In this thesis I describe the work that I have done on the isolation and characterization of a plasminogen activator, Mel-PA, that is released by human melanoma cells cultured in vitro. This enzyme was compared to the urinary plasminogen activator, urokinase. The human melanoma cell line, RPMI-7272, (also referred to as the "Bowes" melanoma cell line) released large amounts of Mel-PA into the surrounding medium when cultured under serum-free conditions. A subline of these cells (Bowes II) was developed that was capable of continuous growth in the absence of serum. These cells released only one type of plasminogen activator with a molecular weight of approximately 70 000 daltons. A technique was developed in which plasminogen activators were separated electrophoretically and detected in polyacrylamide gel slabs containing the co-polymerized substrates, plasminogen and gelatin. The technique was compared with the zymographic procedure developed by Granelli-Piperno and Reich (62) using fibrin-plasminogen-agarose underlays. Mel-PA was concentrated and partially purified by affinity chromatography on benzamidine-sepharose. This preparation was used to prepare rabbit antisera to the enzyme. These antibodies inhibited the activity of plasminogen activators released by all melanoma cells but had no effect on urokinase. Antibodies to urokinase had no effect on Mel-PA. A survey of human plasminogen activators and their distribution by immunochemical and electrophoretic techniques showed that tissue extracts and body fluids, with the exception of normal urine, contained mixtures of Mel-PA- and urokinase-type enzymes. Urine of patients with some types of renal disease also contained a Mel-PA type enzyme. A study of the distribution of plasminogen activators in tissues and body fluids obtained from a number of animals showed that all mammals examined had two immunochemically distinct plasminogen activators that corresponded, in their distribution, to the urokinase-like and Mel-PA-like enzymes of man. Antibodies to human Mel-PA cross-reacted with the corresponding enzyme in all mammals tested, whereas antibodies to human urokinase were species specific. The seeds of the South African legume, Erythrina latissima, contain a 20 000 dalton protein that functioned as an inhibitor of Mel-PA, plasmin, and trypsin, but had no effect on urokinase. During its reaction with the enzymes the inhibitor was cleaved by Mel-PA and trypsin but not by urokinase. The susceptible bond was straddled by an intrachain disulphide bridge. The inhibitor bound reversibly to Mel-PA and could therefore be used to develop an affinity reagent for a one-step purification procedure for Mel-PA in melanoma cell harvest fluids. Purified preparations of Mel-PA c0uld be shown to comprise both active enzyme (two chain form) and pro-enzyme (one chain form). The one chain form could be converted to the two-chain form by treatment with plasmin. It could also be shown that fibrinogen and fibrin contained a contaminating protease that was capable of converting pro-Mel-PA to Mel-PA. A comparative study of the kinetic behaviour of Mel-PA and urokinase showed numerous differences between the catalytic activities of these two enzymes. Mel-PA was capable of binding to fibrinogen insolubilized on a plastic surface whereas urokinase did not. The presence of fibrinogen enhanced the plasminogen activating activity of Mel-PA but had no effect on urokinase activity.

Includes bibliographical references. / It is important to emphasize with the advance of biotechnology and increased global exchange of animals and animal products, the risks of introducing adventitious infections. Previous studies of specitic-pathogen-free mouse colonies have identified the presence of infectious agents in 10-35% of research institutions investigated. Prevalence was higher among non-SPF mice with pinworm reported in 70% of institutions housing rodents under these conditions. Pinworm, a gastrointestinal (GI) nematode is commonly found in laboratory animals. The direct transmission of the parasite by contaminated food, water and bedding result in their continual re-exposure to the host, making the control of pinworm in animal holdings quite difficult. Syphacia obvelata, mouse pinworm, has been shown to interfere with research goals in several experimental models. In this study, we show the consequence of a pinworm outbreak in a transgenic barrier facility and define the immune response elicited in BALB/c mice. Infection with S. obvelata induced a transient Th2-type immune response with elevated cytokine production and parasite-specific IgG1. In contrast, HALB/c mice, deficient for IL-13, lL-4/13 or IL-4Ra showed chronic disease with more than 100-fold higher parasite burden, increased IFN-y production, parasite-specific IgG2b and a default Th2 response. Notably, infected lL-4-/- BALB/c mice showed only slight elevated parasite burden compared to controls, suggesting that IL-13 plays the dominant role in the control of S. obvelata. Furthermore, no significant eosinophilia, mastocytosis or goblet cell hyperplasia was induced. In a well-established ovalbumin (Ova) anaphylaxis model, we show that mice infected with S. obvelata induce a more severe anaphylactic reaction, with consistently greater temperature decline than their non-infected counterparts. Analysis of spleen cells further revealed a marked reduction of Ova-specific Th2 cytokines, highlighting the importance of pinworm free experimental mice. Finally, we generated anti-S. obvelata antibody to optimize the detection ELISA and identified target epitopes for future analysis. In conclusion, we identify the T helper immune response induced to S. obvelata and demonstrate the importance of IL-13 for the expulsion of the GI nematode. We show that S. obvelata induces a non-protective immune response to a common food allergen and confirm that the pinworm-specific ELISA is an effective diagnostic tool for detecting pinworm infected mice.

Tuberculosis (TB), caused by Mycobacterium tuberculosis is the leading infectious disease epidemic that claims over 1.6 million lives, while 10 million fell ill in 2017. South Africa is burdened with the third highest global incidences following India and China with high rates of co-infections with HIV and highest numbers of multi-drug resistant (MDR) and extremely resistant (XDR) TB per capita. The current treatment regimen is decades old and requires a prolonged period of 6 months. The lack of efficient TB therapy and the emergence of MDR and XDR TB, there is an urgent need to find new drug targets for TB therapy through understanding the complex host-pathogen interactions. This may then lead to pathogen, host-directed therapies (HDT) or adjunct therapies as well as the development of effective drugs and drug formulations for the treatment of TB. Here we aimed to investigate potential targets for pathogen-and host-directed therapies for TB. We screened the anti-mycobacterial activity of 172 minor groove binder (MGB) compounds that selectively bind to AT-rich regions of the minor groove of bacterial DNA with the helical structure matching that of DNA in Mtb culture. Of the 172 total compounds screened 17 hits were identified, of which 2, MGB 362 and MGB 364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 μM, respectively, whilst being non-toxic. Encapsulation of MGBs into non- ionic surfactant vesicles (NIVs) demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB alone. Treatment with MGB 364 or MGB 364 formulation did not cause DNA damage in murine infected macrophages as displayed by low expression of γ-H2Ax compared to H2O2 and DMSO. Intranasal administration of MGB 364 and MGB-NIV 364 formulation showed one log reduction in bacterial burden with improved pathology and immune cytokine production when in formulation. However, intranasal administration of 10 mg/kg MGB 362 together with rifampicin had no effect on bacterial loads. In summary, the data demonstrate the potential of MGB as a novel class of drug/chemical entity in anti-TB therapy and NIVs as an effective delivery system in a novel anti-TB formulation. Using deep CAGE and small RNA (CHIP-seq) technologies, International Center for Genetic Engineering and Biotechnology’s Cytokines and Diseases lab in collaboration with the RIKEN Center for Integrative Medical Sciences (Yokohama, Japan) performed a novel transcriptomics study approach by conducting a genome-wide transcriptional analyses of RNA transcripts from classically activated macrophages (caMph) and alternatively activated macrophages (aaMph) during Mtb infection. We identified host target genes that may play a role in host immune subverting mechanism by Mtb to hide away from host effector functions providing a possible target for host-directed therapy for tuberculosis. It is postulated that Mtb modulates the transcriptional landscape of IL-4/IL13 alternatively activated macrophages (aaMph) to escape killing by reactive nitrogen intermediates (NO) and reactive oxygen species (ROS) functions by IFN-γ stimulated classically activated macrophages (caMph). Here we report on the immunoregulatory role of IL-4i1, a candidate gene that was upregulated in aaMph during Mtb infection. IL-4i1 is a secreted L-amino oxidase with antibacterial properties. The enzyme converts Phenylalanine (Phe) into phenylpyruvate releasing toxic products ammonia and hydrogen peroxide (H2O2) which in-turn cause immunosuppression of effector T-cells by directly inhibiting polarization, proliferation and function or by promoting the generation of Foxp3 T-regulatory cells. Thus suggesting that IL-4i1 is involved in immune-regulatory mechanisms and may be implicated in immune evasion mechanisms by the pathogen. Here we report on the role of IL-4i1 on tissue localized T-cell activation and proliferative status thus maintaining immune local immune homeostasis. Thus showing that the absence of IL-4i1 could cause autoimmunity. To determine the functional role of IL-4i1 during Mtb infection, IL-4i1 deficient mice and wild-type littermate controls were infected with H37Rv and hypervirulent HN878 Mtb strain. IL-4i1 deficient mice were highly resistant to both strains of Mtb at 12- and 21-days post-infection as denoted by significant reduction in bacterial loads, reduced inflammation, reduced tissue iNOS expression reduced recruitment of interstitial macrophages, pro-inflammatory cytokines showed a trend for reduction. Interestingly there was a significant increase in NO production in infected tissues. There was an increase in M1-like macrophages that correlated with increased pro-inflammatory cytokines and chemokines. These data suggested that IL-4i1 regulates macrophage-mediated inflammatory responses during acute Mtb infection thus showing potential as an immunomodulatory target for TB HDT therapy. The study thus provides a framework for new drug targets for the development of new effective drugs and vaccines for TB therapy.

Includes abstract. / Includes bibliographical references (leaves 85-97). / Tumor necrosis factor alpha (TNFα) is critical for host protective immunity against Mycobacterium tuberculosis infection. TNFRp55 and TNFRp75 can both bind TNFα and conduct signaling, however the respective roles, in particular that of TNFRp75 in an M. tuberculosis aerosol inhalation infection was poorly defined. In this study the role of signaling through TNFRp55 and TNFRp75 was investigated using TNFR deficient mice in an aerosol inhalation M. tuberculosis infection model.

Includes bibliographical references (leaves 297-327). / Pericardial tuberculosis (TB) offers a relevant human model to study TB at the site of disease and to determine the effect of HIV-1 infection. 96 Patients with pericardial TB were recruited into this study, 68 of whom were HIV-1 infected. Where clinically indicated, pericardiocentesis was performed to drain pericardial fluid and blood was drawn. The data derived from the study provide novel insight into the immune response in the pericardium to TB infection. Furthermore, HIV-1 infection caused a dysregulation of the immune response.

Recent studies have identified a novel 600 kDa neutrophil membrane associated protease which degrades fibrinogen, fibrin and C-reactive protein (CRP) during incubation of these ligands with phorbol 12-myristate 13-acetate (PMA, 5-10 ng/ml) stimulated neutrophils. This proteolysis is predominantly an extracellular event which occurs through a ligand dependent release of this protease from the neutrophil. Degradation products arising from this proteolysis not only become neutrophil associated but influence a number of important processes occurring in inflammation and coagulation. The aim of the present 'study was to purify and further characterize this protease and investigate the location of the neutrophil associated fibrinogen and fibrin degradation products. Whilst enzyme purification procedures were unsuccessful, several observations made during these attempts suggested that the neutrophil membrane associated proteolytic activity displayed similar characteristics to proteases of the azurophil granule. The proteolytic activity of the membrane was concluded from inhibitor profiles, zymography, and the apparent molecular mass values and hydrophobicity of the fibrinogen degradation products that it generated, to be the composite action of the azurophil granule proteases, human neutrophil elastase, cathepsin G and possibly proteinase 3. Electron microscopy analysis of PMA stimulated neutrophils incorporated within fibrin clots revealed morphological changes suggestive of neutrophil degranulation, and the proteolytic activity released by these cells was shown to be identical to that of azurophil granule proteases with respect to the apparent molecular mass values of the fibrin products that it generated. Immunoelectron microscopy revealed minimal internalization of fibrin like material during this process suggesting that neutrophil mediated fibrinolysis under these conditions is predominantly an extracellular event. Immunoelectron microscopy was used to localise fibrinogen degradation products previously reported to be associated with the neutrophil following incubation with fibrinogen. This revealed neutrophil associated fibrinogen products to be intracellular. Internalisation appears to be the result of pinocytosis which is stimulated in the presence of PMA. Although internalisation may be enhanced by an initial interaction of fibrinogen with the neutrophil membrane, a large proportion of uptake occurs via the fluid phase. Both intact and degraded forms of fibrinogen can associate with the neutrophil. Internalised material is rapidly degraded intracellularly into low molecular weight products which are partially released into the surrounding medium. This intracellular degradation, however, contributes minimally to the overall degradation of fibrinogen by neutrophils; the major pathway is extracellular. The demonstration in this· study, that the previously identified fibrinogen- fibrin- and CRP-degrading activity of the neutrophil membrane is due to azurophil granule proteases co-incides with numerous recent reports suggesting that membrane bound forms of these proteases, due to their ability to evade naturally occurring protease inhibitors, are the biologically relevant forms of these proteases. The membrane expression of azurophil granule proteases has recently been shown to be under the control of a variety of inflammatory mediators. Thus, neutrophil mediated degradation of fibrinogen, fibrin and CRP in vivo may be tightly controlled by the regulated expression of azurophil granule proteases on the neutrophil membrane.

Includes abstract. / Includes bibliographical references (leaves 117-129). / Schistosoma mansoni egg passage through intestinal tissue into the faecal stream is a critical event for completion of the life cycle of the helminth and involves induction of a type 2 immune response, which is necessary for the host’s survival. Where as T cell-specific IL-4Rα has been shown not to be essential for survival, macrophage/neutrophil-specific IL-4 receptor α-deficient mice (LysMIcre L-4Rα-/lox ), mice lacking alternative macrophages (AAMФ), had severe intestine pathology and high endotoxin levels due to poor egg excretion and dysregulated granuloma formation. This resulted in increased mortality. To determine the role of AAMФ in granuloma we aimed to describe S.mansoni egg induced granuloma formation in the presence and absence of AAMФ using LysMcreIL-4Rα-/lox mice.

Includes abstract. / In this study, we generated a new mouse model, which allows both inducible and cell-specific deletion and reconstitution of IL-IL4Rα expression. This model has the potential to add a new dimension to our understanding of IL4Rα biology. This has been achieved by using the established Tet System (Goosen and Bujard, 1992) where the crossing of two complementary transgenic mouse lines enable the generation of the final double transgenic model. The first line expresses the transactivator, tTA, from the Tet-Off expression cassette driven by the Vav hemapoeitic specific promoter (Wiesner et al., 2005).

Bibliography: leaves 122-138. / Exposure to Mycobacterium tuberculosis results in clinical tuberculosis only in a small percentage of immunocompetent individuals. In most instances mycobacteria are controlled by the host immune system and survive in a dormant state within granuloma. lmmunosuppression, however, may result in reactivation of active tuberculosis resulting in clinical disease. Using low dose aerosol infection of M. tuberculosis in mice, a short-duration model of rifampicin-isoniazid (RMP-INH)-induced persistent tuberculosis is described. This persistent infection is characterised by undetectable levels of colony-forming units (CFU) in mouse organs and mice being clinically asymptomatic for prolonged periods. Reactivation of persistent tuberculosis can occur spontaneously following short-course chemotherapy or can be achieved by immunosuppression, specifically inhibition of macrophage- specific nitric oxide synthase (NOS2) by a chemical inhibitor, aminoguanidine. This model can therefore be used to characterise spontaneous or drug-induced reactivation of murine tuberculosis, as this is not feasible to study in human subjects. Additionally, this model may serve as a valuable tool for testing novel vaccines and antituberculous drugs, especially those designed to combat persistent infection. Mycobacterial genome copy enumeration and assessment of 168 ribosomal RNA (168 rRNA) and sigma factor A (sigA) gene expression revealed that large numbers of dormant bacilli are present in lung tissue during the persistent phase of infection in this model. This finding opens up the possibility that additional gene expression profiles can be analysed with current technology, unravelling the exact metabolic status of these dormant mycobacteria. Moreover, this model facilitates characterisation of another poorly understood aspect, namely reinfection. Preliminary aerosol reinfection during the persistent phase of tuberculosis revealed that the primary- infected dormant M. tuberculosis strain may be reactivated and may outgrow the primary strain during reinfection. Tumour necrosis factor (TNF) deficient mice are known to be highly susceptible to M. tuberculosis infection. In this study it was asked whether TNF is required for post-infectious immunity in aerosol-infected mice. This model was applied and mice were treated with RlV|P-INH for 4 weeks to reduce the CFU to undetectable levels. While wild-type control mice spontaneously reactivated but controlled the infection upon cessation of chemotherapy, TNF deficient mice developed fatal reactivation of infection. The increased susceptibility of TNF deficient mice was accompanied by diminished recruitment and activation of T cells and macrophages into the lung with defective granuloma formation and reduced inducible nitric oxide synthase expression. Reduced chemokine production in the lung might explain sub-optimal recruitment and activation of T cells and uncontrolled infection. Therefore, despite a massive reduction of the mycobacterial load by chemotherapy, TNF deficient mice were unable to compensate and mount a protective immune response. In conclusion, endogenous TNF is critical to maintain latent tuberculosis infection and in its absence no specific immunity is generated.

Bibliography: leaves 132-155. / Women during pregnancy in holoendemic regions of malaria are at an increased risk for peripheral malaria infections with potential for developing placental malaria. The immunological basis of protection and pathogenesis are incompletely understood. This thesis investigates both processes. Research on maternal placental immune responses necessitates the collection of reliable placental intervillous blood; an appropriate method for placental blood collection was therefore first determined. Five documented methods of collection (perfusion, incision, biopsy, tissue grinding and prick) were compared for foetal blood contamination and mononuclear cell profiles using flow cytometry. Placental blood collection by prick was established as the most appropriate method and was subsequently used for further immunological investigations.