kill against both KS62 and Daudi targets, phenotypic analysis of circulating lymphocytes, assessment of IL-2 levels after mitogen stimulation of lymphocytes, as well as the determination of plasma IL-2 levels. All these above-mentioned parameters were serially monitored over time in an attempt to predict early relapse. A statistically significant reduction in percentage of circulating CD3+ and CD8+ cells and an increase in percentage NK cells was found in patients at diagnosis as compared to normal controls. A reduction in percentage of circulating NK cells over time appeared to be a good prognostic indicator and an increase in percentage NK cells a poor prognostic indicator in this group of patients, although this was not statistically significant. When the in vitro α-IFN enhanced NKA was indicated as a percentage of NKA, a negative correlation appeared to exist between this in vitro response to α-IFN and the in vivo response, although this was not statistically significant (i.e. those patients showing the least α-IFN enhanced NKA were those that responded clinically and those with the highest α-IFN augmented NKA either relapsed or transformed to a higher grade of NHL). By incorporating the percentages of circulating lymphocytes present in the peripheral blood, into the multivariate discriminant analysis, it was possible to derive formulae to enable the prediction of response to either α-IFN or cyclophosphamide. This was a particularly exciting and apparently novel finding. The work presented here has therefore established both a possible negative indicator of α-IFN response (a high in vitro α-IFN response) and a positive indicator (the formula generated in the multivariate discriminant analysis using the flow cytometric phenotypic analysis). By making using of both of these factors, it would increase the chances of patients being selected for appropriate forms of treatment and minimise the chances of patients suffering a relapse. The Kaplan-Meier curve indicated that cyclophosphamide treatment was more beneficial than α-IFN therapy in this group of patients, although this did not attain statistical significance. Verification of all of these above-mentioned findings would need further confirmation in a larger study.

The aim of this study was to investigate the role of PRRs, namely TLR4 and LBP in vivo in the development of an immune response to a Mycobacterium tuberculosis infection. Mice with null mutations of the endotoxin receptors LBP and TLR4 were compared with wild-type mice (PRRs intact) in the context of an aerosol Mtb infection. The following questions were posed: Are the PRR-disrupted animals more susceptible to M.tuberculosis infection? What role do these PRRs play in inflammatory processes and in the development of granuloma? What role do the PRRs play in driving cytokine and chemokine response? Specifically, the following mice were infected aerogenically with Mtb H37Rv to further investigate their roles in vivo in mounting an effective immune response: C3H/HeN and C3H/ HeJ (TLR4 mutant) LBP+/+ and LBP-/-

This thesis is concerned with a study of the proteases released by the cercariae of Schistosoma mansoni while penetrating mammalian skin. The proteases present in secretions collected from the preacetabular glands of cercariae were shown to be active against ¹²⁵I-labelled fibrin but not against undenatured ³H-collagen. A sensitive solid phase radioenzyme assay, with ¹²⁵I-fibrin as the substrate, was used to show that the cercarial protease could be totally inhibited by serine protease inhibitors such as diisopropylfluorophosphate or phenylmethylsulfonyl fluoride, but not by the sulfhydryl reagents iodoacetamide or p-chloromercuribenzoate. Typical trypsin inhibitors such as soy bean trypsin inhibitor, trasylol or benzamidine inhibited the enzyme to a lesser degree. The active-site labels, TLCK, TPCK and AcAAAACK of trypsin, chymotrypsin and elastase respectively had no effect. Calcium and magnesium stimulated protease activity at concentrations below 0,5 mM, but inhibited at higher concentrations, whereas EDTA had no effect. The pH optimum of the protease lay between pH 9,0 and 9,5. From these studies, I have concluded that the major cercarial penetration protease is an alkaline serine protease with trypsin-like specificity, but not acting via the same mechanism. A technique was developed for examining cercarial proteases in polyacrylamide gels containing SDS and copolymerized gelatin substrate. Bands of proteolytic activity could be detected by negative staining. This method was used to show that cercarial secretions contained one major protease with a molecular weight of 35 000 and that crude enzyme preparations are readily contaminated with bacterial proteases. Partial purification of the major cercarial protease was achieved by cation exchange chromatography.

Cytokines released by innate immune cells initiate pathways that drive the differentiation of CD4+ T cells into subsets with defined patterns of cytokine secretion. Type I interferons (IFNs) are cytokines that decrease a virus’s capability to replicate in cells. Prolonged exposure to them, as in chronic viral infections, lead to immune suppression. Little is known about the effects of type I IFNs on cytokine expression by CD4+ T cells. My studies, as described in this thesis, show that the type I IFN, IFNα, can impact cytokine secretion by CD4+ T cells, and timing of IFNα exposure can impact the effect. When isolated mouse CD4+ T cells were pre-treated with IFNα prior to activation, expression of the gene encoding Interleukin-2 (IL-2) significantly increased and expression of the gene encoding IFNγ significantly decreased. When T cell Receptor (TCR) and type I IFN pathways were activated simultaneously, expression of the gene encoding Granulocyte-macrophage colony-stimulating factor (GM-CSF) was decreased. Since timing of IFNα exposure changed the effect, these results demonstrate the sensitivity of the interconnections between the IFNα pathway and pro-inflammatory cytokine expression pathway. These results indicate altered cytokine expression due to prolonged exposure of CD4+ T cells to IFNα as a possible explanation for immune suppression in chronic viral infection. Ikaros is a transcription factor that regulates expression of some pro-inflammatory cytokines. CD4+ T cells that lacked Ikaros expression responded differently to IFNα exposure indicating a possible role for Ikaros in modulating the effect of IFNα on the expression of pro-inflammatory cytokines.