published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Epidemiological studies suggest that both retinoid and carotenoid intakes are inversely correlated with the incidence of human cancers. Animal studies show that both retinoids and carotenoids inhibit tumor cell growth. Both retinoids and carotenoids activate the cytotoxicity function of macrophages in animal experiments. The purpose of this study is to evaluate the molecular mechanism for 13-cis retinoic acid (13-cRA) and beta-carotene (BC) induced immunomodulation which could explain their anti-cancer affects. The effects of 13-cRA and BC were studied on various subpopulations of T-lymphocytes both in vitro and in vivo. For in vitro studies, peripheral blood mononuclear cells (PBMC) were incubated with test compounds at clinically achievable concentrations (10⁻⁸M) for three days. Then the cells were stained with monoclonal antibodies followed by the analysis of flow cytometer. For in vivo studies, PBMC were collected from Barrett's esophagus or oral leukoplakia patients during treatment with 13-cRA (1mg/kg/day) or BC (30 mg/day), respectively. Then the cells were analyzed with monoclonal antibodies and flow cytometry. Both compounds showed the capability of stimulating different subpopulations of T-lymphocytes. 13-cRA predominantly increased the number of T-helper cells, their interleukin 2 (IL-2) receptors and their response to mitogens. Whereas, BC elevated the number of Natural Kill (NK) cells, their IL-2 receptors and their cytotoxicity against K562 target cells. Though these immunomodulatory effects appeared to be unaffected by the presence and cytotoxic functions of macrophages, cytokines seemed to have an important role in the retinoid- and carotenoid-induced immunomodulation. Plasma levels of IL-2 and tumor necrosis factor (TNF) measured by ELISA procedures were increased in patients treated for two months with 13-cRA and BC respectively. Anti-IL-2 and anti-TNF antibodies blocked the retinoic- and carotenoid-induced immunomodulation in in vitro studies. These results indicate that 13-cRA, activating T-helper cells with IL-2 production, and BC, activating NK cells with TNF release, induced immunostimulation which might be able to provide the anti-cancer affects in part seen in epidemiological studies.

To determine the antigenic relatedness of the different developmental stages of Cryptosporidium parvum, monoclonal IgG3 antibody (mAb), Cmg-3, was produced by immunizing mice with partially purified merozoites. The monoclonal Cmg-3 reacted with a 3.5 kDa antigen of sporozoites in western blots and appeared to react with cell surface antigens of air-dried merozoites and sporozoites using immunofluorescence (IF). Additional mAbs, C6B6 (IgG1) and C4A1 (IgM), which react with a 20 kDa and multiple sporozoite antigens, respectively, were employed for immunoelectron microscopic studies with Cmg-3. These mAbs showed similar (surface/cytoplasmic) immunoelectron microscopic colloidal gold labeling patterns with all C. parvum life cycle stages. The three mAbs were also examined for potential modulation of cryptosporidial infections in vivo by daily oral mAb administration to oocyst-inoculated neonatal mice. Monoclonal-treated neonatal mice were sacrificed four and eight days post infection (pi). Differences in infection rates were observed among the treatment groups (p < .05). Suckling mice treated daily with orally administered mixtures of mAbs (ascitic fluids) showed significantly reduced parasite loads compared to control mice at four and eight days pi, while suckling mice receiving mAb Cmg-3 alone showed significant differences only at four days pi. Passive transfer of immunity using hyperimmune bovine colostrum was performed to determine the therapeutic and prophylactic efficacy of daily oral administration of anti-C. parvum antibody on the manifestation of cryptosporidial disease in neonatal mice as a model for treating cryptosporidiosis in immunocompromised patients. Hyperimmune colostrum was found to provide therapeutic and prophylactic efficacy against cryptosporidiosis in neonatal mice. Significantly fewer (p < 0.05) stages of C. parvum were found in mice that received hyperimmune skim colostrum (HSC) or hyperimmune original colostrum (HC) than in those treated with control colostrum (CC) or saline. Using IF, antigen-specific IgG in HSC and HC to C. parvum was 35 times greater than that of CC. There was no significant difference between groups treated with HSC or HC (p < .05), which suggests that the immunoglobulins, other biologically active factors such as cytokines, or both, might be active factors of immunity against cryptosporidiosis.

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Because tumours are comprised of "self" cells and antigens, they escape recognition by the immune system, which discriminates between "self" and "non-self". One such antigen is cathepsin B, a lysosomal cysteine proteinase, that has been implicated as one of the proteolytic enzymes involved in tumour invasion and metastasis. Cathepsin B autoantibodies could open possibilities which may be useful in cancer immunotherapy. In this study generation of cathepsin B autoantibodies was attempted by manipulating the immune system into recognising and responding to cathepsin B in complex with a "foreign" protein, bovine serum albumin (BSA). Cathepsin B was isolated from rabbit liver using the three phase partitioning (TPP) method, modified by adding t-butanol in the homogenisation buffer. Isolation of cathepsin Band cathepsin L, using this novel method, minimised the formation of artefacts such as a covalent cathepsin L-stefin B complex and produced higher yields of enzyme. Pure rabbit liver cathepsin B was conjugated to BSA, using glutaraldehyde as coupling agent, and administered intramuscularly into rabbits. Another three inoculation protocols, which functioned as controls were: i) free cathepsin B administered intramuscularly, ii) complexed cathepsin B administered intravenously, and iii) free cathepsin B administered intravenously. IgGs isolated from inoculated rabbits' serum were assayed by a three layer ELISA system, immunoinhibition assays and dot blots. The anti-complex (intramuscular) antibodies showed the highest recognition for cathepsin B and were the only antibodies that were immunoinhibitory. This suggests that the immune system was, to some extend, successfully manipulated into recognising the complexed "self" cathepsin B. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

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Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses.

Magister Public Health - MPH / Background: The goal of ART therapy is sustained viral load suppression with good immunological and clinical response. This optimal response to therapy results in the prevention of emergent ART drug-resistant mutations, decrease morbidity, and AIDS-related mortality and sustained retention on ART. Kenya, like most countries in Sub-Saharan Africa, has scaled-up the use of ART and is currently implementing a “Test and Treat” strategy in which any client identified and confirmed with an HIV diagnosis is initiated ART. Few studies have been carried out to ascertain the response of HIV patients initiating treatment in resource-limited settings. Moreover, it has been demonstrated that a certain proportion of patients fail to adequately respond to therapy and therefore require therapy modification. Aim: To assess treatment outcomes and calculate retention of HIV infected adult patients’ (15 years and above) initiating ART at Kakamega County General Hospital. The primary study outcome was the treatment outcome of patients-initiated ART two to three years prior to the study; while, the role of WHO criteria for screening treatment failure was assessed as a secondary outcome. Methods: This was a retrospective cohort study in which patients initiating ART between June 2014 and March 2015 were followed up until they were censored or study closed in August 2017. 284 patients were enrolled in the study after accurately matching information in their clinic files and the electronic medical record. Data were collected from patient records using a chart abstraction tool and transferred to an Access database from where the cleaning and validation of entries were done. Data from Access was transferred to STATA 15.1 for analysis. Descriptive statistics and inferential statistics were then performed to answer the research questions.

Estudamos a impostação de mãos sobre camundongos, avaliando parâmetros hematológicos e imunológicos. Nossos resultados demonstraram nos animais que receberam a impostação de mãos uma diminuição significativa do número de plaquetas, elevação do número de monócitos na leucometria específica, elevação da atividade citotóxica de células não-aderentes com atividade NK e LAK. Os grupos controle e placebo não mostraram qualquer alteração Os resultados encontrados nos levam a concluir que há uma alteração fisiológica decorrente à impostação de mãos e que há que se estudar por que ela ocorre / It has been investigated whether the hands imposition on male mice, produces physiological effects through hematological and immunological parameters. The results demonstrated that animals receiving hands imposition have presented a reduction of the platelet count, increase in monocytes count in specific white blood cells counts, increase in cytotoxic activity of non-adherent cell with NK and LAK activities.Control and placebo groups presented no response. The results found lead us to the conclusion that there is a physiological alteration due to the hands imposition and it should be investigated how it does occur