Made available in DSpace on 2014-08-20T13:32:48Z (GMT). No. of bitstreams: 1 dissertacao_karine_begnini.pdf: 1622668 bytes, checksum: 238f06c82a5dce8f22542c15f37944f4 (MD5) Previous issue date: 2012-02-15 / Bacillus Calmette-Guerin (BCG) is one of the great success stories of immunotherapy as a treatment for superficial urothelial carcinoma of the bladder. The high incidence of local side effects and presence of non-responder diseases has led to efforts to improve the therapeutic vaccine. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ΔleuD/Ag85B), could enhance cytotoxicity to the human bladder carcinoma cell line (5637). This rBCG was generated by incorporating an expression plasmid encoding the mycobacterial antigen Ag85B into the BCG ΔleuD strain. The inhibitory effect of BCG ΔleuD/Ag85B in 5637 cells was determined by the MTT method, morphology observation and the LIVE/DEAD assay. Gene expression profiles for apoptotic genes, cell cycle-related genes and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ΔleuD/Ag85B treatment were evaluated by Western blotting. BCG ΔleuD/Ag85B revealed a superior cytotoxicity effect than the strains used as controls in this study. The results demonstrated that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ΔleuD/Ag85B treatment, whereas mRNA levels of antiapoptotic genes decreased. Interestingly, BCG ΔleuD/Ag85B also increased the mRNA level of antioxidant enzymes in bladder cancer cell line. Bax and p53 protein levels were increased by BCG ΔleuD/Ag85B treatment. In conclusion, these results suggested that BCG ΔleuD/Ag85B enhanced cytotoxicity on superficial bladder cancer cells in vitro. The therapeutic model using rBCG may have potential for future clinical application in the treatment of bladder cancer. / O Bacilo Calmette-Guérin (BCG) constitui uma das grandes histórias de sucesso da imunoterapia como tratamento para carcinoma superficial da bexiga. Porém, a alta incidência de efeitos colaterais locais e a ocorrência de tumores resistentes ao tratamento têm impulsionado estudos visando melhorias da vacina terapêutica. Neste trabalho, propusemos que uma cepa auxotrófica de BCG superexpressando o antígeno Ag85B (BCG ΔleuD/Ag85B), é capaz de aumentar a citotoxicidade na linhagem celular humana de carcinoma superficial de bexiga (5637). A cepa de BCG recombinante foi gerada através da incorporação da sequencia do antígeno Ag85B em um plasmídeo de expressão micobacteriano na cepa de BCG ΔleuD. O efeito inibitório do BCGΔleuD/Ag85B em células 5637 foi determinada através das técnicas colorimétricas MTT e LIVE/DEAD, além de observação morfológica. Os perfis de expressão gênica para genes apoptóticos, genes relacionados ao ciclo celular e genes de estresse oxidativo foram avaliados por qRT-PCR. Os níveis protéicos de bax, bcl-2 e p53 foram avaliados por western blot. O BCG ΔleuD/Ag85B revelou citotoxicidade superior às cepas utilizadas como controle neste estudo. Os resultados obtidos demonstram níveis superiores de expressão de genes pró-apoptóticos e de genes relacionados com o ciclo celular após tratamento com BCG ΔleuD/Ag85B. Níveis inferiores de mRNA de genes antiapoptóticos foram detectados após o mesmo tratamento. Ainda, o tratamento com BCG ΔleuD/Ag85B também elevou os níveis de mRNA de enzimas antioxidantes em linhagem de células de câncer superficial de bexiga. As proteínas Bax e p53 mostraram-se elevadas após tratamento com BCG ΔleuD/Ag85B. Em conclusão, estes resultados sugerem que a cepa de BCG superexpressando Ag85B é capaz de aumentar a citotoxicidade sobre as células de câncer superficial de bexiga in vitro. Este modelo terapêutico usando BCG recombinante possui potencial para uma futura aplicação clínica em tratamento de câncer de bexiga.

Tuberculosis is one of the most prevalent infectious diseases globally due to the successful survival mechanisms displayed by Mycobacterium tuberculosis (Mtb). Mtb primarily infects alveolar macrophages (AMs) and is able to live intracellularly for extended periods of time due to a number of virulence factors which inhibit the antibacterial mechanisms of the AMs. This aspect of the Mtb life cycle means TB treatments suffer from poor bioavailability and efficacy. Additionally, the rise in resistant strains of Mtb means the use of higher doses and the use of alternative second and third line drugs which increase the risk of systemic toxicity. Drug encapsulation is a novel approach that can provide more favourable drug pharmacokinetics and pharmacodynamics. The aim of this project was to develop a liposomal drug delivery system to target Mtb infected alveolar macrophages. The system involved the encapsulation of two drugs; the antibiotic gatifloxacin (GFLX) and Mtb virulence factor inhibitor CV7. The hypothesis was that the two different antibacterial mechanisms would work in synergy and increase the efficacy of the treatment. AM targeting and receptor-mediated endocytic uptake was encouraged by the presence of a ligand attached to the surface of the liposome. Furthermore a pH-sensitive release mechanism was to be incorporated into the liposome to encourage the release of the encapsulated drugs in the vicinity of the intracellular bacteria. The intention was to produce a drug delivery system to enable a TB therapy regime of fewer, lower doses to increase compliance and reduce systemic toxicity by increasing efficacy through improved bioavailability. GFLX was successfully encapsulated using a weak base active loading method. To establish encapsulation efficiency, a homogeneous fluorescence assay able to quantify intra- and extra-liposomal gatifloxacin simultaneously was developed. pH-sensitive release of the payload could be achieved using a pH-sensitive peptide with a novel design based on chimeric structure, namely P3. CV7 was successfully encapsulated using a weak acid active loading method. CV7 liposomes were able to be functionalised by the incorporation of a mannose ligand on the surface of the liposome. An inhibition assay using the target enzyme of CV7, MptpB, was optimised to assess efficacy of liposomally encapsulated and released CV7. Flow cytometry and confocal microscopy studies confirmed that the liposomal formulations were internalised by the target macrophage cell line, J774a.1. Mannose liposomes conveyed superior uptake kinetics. Further confocal microscopy showed that after internalisation the liposomes entered the endolysosomal pathway and colocalised with BCG. A BCG-macrophage infection model was used to determine the intracellular efficacy of the liposomal formulations. Encapsulated CV7 displayed increased efficacy over free CV7, while encapsulation in functionalised liposomes showed better efficacy still. The encapsulation of GFLX did not increase the efficacy of GFLX and synergy between the two drugs was not achieved. In conclusion, the liposomal encapsulation of CV7 increased uptake of the drug by the target cell line and facilitated colocalisation of the drug with the target pathogen thereby increasing efficacy. Such a formulation could potentially increase bioavailability and efficacy in vivo for a more tolerable TB therapy.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Background: There are critical unmet needs for improved strategies in the detection and diagnosis of M.tuberculosis infection in children, and for prevention of tuberculosis disease in children. Bacillus Calmette-Guérin (BCG) vaccination has limited the utility of tuberculin skin testing (TST) in areas with high vaccine coverage. Objectives: The aim of this study was to estimate the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. Methods: This study was a cross-sectional design to assess the performance of a new T-cell based blood test, namely QuantiFERON-TB Gold In Tube (QFT-GIT), for diagnosis of tuberculosis infection in the children (n=182) of adults (n=124) with pulmonary tuberculosis, additionally to determine the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. The study was carried out at Chris Hani Hospital. For children involved in the study, tuberculosis exposure information was obtained, together with TST, QFT-GIT, and HIV testing. Data obtained from both experiments was statistically analysed using SPSS version 24 to determine whether there was a significant agreement between QFT-GIT and TST on the detection of M.tuberculosis prevalence in children with house hold contacts with confirmed M.tuberculosis infection. Results: This study examined the sensitivity and specificity of the QFT-GIT tests compared with the standard TST for diagnosing latent tuberculosis disease in paediatric contacts. Because of the lack of a latent tuberculosis “gold standard”, the specificity and sensitivity of QFT-GIT was calculated with a two-by-two table method. The specificity of the QFT-GIT was 84% and the sensitivity was 85%. There was a good correlation between QFT-GIT and TST (Cohen’s kappa of 0.705). Seventeen percent (17%) of the 182 children tested by QFT-GIT yielded indeterminate results. Age was associated with indeterminate QFT-GIT results in paediatric tuberculosis contacts. Point prevalence for QFT-GIT was recorded as 31% at baseline and 39.5% after six months indicating variability between QFT-GIT results at baseline and after six months. Conclusion: It was concluded that the prevalence of tuberculosis infection was common among South African children who live with an adult with active tuberculosis. The agreement between QFT-GIT assay and TST for the diagnosis of latent tuberculosis in children was high. Although TST and QFT-GIT assays appeared comparable, QFT-GIT showed higher positivity rate amongst those contacts with reported household tuberculosis exposure compared to TST. The QFTGIT assay was a better indicator of the risk of M.tuberculosis infection than TST in a BCG-vaccinated population.