Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 87-103).

Over the past several years there has been a steady increase in the incidence of immunologically compromised patients. This has been the result of both chemical agents, such as those used in cancer chemotherapy, and biological agents such as HIV, the cause of Acquired lmmunodefeciency Syndrome (AIDS). The increase in immune-suppressed patients has lead to an increase in life-threatening mycoses requiring treatment with antifungal agents. Pharmaceutical companies have increased research for the development of new antifungal agents which are more effective and less toxic than those that are currently used. Several researchers have reported on antifungal agents that demonstrate both positive and negative effects on the immune system. Because antifungal therapy relies on host immune defenses in eliminating diseases, more emphasis is being placed on how antifungal agents interact with the immune system. The purpose of this study was to evaluate the effects of Ketoconazole, ltraconazole and Fluconazole on T- and B-cell proliferation and natural killer cell activity using normal, cyclosporine-compromised and cyclophosphamide-compromised immune models in mice. T and B cells obtained from the spleens Balb/c mice were mitogen stimulated and grown in the presence of 0, 1, 2, 4, 8 and 16 µg/ml of these 3 antifungal agents. Cell proliferation was determined by the uptake of 3[H]-thymidine and was measured as counts per minute. Natural killer cell activity was measured by the release of 51-sodium chromate (51Cr) into the supernatant by 51Cr-labeled Yac cells. Ketoconazole caused a significant reduction in cell proliferation in all immune models in both T and B cells. Itraconazole also significantly inhibited cell proliferation in all models in both T and B cells as well as natural killer (NK) cell activity in the immune-normal model. Viability studies on mitogen-stimulated lymphocytes suggest that inhibitory effects of Katoconazole and Itraconazole on lymphocyte proliferation are due to toxic effects. Fluconazole appears to have few if any inhibitory effects on either cell proliferation or natural killer cell activity.

The complement system is considered as the chief recognition and effector component of innate immunity; it is involved in inflammation and enhances the adaptive immune response. Factor I (fI) is a heterodimeric serine protease consisting of a heavy (HC) and a light-catalytic (LC) chain; it circulates in an active form regulating complement by selectively cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), CR1, MCP or C4bp. The cleavage of C3b occurs through a ternary complex formed between fI, C3b and a cofactor like fH and yields iC3b, a major opsonin. The structural and functional properties of fI were investigated. The interchain disulphide bond formed between C³⁰⁹-C⁴³⁵ tnat links the HC and LC of fI as well as the composition of the TV-linked carbohydrates of fI were determined. By using two independent assays, the proteolytic and amidolytic assays, the catalytic properties of human fI were characterised in detail. The catalytic subunit, the SP domain, was shown to have a native conformation that accommodates substrate recognition and cleavage, fI has specificity similar to thrombin, but exhibits lower catalytic activity. fI amidolytic activity reaches optimum at pH 8.25 and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide AMC substrates by fI was unaffected by fH or C3(NH₃) at optimum pH. fI and the isolated SP domain were found to have similar amidolytic activities, but strikingly different proteolytic activities on C3(NH 3 ). fl did not cleave C3(NH₃) in the absence of fH, but cleaved it rapidly at two sites in its presence. The SP domain however, cleaved C3(NH₃) slowly in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. These results suggested that the HC domains and/or the cofactor must orient the natural substrates and restrict cleavage by fI to the two sites which yield iC3b. The implications of these findings are discussed.

Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

This study was undertaken to examine the specificity of the cell-mediated immune response to vesicular stomatitis virus in mice, using the recombinant adenovirus vector AdG12. AdG12 contains the coding region for VSV glycoprotein (G) within the genome of adenovirus type 5. Ultimately, these studies attempted to provide a model for the use of adenovirus vectors to elicit specific CTL responses in mice against an inserted foreign protein. Cell-mediated immunity was examined using a standard ⁵¹Cr release assay. Splenocyte effectors from VSV or AdG12 primed mice were tested for their ability to lyse labelled infected target cells. A number of target cell lines were analyzed for productive infection by AdG12 and expression of VSV-G. Of the lines tested, B10.D2 (H-2ᵈ) and PAK (H-2ᵇ) lines were shown to be infectible with AdG12 and expressed VSV-G 36 hours post infection. Cell lines P815 (H-2ᵈ) and EL-4 (H-2ᵇ) did not appear to be AdG12 infectible. Responses were measured in mice intravenously infected with AdG12. Results demonstrated that peak cytotoxic activity from AdG12 primed mice occurred six days post-infection against syngeneic target cells infected with AdG12, Ad5 wt or VSV. However, these effectors also significantly lysed allotargets infected with VSV, implying that VSV infected targets were lysed in a non-MHC restricted manner. In subsequent experiments, it was discovered that VSV infected B10.D2 and PAK targets were markedly lysed by effectors from immunized and non-immunized Balbic, C57B1/6 and CBA/J mice. Thus, it appeared that these mouse strains contained an inherent or natural cytotoxic activity against VSV infected targets that was unlike classical CTL killing. Depletion experiments showed that this activity was not due to adherent or Thy1 bearing cells within spleen cell populations. To further characterize this activity, splenocyte effectors were tested for their ability to lyse NK-sensitive YAC-1 targets, but no significant lysis was demonstrated. However, despite these results, it appeared that this activity was that of an NK-like effector. The presence of NK-like cytotoxicity against VSV infected targets precluded efforts to define specific anti-VSV responses in these mice. / Thesis / Master of Science (MS)

Alginate is a well-known naturally-derived biomaterial that has been widely used in preparing microparticles for drug delivery and in preparing scaffolds for tissue engineering. Despite desirable properties, alginate has been shown to activate inflammatory cells in vivo. The mechanisms are still unclear. In this thesis, the mechanism by which alginate caused innate immune responses was investigated in vitro by using RAW264.7 cells, a macrophage-like cell line. The NF-(kappa)B pathway, an important signaling pathway in macrophages, has been tracked to identify cellular responses. The secretion of cytokines IL-1(beta), IL-6, IL-12(p40) and TNF-(alpha) was quantified to determine the activation outcomes. Also the interaction between alginate and serum was studied. Experimental results indicated that alginate induced the activation of RAW264.7 cells with a time and dose dependent behavior. Like lipopolysaccharide, a bacterial product and known activator of innate immunity, alginate induced macrophage activation through the NF-(kappa)B pathway and eventually led to detectable IL-1(beta), IL-6 and TNF-(alpha) cytokine secretion. Serum influenced alginate recognition by macrophages in an unknown mechanism. Also, alginate promoted cell survival in a nutrition starvation condition. These results revealed in vitro alginate stimulation, and provided much information for further research. / Thesis / Master of Applied Science (MASc)

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