Introdução: A imunoterapia intravesical com o bacilo de Calmette-Guérin (BCG) é o tratamento adjuvante de escolha no câncer superficial de bexiga. Recentemente, os estudos do mecanismo imunoterápico do BCG têm permitido identificar as reações imunológicas e os genes associados ao efeito antitumoral, possibilitando a produção de vacinas recombinantes, possivelmente mais efetivas e com menos efeitos colaterais. Com esses objetivos, associou-se o componente pertussis (S1PT) ao BCG, criando uma variante recombinante (rBCG-S1PT) com capacidade para promover uma resposta imune direcionada ao tipo T helper 1 (Th1), o que poderá elevar a eficácia antitumoral do imunoterápico. Objetivo: Avaliar comparativamente o efeito antitumoral do rBCG-S1PT e do BCG no modelo experimental de carcinoma urotelial de bexiga. Métodos: O estabelecimento do modelo murino ortotópico e singênico de tumor vesical foi realizado através da implantação transuretral das células tumorais de bexiga da linhagem MB49 de camundongo C57BL/6. Experimento I Os animais (modelo experimental) foram divididos em três grupos, os quais receberam 4 aplicações semanais de rBCG-S1PT, BCG, ou soro fisiológico (grupo controle), por via intravesical. Após 7 dias da última aplicação, foram extraídos o baço e a bexiga, com o intuito de inferir o peso tumoral. Em seguida, as bexigas foram submetidas à avaliação do padrão de resposta imunológica e exame anátomo-patológico e imunohistoquímico. Experimento II Realizado como descrito no Experimento I, porém os animais foram acompanhados por 60 dias para análise de sobrevida. Experimento III Este ensaio foi realizado como descrito anteriormente, porém não foi realizada a implantação tumoral, para controle dos achados imunológicos e anátomo-patológicos. Resultados: A taxa média de implantação tumoral foi de aproximadamente 90% dos animais inoculados. Obtivemos redução das médias dos pesos vesicais dos grupos BCG e rBCG-S1PT (p<0,001). Nos dois grupos tratados com os imunoterápicos observou-se aumento significativo da expressão de TNF-, a qual foi mais intensa com o uso do rBCGS1PT (p<0,05). A IL-10 também teve aumento significante de sua expressão no grupo BCG recombinante (p<0,01). Os esplenócitos provenientes dos camundongos que foram tratados com imunoterápicos diminuíram a viabilidade das células tumorais MB49, sendo que este efeito foi mais intenso no grupo rBCG-S1PT. O grupo de animais tratados com rBCG-S1PT apresentou aumento significativo da sobrevida em relação aos outros grupos (Experimento II). As aplicações dos imunoterápicos em animais sem tumor (experimento III) não revelaram diferenças histológicas em relação ao grupo controle e o padrão de resposta imunológica encontrado sugere uma tendência à resposta Th1. Conclusão: Obtivemos sucesso no estabelecimento do modelo murino ortotópico singênico de tumor vesical. O imunoterápico rBCG-S1PT apresentou mais benefícios no tratamento do tumor vesical ortotópico em camundongos em relação ao BCG, como também maior redução da viabilidade das células tumorais in vitro. A cepa rBCG-S1PT apresentou elevação significativamente maior das citocinas da resposta imune Th1 em relação aos demais grupos. Concluimos, então, que os dados apresentados sugerem a possibilidade deste recombinante proporcionar melhor controle clínico do tumor vesical em humanos que a imunoterapia com BCG / Introduction: The intravesical immunotherapy with bacillus Calmette-Guérin (BCG) is the adjuvant treatment of choice in superficial bladder cancer. Recently, studies of the mechanism of BCG have identified the immune reactions favorable and the genes responsible for the antitumor effect, enabling the production of recombinant vaccines, possibly more effective and with fewer side effects. With those goals, the pertussis toxin (S1PT) was combined to BCG, creating a recombinant variant (rBCG-S1PT) with the capacity to promote an immune response targeted to the T helper type 1 (Th1), which may increase the effectiveness of its antitumor effect. Objective: Compare the antitumor effects of rBCG-S1PT and BCG in an experimental model of bladder cancer. Methods: The development of the animal model of bladder cancer was conducted by transurethral instillation of bladder tumor cell line MB49 of the mouse strain C57BL/6, setting the orthotopic and syngeneic murine model. Experiment I - The animal models were divided into three groups, which received 4 weekly intravesical applications of rBCG-S1PT, BCG, or saline (SF - control group). After 7 days of the last instillation, splenectomy was performed for splenocyte culture and the bladders extracted and weighed in order to infer the tumor weight. Then, the bladders were divided into two pieces. The first was used for molecular analysis to assess the pattern of immune response. The second was sent to histopathological analysis. Experiment II - Held as described in Experiment I, but the animals were monitored for 60 days for analysis of survival. Experiment III - This test was carried out as previously described (Experiment I), but with no tumor cells instillation. Results: The rate of tumor implantation was 90% of the animals submitted to tumor inoculation. We obtained reduction of the average weights of bladder in groups BCG and rBCG-S1PT ((p<0,001). In both groups treated with immunotherapy, there was an increase of expression of interleukins TNF-, which was more intense in the group treated with rBCG-S1PT (p<0,05). There was also increased expression of IL-10 in the recombinant BCG (p<0,01). The splenocytes from animals that received immunotherapies had reduced tumor cells viability, more intensely demonstrated in the rBCG-S1PT group. The analysis of survival showed a significant increase in the group of animals treated with rBCGS1PT (Experiment II). The instillation of immunotherapeutic agents in animals without tumor did not demonstrate histological differences when compared to the control group and the immunological response pattern was similar to that of Experiment I (Experiment III). Conclusion: The establishment of the syngeneic orthotopic animal model was successful. The immunotherapy with rBCG-S1PT demonstrated more benefits than BCG in the treatment of bladder cancer in mice, reducing the bladder weight, increasing survival, and reducing tumor cells viability in vitro. The immune response obtained with the rBCG-S1PT expressed higher cytokines related to Th1. All this data may indicate that this recombinant agent may promote better bladder tumor control than BCG imunotherapy

La tuberculose bovine est une maladie infectieuse contagieuse causée par Mycobacterium bovis. Cette maladie touche les bovins et de nombreuses espèces de mammifères domestiques et sauvages, ainsi que l’homme. La circulation de la bactérie dans des systèmes multi-hôtes variés favorise l’entretien de la maladie et la contamination des bovins vivant à proximité des animaux sauvages infectés. En marge de la transmission directe de M. bovis par voie respiratoire, la transmission indirecte aux bovins, liée à l’inhalation ou à l’ingestion de matrices environnementales contaminées par un animal infecté excréteur, est suspectée dans plusieurs régions du monde. L’existence de réservoirs environnementaux où le bacille M. bovis est capable de persister, pourrait donc être un facteur important de la réémergence puis du maintien de la maladie dans les systèmes multi-hôtes.En Côte d’Or, département fortement touché par la tuberculose bovine depuis 2004, la transmission indirecte de la bactérie entre la faune sauvage infectée et les bovins est suspectée dans plusieurs élevages. Pour évaluer la présence et la survie de cette bactérie dans l’environnement, nous avons analysé un grand nombre d’échantillons prélevés dans des zones partagées par les bovins et/ou la faune sauvage infectés dans le but de déterminer la distribution environnementale de M. bovis. Pour ce faire, nous avons développé ou modifié des systèmes de détection moléculaire adaptés aux matrices environnementales complexes. Nous avons également évalué l’impact de la température et des propriétés physico-chimiques de deux sols sur la survie de M. bovis, ainsi que le rôle de la mésofaune du sol (lombrics en particulier) dans la dissémination de la bactérie à partir de matière organique contaminée. L’étude environnementale a mis plus particulièrement en évidence la contamination de deux biotopes: les zones humides des pâtures et les sols de terriers de blaireaux. De plus, les études expérimentales ont montré que M. bovis pouvait survivre plusieurs mois dans le sol à 4°C et que les lombrics pouvaient disséminer la bactérie dans le sol, voire jouer un rôle potentiel de vecteur pour les animaux qui les consomment. Ces résultats apportent de nouvelles connaissances sur la persistance et la circulation de M. bovis dans l’environnement en Côte d’Or et permettront de proposer des améliorations aux mesures de biosécurité déjà existantes dans les élevages bovins. / Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis. This disease affects cattle, and many species of domestic and wild mammals, and humans. The circulation of the bacteria in various multi-host systems promotes the maintenance of the disease and the contamination of cattle in the vicinity. Beside direct transmission of the bacteria through the respiratory route, indirect transmission, through inhalation or ingestion of environmental matrices contaminated by an infected animal excretory, is suspected in several countries. Environmental contamination with M. bovis appears to be a crucial factor in the persistence of the infection in multi-host systems.In Côte d'Or, a French department affected by bovine tuberculosis since 2004, the indirect transmission of the bacteria from infected wildlife to cattle is suspected in several cases. To assess this type of transmission of the bacillus, we evaluated the environmental contamination with M. bovis on a large number of samples taken in areas shared by cattle and / or wildlife infected. For this purpose, we developed or modified molecular detection systems adapted for environmental complex matrices. We also assessed the impact of physicochemical properties of both soil and temperature on survival of M. bovis and the role of earthworms in the spread of the bacteria from contaminated organic material. The environmental study showed the contamination of two media in particular: wetlands pastures and soil badger setts. Moreover, experimental studies have shown that M. bovis can survive in soil for several months at 4 ° C and the worms could spread the bacteria in the soil, or even play a potential role for vector animals that consume them. These results will propose improvements to existing biosecurity measures on cattle farms and provide new knowledge about the persistence and circulation of M. bovis in the environment in Côte d'Or.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Protective immunity against pathogenic mycobacteria depends principally on cell-mediated immunity executed by efficient anti-infectious functions of type 1 T helper (Th1) subset of CD4+ T cells. The polarization of Th1 responses is orchestrated by IL-12 secreted by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). A hallmark of Th1 type CD4+ T cells is the production of IFN-γ that activates plethora of innate cell-mediated immunity. It is well known that cytokines such as IFN-γ, IL-12 and TNF-α are required for control of mycobacterial infection in humans as well as in mice. However, it remains unclear that why the immune response controls mycobacteria, but does not eradicate infection suggesting critical roles for series of survival strategies employed by pathogenic mycobacteria. In general, these evasion strategies include blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, induced secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. that ultimately suppress the secretion of IL-12 and IFN-γ from APCs and T cells respectively, culminating in a skewed Th1/Th2 balance towards unprotective Th2 responses. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 but are deficient in clearing intracellular infections including pathogenic mycobacteria. This eventually leads to inhibition of host’s immuno-protective responses with concomitant increase in the vulnerability to chronic mycobacterial infection. In this intricate process, modulation of cyclooxygenase-2 (COX-2) levels, a key enzyme catalyzing the rate-limiting step in the inducible production of prostaglandin E2 (PGE2), by mycobacteria like Mycobacterium bovis BCG assumes critical importance in influencing the overall host immune response. PGE2, an immunosuppressive member of prostaglandin family, is known to restrain production of IL-12, as well as reactive oxygen intermediates. PGE2-mediated inhibition of IL-12R, diminishes IL-12 responsiveness of macrophages and dendritic cells. PGE2 also inhibits the secretion of IFN-γ, which is important in activating T cells and macrophages. In contrast, PGE2 promotes IL-10 production by macrophages, dendritic cells and Th1-to-Th2 shift of acquired immune responses by inhibiting IL-2 and enhancing IL-4 production. Albeit, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways are generally believed to be involved, little is known about the signaling molecules playing significant roles upstream of MAPK and NF-κB pathways during mycobacteria triggered COX-2 expression. Further, information on early receptor proximal signaling mechanisms essential during mycobacteria mediated induction of COX-2 remains scanty. In this regard, signaling cascade triggered upon recognition of mycobacterial components by pattern recognition receptors (PRR) signify as critical event in overall regulation of cell fate decisions. PRR like Toll like receptor (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are two nonredundant recognition mechanisms of pathogenic mycobacteria. Several components of mycobacteria have been identified as being responsible for TLR2-dependent activation including 19-kDa lipoprotein, lipomannan etc.; while NOD2 recognizes mycobacterial peptidoglycans through its interaction with muramyl dipeptide (MDP). Interestingly, although mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), trehalose 6,6′-dimycolate (TDM; cord factor), PE/PPE family proteins etc., are released and traffic out of the mycobacterial phagosome platform into endocytic compartments. Importantly, these antigens could gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represents a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. Further, PIM2 is a known TLR2 agonist and reported to activate NF-κB, AP-1, and MAPK suggesting that mycobacterial envelope antigen PIM2 could modulate the inflammatory responses similar to mycobacteria bacilli. In this context, we explored the signaling events modulated by M. bovis BCG, and role for TLR2 and NOD2 in this intricate process, to trigger the expression of COX-2 in macrophages. Our studies demonstrated that M. bovis BCG triggered TLR2-dependent signaling leads to COX-2 expression and PGE2 secretion in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or CSF of tuberculosis patients. Similarly, mycobacterial TLR2 agonist PIM2 and NOD2 ligand MDP triggered COX-2 expression in macrophages. The induced COX-2 expression in macrophages either by M. bovis BCG or PIM2 or MDP was dependent on NF-κB activation, which was in turn mediated by iNOS/NO and Wnt-β-Catenin dependent participation of the members of Notch1-PI3K signaling cascade. Importantly, loss of iNOS activity either in iNOS null macrophages or by pharmacological intervention in wild type macrophages severely abrogated M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD) as well as activation of PI3K signaling cascade. On contrary, treatment of macrophages with SIN-1, an NO donor, resulted in a rapid increase in generation of NICD, activation of PI3K pathway as well as the expression of COX-2. Interestingly, pharmacological inhibition as well as siRNA mediated knockdown of Wnt-β-Catenin signaling compromised ability of M. bovis BCG to induce activation of Notch1-PI3K signaling and drive COX-2 expression. Concomitantly, activation of Wnt-β-Catenin signaling by LiCl triggered activation of Notch1 and PI3K pathway as well as COX-2 expression. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbation experiments suggested involvement of the cross-talk of Notch1 with PI3K signaling cascade. In this perspective, we propose TLR2 and NOD2 as two major receptors involved in mycobacteria mediated activation of Notch1PI3K signaling, and the activation of iNOS/NO and Wnt-β-Catenin signaling axis as obligatory early receptor proximal signaling events during mycobacteria induced COX-2 expression in macrophages. Functional characterization of mycobacterial antigens that are potent modulators of host immune responses to pathogens by virtue of induced expression of COX-2 assumes critical importance for deciphering pathogenesis of mycobacterial diseases as well as to identify novel therapeutic targets to combat the disease. In this context, a group of novel antigens carried by M. tuberculosis that are expressed upon infection of macrophages belong to PE and PPE family of proteins. Ten percent of the coding capacity of M. tuberculosis genome is devoted to the PE and PPE gene family members, exemplified by the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many members of the PE family exhibit multiple copies of polymorphic guanine-cytosine– rich sequences (PGRS) at the C-terminal end, which are designated as the PE_PGRS family of proteins. A number of PE/PPE proteins associate with the cell wall and are known to induce strong T & B cell responses in humans. However information related to effects of PE/PPE antigens on the maturation and functions of human dendritic cells and eventual modulation of T cell responses as well as underlying signaling events remains obscure. Our results demonstrated that two cell wall associated/secretory PE_PGRS proteins PE_PGRS 17, PE_PGRS 11 and PPE family protein PPE 34 recognize TLR2, induce maturation and activation of human dendritic cells and enhance the ability of dendritic cells to stimulate CD4+ T cells. In addition, tuberculosis patients were found to have a high frequency of T cells specific to PE_PGRS and PPE antigens. We further found that PE/PPE proteins-mediated activation of dendritic cells involves participation of ERK1/2, p38 MAPK and NF-κB signaling pathways. While, PE_PGRS antigens-matured dendritic cells secreted high amounts of inflammatory cytokine IL-12, PPE 34 triggered maturation of dendritic cells was associated with secretion of high amounts of anti-inflammatory cytokine IL-10 but not the Th1-polarizing cytokine IL-12. Consistent with these results, PPE 34-matured dendritic cells favored secretion of IL-4, IL-5 and IL-10 from CD4+ T cells and contributed to Th2 skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, PPE 34-skewed Th2 immune response involved induced expression of COX-2 in dendritic cells. Our results suggest that by inducing differential maturation and activation of human dendritic cells, PE/PPE proteins could potentially modulate the initiation of host immune responses against mycobacteria. Taken together, our observations clearly signify the potential role for TLR2 and NOD2 triggering by M. bovis BCG in activating receptor proximal Notch1-PI3K signaling during induced COX-2/PGE2 expression which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, differential maturation of human dendritic cells by PE_PGRS and PPE antigens as well as their ability to stimulate CD4+ T cells towards Th1 and Th2 phenotype respectively, improves our understanding about host-mycobacteria interactions and clearly paves a way towards the development of novel combinatorial therapeutics.

La tuberculose bovine causée par Mycobacterium bovis (M. bovis) est une zoonose multi-hôtes. Outre les bovins, elle peut être transmise à des populations sauvages variées dont certaines peuvent entretenir l'infection et/ou la retransmettre aux bovins, pouvant empêcher leur assainissement. Les conditions de persistance de M. bovis dans les populations sauvages ont été étudiées dans différentes régions du monde, mettant en lumière des statuts épidémiologiques variables selon les espèces et les contextes géographiques, cynégétiques et zootechniques. Notre objectif était de déterminer le rôle des différentes populations-hôtes sauvages impliquées dans le système multi-hôtes de M. bovis en Côte d'Or où, en plus des bovins, la tuberculose bovine, circule chez le blaireau, le sanglier, le cerf et le renard. Pour cela, nous avons estimé les niveaux d'infection et d'excrétion, les niveaux de densité, et au moyen d'un suivi télémétrique et de dispositif de vidéosurveillance, nous avons estimé les contacts entre les populations sauvages et les bovins. Nos résultats suggèrent que les trois populations ayant le rôle le plus important et aptes à retransmettre l'infection aux bovins sont le blaireau, le sanglier et le cerf, mais d'autres études sont encore nécessaires pour confirmer ces hypothèses et savoir si certaines d'entre elles peuvent entretenir l'infection de façon autonome. Elles pourraient, également, conjointement, constituer une communauté d'hôtes réservoir. Enfin, nos résultats nous ont permis de caractériser le risque de transmission de tuberculose bovine de ces populations sauvages vers les bovins et de proposer des mesures de lutte visant à réduire ce risque / Bovine tuberculosis, caused by Mycobacterium bovis is a multi-host zoonosis. Besides cattle,it can be transmitted to various wild populations and some of them are able to maintain or to spillback the infection to cattle, thus hampering control strategies. The maintenance of M. bovis by wild populations is dependent on species, on geographical configurations, and on hunting and husbandry practices. Our objective was to investigate the role the different wild populations involved in the M. bovis multi-host system of Côte d’Or, where bTB has been reoccuring in cattle since 2002 and has also been found in badgers, wild boar, red deer and foxes. To do so, we have assessed different risk factors. These include infection rate, ability to 10 shed M. bovis, populations densities and level of indirect contact between wild populations and cattle. For the latter factor, we have tracked 11 wild boars and 10 badgers and used remote surveillance in cattle farms. Our results suggest that, in the study site, badgers, wild boar and red deer may be able to spillback the infection to cattle. Nevertheless, further studies are required to confirm these hypotheses and to investigate whether some of these wild populations can act, individually as reservoirs. It might also be the case that, taken jointly, these wild populations could constitute a maintenance community. On the basis of our results, we made recommandations aiming at reducing the risk of spillback transmission

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies. / AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ʼn poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ʼn bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ʼn nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ʼn filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.

A study was conducted to determine the epidemiological status of bovine tuberculosis in the state of Rio Grande do Sul. The state was divided in seven regions, and in each of them, a pre-established number of farms was randomly sampled. In each farm, cows with age equal to or greater than 24 months were selected at random and submitted to the comparative cervical tuberculin test. The animals whose tests were inconclusive were retested with the same diagnostic procedure within a minimum interval of 60 days. In all, 9,895 animals from 1,067 farms were tested. An epidemiological questionnaire was applied in the farms in order to identify risk factors associated with bovine tuberculosis. The prevalence of infected herds in the state was 2.8% [1.8; 4.0] and that of infected animals was 0.7% [0.4; 1.0]. There was a trend towards a concentration of infected herds in the northern part of the state, with a predominance of dairy and mixed herds. The risk factors associated with the condition of infected herds were being a dairy herd (OR = 2.90 [1.40; 6.13]) and herds with 16 or more cows (OR = 2.61 [1.20; 5.49]). Thus, the best strategy to be adopted by the state is the implementation of surveillance systems to detect and remediate the infected herds, preferably incorporating elements of risk-based surveillance. In addition, the state must carry out a solid action of health education so that the producers test animals for bovine tuberculosis before introducing them in their herds / Um estudo foi realizado para determinar a situação epidemiológica da tuberculose bovina no Estado de Rio Grande do Sul. O Estado foi dividido em sete regiões e em cada uma delas foi aleatoriamente amostrado um número pré-estabelecido de propriedades. Dentro de cada propriedade, fêmeas com idade igual ou superior a 24 meses foram escolhidas aleatoriamente e submetidas ao teste tuberculínico cervical comparativo. Os animais que resultaram inconclusivos foram testados novamente com o mesmo procedimento diagnóstico em intervalo mínimo de 60 dias. Ao todo foram testados 9,895 animais provenientes de 1,067 propriedades. Nas propriedades, foi aplicado um questionário epidemiológico para identificar fatores de risco associados à tuberculose bovina. A prevalência de focos no estado foi de 2.8% [1.8; 4.0] e a de animais 0.7% [0.4; 1.0]. Houve tendência de concentração de focos na parte Norte do estado, caracterizada pelo predomínio de propriedades de leite e mistas Os fatores de risco associados à condição de foco foram exploração leiteira (OR = 2.90 [1.40; 6.13]) e rebanhos com 16 ou mais vacas com pelo menos 24 meses de idade (OR = 2.61 [1.20; 5.49]). Assim, a melhor estratégia a ser adotada pelo estado é a implementação de sistema de vigilância para detecção e saneamento dos focos, de preferência incorporando elementos de vigilância baseada em risco. Além disso, o estado deve realizar uma sólida ação de educação sanitária para que seus produtores passem a testar os animais para tuberculose bovina antes de introduzi-los em seus plantéis

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-25T16:00:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:38Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:46Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T20:58:56Z (GMT). No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O Mycobacterium bovis BCG é utilizado mundialmente como vacina contra tuberculose há mais de meio século, com altos níveis de segurança. O BCG possui características que o tornam um promissor vetor para a produção de vacinas recombinantes multivalentes. Modificações genéticas podem aprimorar as propriedades imunogênicas e adjuvantes do BCG. Uma dessas modificações permite que o BCG escape do endossoma de células apresentadoras de antígenos. Desta maneira, os imunógenos de BCG acessam vias de apresentação cruzada de antígenos, ampliando a sua imunogenicidade. Este trabalho teve como objetivo desenvolver uma cepa de BCG ΔleuD expressando listeriolisina (Hly) de Listeria monocytogenes e avaliar seu potencial citotóxico na linhagem celular 5637 de câncer de bexiga. Para isso, o BCG ΔleuD foi transformado com o plasmídeo recombinante pUP410 contendo o gene hly. A cepa obtida demonstrou um nível de citotoxicidade similar à cepa BCG Pasteur parental, quando avaliada na linhagem celular de câncer de bexiga. Esta cepa necessita ser avaliada em outras linhagens de câncer para comprovar seu possível efeito citotóxico potencializado. / Mycobacterium bovis BCG has been used worldwide as a vaccine against tuberculosis for over fifty years with an outstanding safety record. BCG has features that make it a promising vector for production of recombinant multivalent vaccines. Genetic modification can enhance the adjuvant and immunogenic properties of BCG. One of these modifications would allow BCG to escape the endosome of antigen presenting cells. Therefore, theimmunogens of BCG can access antigens cross priming routes, increasing their immunogenicity. The aim of this work was to develop a strain of BCG ΔleuD expressing listeriolysin (Hly) of Listeria monocytogenes and evaluate its cytotoxic potential on cell line 5637 of bladder cancer. Thus, BCG ΔleuD was transformed with recombinant plasmid pUP410 containing the hly coding sequence. The recombinant BCG strain expressing listeriolysin showed a similar level of cytotoxicity when compared to the parental BCG Pasteur. This strain needs to be evaluated in other cancer lines in order to confirm its possible potentiated cytotoxic effect.

This thesis is about making inference on the host status of feral ferrets in New Zealand for Mycobacterium bovis, the aetiological agent of bovine tuberculosis. The central question addressed is whether the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to persist in ferret populations in the absence of external, non-ferret sources of infection (inter-specific transmission). The question is tackled in three parts�firstly using model selection to identify suitable models for estimating the force of M. bovis infection in ferret populations; secondly applying statistical hypothesis testing to the results of planned manipulative field experiments to test the relationship between M. bovis infection in brushtail possums and that in ferrets; and thirdly using modelling to estimate intra-specific disease transmission rates and the basic reproductive rate (Ro) of M. bovis infection in ferrets. The model selection approach clearly identified the hypothesis of oral infection related to diet was, as modelled by a constant force of infection from the age of weaning, the best approximation of how M. bovis infection was transmitted to ferrets. No other form of transmission (e.g., during fighting, mating, or routine social interaction) was supported in comparison. The force of infection (λ) ranged from 0.14 yr-1 to 5.77 yr-1, and was significantly higher (2.2 times) in male than female ferrets. Statistical hypothesis testing revealed transmission of M. bovis to ferrets occurred from both brushtail possums and ferrets. The force of M. bovis infection in ferrets was reduced by 88% (λ=0.3 yr-1 vs. λ=2.5 yr-1) at sites with reductions in the population density of sympatric brushtail possum populations. A smaller decline in the force of infection resulting from the lethal cross-sectional sampling of the ferret populations was also demonstrated. The modelling approach estimated the basic reproductive rate (Ro) of M. bovis infection in ferrets in New Zealand to vary from 0.17 at the lowest population density (0.5 km-2) recorded to 1.6 at the highest population density (3.4 km-2) recorded. The estimates of Ro were moderately imprecise, with a coefficient of variation of 76%. Despite this imprecision, the Ro for M. bovis infection in ferrets was significantly less than unity for all North Island sites surveyed. Hence it is inferred ferrets are spillover hosts (0<Ro<1) for M. bovis infection in these environments. That is, M. bovis infection will progressively disappear from these ferret populations if the source of inter-specific transmission is eliminated. The estimates of Ro for M. bovis infection in South Island ferret populations were above one (the level required for disease establishment) for a number (5/10) of populations, though the imprecision made it impossible to ascertain whether Ro was significantly greater than one. The estimated threshold population density (Kt) for disease establishment was 2.9 ferrets km-2. It is inferred that, given sufficient population density (>Kt), the rate of intra-specific transmission of M. bovis among ferrets is sufficient for the disease to establish in ferrets in the absence of interspecific transmission. In these areas, ferrets would be considered maintenance hosts for the disease. Active management (e.g., density reduction or vaccination) of ferrets would be required to eradicate M. bovis from ferret populations in these areas, in addition to the elimination of sources of inter-specific transmission, particularly brushtail possums.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "December 2000." Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 114-120). Also available on the Internet.