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Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection without showing signs of illness. Steroid hormones such as glucocorticoids (GCs) however can lead to reactivation of TB. Currently two different injectable contraceptives are available in South Africa, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET). MPA is able to bind to and activate the glucocorticoid receptor (GR) and possesses selective GC activity, a pharmacological characteristic absent in NET. The aim of this study was to investigate the immune modulatory effects of the two contraceptives MPA and NET on immune responses to mycobacteria in vitro and in vivo. Human peripheral blood mononuclear cells (PBMCs) were infected with BCG (M. bovis Bacille Calmette-Guérin) and treated with MPA, NET, progesterone or cortisol and cytokine expression was measured in order to determine whether the synthetic progestins mimic endogenous progesterone or the GC cortisol. MPA, but not NET suppressed the expression of IFN-γ, IL-1α, IL-1β, IL-2, IL-12p40 and IL-13 similarly to cortisol. Furthermore expression of miRNAs, small double stranded RNA molecules that bind to complementary sequences in mRNAs of target genes and cause their degradation, was determined under the different experimental conditions. The expression of several miRNAs including miR-30c, miR-222, miR-454 and miR-331-3p were differentially influenced by MPA, cortisol and/or NET in PBMCs stimulated with BCG. For example, BCG induced the expression of miR-454 (p=0.003) which was then inhibited to basal levels by cortisol (p=0.008), MPA (p=0.002) and NET (p=0.002). These results demonstrate that steroid hormones including the contraceptives MPA and NET can modulate immune responses to mycobacteria at the miRNA level. To determine the effect of MPA and NET on BCG-induced expression of miRNAs in vivo a mouse model was used. C57BL/6 mice were injected weekly with either MPA or NET using a dose equivalent to humans and then infected with BCG. Mice treated with MPA had a higher spleen bacterial load 21 days after infection compared to both NET treated and control mice (p=0.023). In whole blood, MPA and NET treatment suppressed the BCG-induced production of miR-100 and miR-509-3p to basal levels. In contrast to NET, MPA induced expression of miR-99a expression independent of BCG infection. In the lung, the site of disease, a total number of 22 out of 377 miRNAs tested were differentially expressed 21 days after infection. The results of this study indicate that both synthetic progestins altered the immune response to BCG at the level of cytokine expression as well as the miRNA level. MPA was found to mimic cortisol by inhibiting secretion of inflammatory cytokines whereas NET appeared to have more progestogenic properties. Each of the steroid hormones was observed to induce a characteristic miRNA expression profile, both in vitro as well as in vivo, although not identical. These results highlight that the two contraceptives – although used interchangeably by women in developing countries - are pharmacologically unique and differentially modulate immune responses to mycobacteria. These data support the urgent need for further research into the link between hormonal contraceptives and susceptibility to infectious diseases. / AFRIKAANSE OPSOMMING: Individue wat latent met Mycobacterium tuberculosis ( M.tb ) geïnfekteer is, onderdruk die infeksie en wys geen simptome van die siekte nie. Steroïed hormone soos glukokortikoïede (GKe) kan egter tot die heraktivering van TB lei. Daar is tans twee verskillende inspuitbare voorbehoedmiddels beskikbaar in Suid-Afrika, medroksiprogesteroon-asetaat (MPA) en noretisteroon enantaat (NET). MPA is staat om aan die glukokortikoïed reseptor (GR) te bind en dit te aktiveer. MPA beskik ook selektiewe GK aktiwiteit, 'n farmakologiese eienskap wat afwesig is in NET. Die doel van hierdie studie was om die immuun-regulerende effekte van die twee voorbehoedmiddels, MPA en NET, op immuunresponse teen mikobakterieë in vitro en in vivo te ondersoek. Menslike perifêre bloed mononukleêre selle (PBMSe) is met BCG geïnfekteer en met MPA, NET, progesteroon of kortisol behandel. Sitokien uitdrukking was gemeet om vas te stel of die sintetiese progestiene, endogene progesteroon of die GK kortisol naboots. MPA, maar nie NET, onderdruk die produksie van IFN-γ, IL- 1α, IL- 1β, IL- 2, IL- 12p40 en IL- 13 soortgelyk aan kortisol. Verder is uitdrukking van miRNAs, klein dubbelstring RNS molekules wat aan komplimentêre volgorde in mRNAs van teiken gene bind en wat hul degradering veroorsaak, bepaal in elk van die verskillende eksperimentele toestande. Die uitdrukking van verskeie miRNAs insluitende miR-30c, miR-222, miR-454 en miR-331-3p is differensieël beïnvloed deur MPA, kortisol en/of NET in PBMSe wat met BCG gestimuleer is. Byvoorbeeld, BCG veroorsaak die uitdrukking van miR-454 wat dan geïnhibeer word tot agtergrondvlakke deur kortisol, MPA en NET. Hierdie resultate toon dat steroïed hormone, insluitend die voorbehoedmiddels MPA en NET, die immuunrespons teen mikobakterieë op miRNA-vlak affekteer. Om die effek van MPA en NET op BCG-geïnduseerde uitdrukking van miRNAs in vivo te bepaal, is ʼn muismodel gebruik. C57BL/6 muise is weekliks met 'n dosis van MPA of NET, ekwivalent aan dosisse gebruik in die mens, ingespuit en met BCG geïnfekteer. Muise wat met MPA behandel is, het 'n hoër bakteriële lading in die milt 21 dae na infeksie in vergelyking met NET-behandelde muise en kontrole muise. In hul bloed, onderdruk MPA en NET behandeling die BCG-geïnduseerde produksie van miR-100 en miR-509-3p tot basale vlakke. In teenstelling met NET, induseer MPA die uitdrukking van miR-99a onafhanklik van BCG infeksie. In die long is 'n totaal van 23 miRNAs differensieël uitgedruk 21 dae na die infeksie. Die resultate van hierdie studie dui daarop dat beide sintetiese progestien die immuunrespons teen BCG verander op sitokien sowel as miRNA vlak. MPA boots hoofsaaklik kortisol na deur inhibering van sitokien-produksie terwyl NET meer progesterone eienskappe het. Op miRNA vlak veroorsaak elk van die steroïed hormone 'n kenmerkende miRNA uitdrukkingsprofiel, beide in vitro asook in vivo. Hierdie resultate beklemtoon dat die twee voorbehoedmiddels - alhoewel hulle afwisselend gebruik word deur vroue in ontwikkelende lande - farmakologies uniek is en differensieël die immuunrespons reguleer teen Mycobacterium. Hierdie data ondersteun die dringende behoefte aan verdere navorsing in verband met hormonale voorbehoedmiddels en vatbaarheid vir aansteeklike siektes.

Two epidemiological studies, designed to expand our knowledge of morbidity and mortality indicators concerning malaria and Bacillus Calmette-Guerin vaccination in the Hospital Albert Schweitzer health district located in Central Haiti's Artibonite Valley, are presented. The hospital serves a rural population of 190,000 in its 610 square mile district. A large proportion of the rural population still relies on traditional healers in times of illness. Consequently, accurate morbidity and mortality data from individuals and communities in the district are scarce. The first study investigated an outbreak of axillary lymphadenitis and abscesses after Bacillus Calmette-Guerin vaccination among rural Haitian children treated at Hospital Albert Schweitzer from January 1986 through March 1991. Seventy-seven cases of vaccine-related complications were identified, all among children immunized before the age of 1 year. The proportions of children with complications were 0.017% for 1986 through 1989, 0.91% for 1990, and 2.2% for January through March 1991. The probable explanation for the increase is a change in the BCG strain or in the reactogenicity of the Pasteur strain. / Graduation date: 1994

Orientadores: Maria Helena Andrade Santana, Lucimara Gaziola de la Torre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-16T11:16:49Z (GMT). No. of bitstreams: 1 Trevisan_JuliaExaltacao_M.pdf: 7656590 bytes, checksum: c2da607d637d0d57546aa48d6017dcbe (MD5) Previous issue date: 2010 / Resumo: Este trabalho apresenta um estudo comparativo de processos escalonáveis dos tipos "top-down" (cominuição) e "bottom-up" (síntese), visando a produção de lipossomas funcionais para a vacinação gênica contra a tuberculose. Na abordagem "top-down" os lipossomas foram produzidos através da alimentação de uma dispersão de fosfolipídios em água e simultânea cominuição através de cisalhamento produzido por agitadores mecânicos dos tipos Caules (C) e Ultra-turrax® (U-T). Os lipossomas provenientes do U-T foram posteriormente submetidos a maiores níveis de cisalhamento, pela passagem através de microcanais tortuosos a pressões da ordem de 100 atm, em microfluidizador (M). A influência das variáveis operacionais sobre o diâmetro, polidispersidade e potencial zeta foi analisada através de planejamento estatístico fatorial e superfícies de resposta. Para C e U-T, os efeitos do cisalhamento prevaleceram em relação à vazão de alimentação. Para M, a pressão exerceu maior influência que o número de passagens. A taxa de cisalhamento aplicada variou na faixa de 102 a 7.106 s-1, produzindo diâmetros médios de 957 a 90 nm, polidispersidades de 0,60 a 0,25 e potenciais zeta de -60 a -44 mV. O diâmetro médio dos lipossomas variou com o inverso da raiz quadrada da taxa de cisalhamento. No processo "bottom-up" os lipossomas foram produzidos continuamente na interface etanol/fosfolipídio e água, formada por focalização hidrodinâmica em microcanal reto de 0,14 mm de largura. A velocidade de formação dos lipossomas variou com a razão água/etanol, concentração e composição lipídica. As propriedades físico-químicas originais dos lipossomas funcionais foram aproximadamente reproduzidas em ambos os processos escalonáveis. Comparativamente, o processo "top-down" é mais robusto, de fácil operação, permite utilizar altas concentrações lipídicas (400 mM), com obtenção de maior produtividade. Porém é descontínuo e requer maior consumo energético. O processo "bottom-up" é menos robusto, opera de forma estável a mais baixas concentrações lipídicas (100 mM), porém a produção é contínua e com menor consumo energético / Abstract: The work presents a comparative study of scalable process with a top-down (comminution) and bottom-up (synthesis) approach, aiming the production of functional liposomes for the genetic vaccination against tuberculosis. For the top-down approach, liposomes were produced through the feed of a phospholipid in water solution and simultaneous comminution by cowls (C) and Ultra-Turrax® (U-T) mechanical dispersers. The liposomes obtained by U-T were then submitted to greater levels of shear through the processing in tortuous microchannels under a pressure in the order of 100 atm, in a microfluidizer (M). The influence of the operational variables on the diameter, polydispersity and zeta potential was analyzed through statistics factorial planning and response surface. For C and U-T the effects of shear overcame the effects of feed flow rate, and for M the effects of shear had greater influence than the number of passes.The applied hear rate varied in the range of 102 a 7.106 s-1, resulting in liposomes with mean diameter of 957 to 90 nm, polydispersity of 0,60 to 0,25 and zeta potential of -60 to -44 mV. The mean diameter varied with the inverse of the square root of the shear rate applied. In the bottom-up process liposomes were produced continuously at the interface ethanol/phospholipid and water, formed by the hydrodynamic focusing in a straight microchannel with 0,14 mm of width. The liposome formation velocity varied with the ethanol/water flow ratio, the lipid concentration and composition. The original physico-chemical properties of the functional liposomes were approximately reproduced in both scalable approaches. Comparatively, the top-down approach was more robust, easy to operate and allows the use of high lipid concentration (400 mM), achieving a greater productivity. However, it is discontinuous and requires a higher energetic consumption. The bottom-up approach is less robust, is stably operated with lower lipid concentration (100 mM), but it is a continuous production with a low energetic consumption / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

MSc (Chemistry) / Department of Chemistry / Identification of unique leads represents a significant challenge in drug discovery. This challenge is widely visible in neglected diseases such as tuberculosis, which is an infectious disease caused by bacillus Mycobacterium tuberculosis. The urgent need in search of new biological entities to fight back TB and drug resistant TB is a drive behind this project. Several specific synthetic protocols have been developed using 1,3,5-triazines due to the important biological properties which they display. The chemistry and an extensive spectrum of biological activities of s-triazines have been examined since several decades and this heterocyclic core has received emerging consensus. Hence, the aim of this project was to synthesize novel anti-TB drugs total with the usage of 1,3,5-triazine as a linker between known anti-TB drugs together with different types of amines. A total of 20 compounds were synthesized, 3 compounds were mono-substituted with an average yield of 75 %, 6 compounds were di-substituted with an average yield of 63 % and 11 compounds were tri-substituted with an average yield of 93 %. Out of 10 compounds which were analysed for biological activity 8 of which showed biological activity against M.smegmatis. Furthermore compound 26 which was hybridized with an amine and a known anti-TB drug inhibited better biological activity. In conclusion the influence of cyanuric chloride in combination with pyrrolidine and anti-TB drugs deserves further study. The newly synthesized compounds were characterized by IR, melting point, GC-MS, biological testing, 1H and 13C NMR. / NRF

La tuberculose est une maladie contagieuse causée par les bactéries appartenant au complexe Mycobacterium tuberculosis. On estime près de neuf millions de nouveaux cas et un million de décès chaque année dans le monde. De plus, approximativement un tiers de la population mondiale est infecté de manière latente, donc à risque de développer la maladie. Le seul vaccin préventif jusqu’à présent disponible est le Bacille de Calmette-Guérin (BCG). Cependant, son efficacité contre la forme pulmonaire de la maladie, contagieuse et plus fréquente chez l’adulte, est extrêmement variable. Le développement de nouveaux vaccins prophylactiques contre la tuberculose est basé sur une stratégie de remplacement ou d’amélioration de l’actuel vaccin BCG. De nombreux candidats vaccins sous-unitaires sont évalués dans un protocole de vaccination de rappel après le BCG. Ce dernier est en effet administré à plus de 80% des nouveau-nés et des nourrissons des populations à haut risque.<p>Le présent travail a eu pour but principal d’étudier une nouvelle approche de vaccination combinant le Bacille de Calmette-Guérin avec des vaccins sous-unitaires à ADN plasmidique dans différents modèles précliniques.<p>Plusieurs hypothèses tentent d’expliquer la faible efficacité du vaccin BCG, comme la faible induction de réponses immunitaires de type cellulaire T CD8+, le déclin de l’immunité protectrice induite au cours du temps, ou son répertoire antigénique limité. Les vaccins à ADN plasmidique induisant de telles réponses, le travail proposé a consisté au développement d’un nouveau protocole de vaccination basé sur la coadministration par la voie intradermique du vaccin BCG formulé avec un vaccin à ADN plasmidique codant pour un antigène mycobactérien. Nous avons observé dans plusieurs modèles murins (adulte et néonatal) une augmentation significative des réponses cellulaires de type CD4+ Th1 et CD8+, ainsi que de la réponse humorale spécifique. L’immunogénicité de cette approche a également été analysée dans un modèle animal de grande taille, à savoir le modèle porcin. Les résultats obtenus indiquent que les vaccins à ADN plasmidique sont capables d’augmenter les réponses spécifiques à l’antigène codé par le plasmide mais également celles spécifiques à d’autres antigènes exprimés par le vaccin BCG. Enfin, dans la deuxième partie du travail, nous avons développé des vaccins plasmidiques codant pour des combinaisons d’antigènes phase-spécifiques de M. tuberculosis et nous avons analysé leur immunogénicité en modèle murin.<p>En conclusion, nous avons montré que la stratégie de coadministration par la voie intradermique du vaccin BCG avec un vaccin à ADN plasmidique encodant des antigènes mycobactériens s’avère être un protocole de vaccination réaliste et efficace pour améliorer l’immunité induite par le vaccin BCG. Elle offre par ailleurs des perspectives pour être appliquée avec des plasmides codant pour des antigènes caractéristiques de la tuberculose latente, peu reconnus après vaccination BCG, pour protéger à la fois contre la tuberculose active d’une primo-infection et contre la réactivation d’une infection latente. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished