O Fator de Necrose Tumoral-alfa (TNF-alfa) possui um importante papel na imunopatogênese da psoríase e agentes biológicos, como os inibidores de TNF-alfa, têm apresentado bons resultados no tratamento desta. No entanto, estes agentes foram associados ao aumento de casos de reativação de tuberculose entre os pacientes que os utilizaram. Este estudo foi realizado com o intuito de avaliar a resposta imune de pacientes com psoríase grave, ativa, sem tratamento, frente a antígenos de Mycobacterium tuberculosis (Mtb), e o efeito dos inibidores de TNF-alfa nesta resposta. Estudamos 24 pacientes com psoríase grave divididos em 2 grupos: não reatores (n = 14) e reatores (n = 10) ao teste intradérmico com PPD. Como controle, utilizamos um total de 26 indivíduos sadios, também separados em 2 grupos segundo a reatividade ao PPD (PPD-, n = 13; PPD+, n = 13). Em uma segunda etapa estudamos 11 pacientes com psoríase leve a moderada, também sem tratamento, PPD (-) para avaliarmos a importância da gravidade da psoríase na resposta aos antígenos micobacterianos. Avaliamos a resposta imunológica in vitro através da linfoproliferação, quantificação da produção de IFN-gama (ELISA) e quantificação de células produtoras de IFN-gama (ELISPOT), na presença e ausência dos inibidores de TNF-alfa (infliximab e etanercepte), utilizando os antígenos purificados ESAT-6, Ag85B e o antígeno bruto sonicado da cepa H37Rv (AgSMtb), e o mitógeno fitohemaglutinina (PHA). Os pacientes com psoríase grave PPD (-) apresentaram reposta linfoproliferativa e níveis de IFN-gama menores que nos controles PPD (-). Os pacientes com psoríase leve a moderada apresentaram resposta imune intermediária entre controles e pacientes graves. Em relação aos inibidores de TNF- alfa, verificou-se que infliximab e etanercepte apresentaram diferença em suas capacidades de inibição, sendo que somente o infliximab ocasionou a inibição total de TNF-alfa. Em contrapartida o etanercept manteve a produção de TNF-alfa, e em alguns casos elevou sua produção. Estes diminuíram apenas parcialmente a reatividade in vitro dos pacientes com psoríase, uma vez que a secreção de IFN-gama e o número de células produtoras de IFN-gama não foram alterados na presença dos inibidores. A secreção de IL-10 foi diminuída tanto na presença do infliximab, quanto na presença do etanercepte. Os dados obtidos permitem concluir que (a) os pacientes com psoríase grave PPD (-) apresentam uma baixa reatividade in vitro, principalmente das respostas que avaliam linfócitos T de memória central, aos antígenos de Mtb, sendo que essa baixa reatividade não está totalmente relacionada com a gravidade da doença, uma vez que os pacientes com psoríase leve a moderada apresentaram resposta intermediária a dos controles e pacientes com psoríase grave; (b) e que apesar dos inibidores de TNF- alfa promoverem uma inibição parcial da resposta imune, a reativação da tuberculose estaria mais relacionada à própria ausência de TNF-alfa, não compensada pela atuação isolada, e provavelmente insuficiente, de IFN-gama na manutenção do granuloma, do que a outras substanciais modificações na resposta imunológica frente aos antígenos micobacterianos. / Tumor necrosis factor alpha (TNF-alpha) has a pivotal role in psoriasis pathogenesis and biologic agents, such as TNF-alpha inhibitors, have provided good results in its treatment. However, the use of these agents has been associated with an increase in the number of cases of tuberculosis reactivation. This study aimed to evaluate the immune response of severe psoriasis patients, with active, untreated disease to relevant Mycobcterium tuberculosis antigens, and the effect of the TNF-alpha inhibitors (infliximab and etanercept) in this response. Twenty four severe psoriasis patients were enrolled and divided in two groups according to their reactivity to the tuberculin skin test: TST (n= 14) and TST + (n=10). As controls, we studied 26 healthy donors, also divided in two groups to the TST reactivity (TST -, n=13; TST+, n=13). Eleven mild to moderate psoriasis patients, untreated, TST (-) were studied to evaluated the role of psoriasis severity in the immune response to the mycobacterial antigens. Immune responses were evaluated in vitro by the lymphocyte proliferative response (LPR) assay, ELISA for IFN-? secretion by peripheral blood mononuclear cells and enumeration of IFN-? secreted cells (ELISPOT) induced in response to the purified antigens ESAT-6, Ag85B and a crude sonicated antigen preparation from H37Rv Mtb strain (AgSMtb), as well as to the mitogen phytohemagglutinin (PHA), in the presence or absenceinflimab/etanercept. The LPR and IFN-g secretion to Mtb antigens were lower in TST- severe psoriasis patients than TST- controls. Mild to moderate psoriasis patients had intermediate responses, between controls and severe psoriasis patients. The TNF-a inhibitors infliximab and etanercept showed differences in their inhibitiory activity, since only infliximab was capable to neutralize all TNF-a. On the other hand, etanercept kept TNF-alpha production, and in some cases even increased its production. The TNF-alpha inhibitors diminished partially the in vitro patients immune responses, since the IFN-? secretion and enumeration of IFN-? secreted cells were not affected. IL-10 secretion was diminished with both TNF-a inhibitors. In conclusion: (a) TST(-) severe psoriasis patients have decreased in vitro reactivity, mainly in those responses that evaluate central memory T-cell responses, to Mtb antigens, and this decrease could not be fully explained by disease severity, since mild psotiasis patients had intermediate responses; (b) and despite the fact that TNF-alpha inhibitors promote a partial immune response inhibition, tuberculosis reactivation could be related more with the lack of TNF-alpha, which was probably not compensated by the IFN-g activity alone, probably insufficient, to the support granuloma formation, than other defects of the immune response to Mtb antigens.

Realizou-se estudo comparando o uso da reação em cadeia da polimerase à evolução clínica e painel de exames tradicionais para diagnóstico em pacientes com suspeita clínica de tuberculose cutânea e micobacteriose atípica. Observou-se sensibilidade da reação em cadeia da polimerase de 88%, especificidade de 83%, valor preditivo positivo de 82%, valor preditivo negativo de 88% e acurácia de 85% com concordância pelo teste de McNemar (p= 0,655). Os exames do painel de maior acurácia, após a reação em cadeia da polimerase, foram o teste tuberculínico com acurácia de 79% e a presença de dermatite crônica granulomatosa com reação em cadeia da polimerase positiva com acurácia também de 79%, ambos com concordância pelo teste de McNemar (p= 0,179 e p= 0,655, respectivamente) / A study was performed comparing the polymerase chain reaction and the traditional panel of exams for the diagnosis in patients with a clinical suspicion of cutaneous tuberculosis and atypical mycobacteria infection to the clinical evaluation. It was observed that the sensitivity of the PCR was 88%, the specificity was 83%, the positive predictive value was 82%, the negative predictive value was 88% and the accuracy was 85% in agreement with the McNemar test (p=0.655). The panel exams of second highest accuracy, were the tuberculin test with an accuracy of 79% and the chronic granulomatous dermatitis with positive PCR, also with an accuracy of 79%, both in agreement with the McNemar test (p=0.179 and p=0.655, respectively)

FtsZ, the principal cell-division protein, polymerizes in GTP-dependent manner in vitro (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994; Rivas et al., 2000). FtsZ polymerization at the mid-cell site of bacterium leads to formation of a guiding scaffold, the Z-ring, for bacterial cytokinesis (Bi and Lutkenhaus, 1991; Sun and Margolin, 1998). GTP-induced polymerization process of FtsZ can be monitored in vitro Using 90º light scattering (Mukherjee and Lutkenhaus, 1999) and polymers formed can be visualized using transmission electron microscopy (Lu and Erickson, 1998) or quntitated in terms of the amount of FtsZ polymer pelleted during ultracentrifugation (Mukherjee and Lutkenhaus, 1998). The research work presented in this thesis focused on structure-function correlative analysis of Mycobacterium tuberculosis FtsZ(MtFtsZ0 and FtsZ proteins of Mycobacterium leprae (M1FtsZ), Mycobacterium smegmatis(MsFtsZ), and Streptomyces coelicolor (ScFtsZ) (as it is from Actinomycetes family to which mycobacteria belong) in vitro. It was initiated with investigation on the biochemical properties of Mycobacterium leprae FtsZ (M1FtsZ) in vitro. In comparison with those of MtFtsZ. Subsequently, the role of C-terminal stretch of amino acid residues of MtFtsZ in polymerization was investigated. Finally, a comparative analysis of the biochemical properties of MtFtsZ, MsFtsZ, and ScFtsZ was carried out in order to find out whether a correlation exists between the time taken by the FtsZ of a bacterium to polymerise and the generation time of the organism. The thesis is presented in five chapters. First Chapter gives an exhaustive introduction on the structure-function aspects of FtsZ. Second Chapter deals with materials used in this research work and details of various experimental methods [cloning and expression of FtsZ (White et. Al., 2000), decision and point mutagenesis, preparation of His-tag free MtFtsZ and M1FtsZ by thrombin cleavage method, 90º light scattering (Mukherjee and Lutkenhaus, 1999), White, et al., 2000), transmission electron microscopy (Lu and Erickson, 1998), pelleting assay for polymeric FtsZ (Mukherjee and Lutkenhaus, 1998), GTP-binding by UV-crosslinking (RayChaudhuri and Park, 1992; de Boer et al.,) GTPase assay(RayChaudhuri and Park, 1992); de Boer et al., 1992), Circular Dichroism (Saxena and Wetlaufer, 1971) and ANS fluorescence emission spectroscopy (Semisotnov, et al., 1991)]. The Chapters three to five contain all the data related to the research work, the outlines of which are given below. Chapter 3. Biochemical Characterisation of FtsZ Protein of Mycobacterium leprae In Comparison with the Biochemical Properties of FtsZ Protein of Mycobacteriulm tulberculosis In Vitro The major finding in this part of thesis work is on the demonstration that single reciprocal point mutation partially revives polymerization-inactive M1FtsZ and Inactivates polymerization-active MtFtsZ in vitro. In brief, soluble, recombinant M1FtsZ did not show detectable polymerization in vitro, in contrast to MtFtsZ, which showed appreciable polymerization, under standard conditions, when monitored using 90º light scattering assay and transmission electron microscopy. This was a surprising result, as M1FtsZ and MtFtsZ has 96% protein sequence identity. Mutation f T172 in the N-terminal domain of M1FtsZ to A172, as it exists in MtFtsZ, showed dramatic levels of polymerization in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in M1FtsZ, abolished polymerization in vitro. Further, M1FtsZ showed weak GTPase activity, in contrast to MtFtsZ, which showed appreciable GTPase activity. While T172A mutation enhanced GTPase activity of MtFtsZ in vitro. Circular dichroism spectroscopy and ANS fluorescence emission spectroscopy showed that there were no major secondary or tertiary structural changes in these point mutants. These observations demonstrate that the residue at position 172 plays a critical role in the polymerization of M1FtsZ and MtFtsZ, without appreciably affecting their respective GTpPase activity. Further, this result might have implications on evolution of a slow polymerizing FtsZ in slow growing bacteria. Further details of evolution related questions are addressed in Chapter 5. Chapter 4. Role of Carboxy Terminal Residues in the Biochemical Properties of FtsZ Protein of Mycobacterium tuberculosis In Vitro The major finding in this part of thesis work is the demonstration that the C-terminal end residues are critically required for polymerization of MtFtsZ in vitro, which is in direct contrast to the dispensability of C-terminal residues of Escherichia coli FtsZ(EcFtsZ), Bacillus subtilis FtsZ (BsFtsZ), and Pseudomonas aeruginosa (PaFtsZ) for polymerization. FtsZ protein from several bacterial species namely, Methanococcus jannaschii (MjFtsZ), Bacillus subtillis(BsFtsZ), Pseudomonas aeruginosa (PaFtsZ), and Aquifex aeolicus (AaFtsZ) (Lowe and Amos, 1998; Oliva et al., 2007), and Mycobacterium tuberculosis H37Rv (mtFtsZl Leung et al., 2004), whose crystal structures have been solved so far, were found to possess an N-terminal domain and a C-terminal domain that were connected to each other through a helix. The extreme C-terminal portion of all these FtsZ proteins is constituted by an unstructured tail (Lowe and Amos, 1998; Oliva et al., 2007l Leung et al., 2004), which is not found in the respective crystal structure of the protein. We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (mtFtsZ) have any role in MtFtsZ polymerization in vitro. Deletion of C-terminal 66 residues (313-379) was found to abolish polymerization. Replacement of the C-terminal 66 residues with the extreme C-terminal 13-residue stretch (DDDDVDVPPFMRR) did not restore polymerization. Although the terminal R in DDDDVDVPPFMRR is dispensable for full-length MtFtsZ polymerization, the terminal R in DDDDVDVPPFMR is indispensable for polymerization. Neither replacement of this R, in the terminal R deletion mutant DDDDVDVPPFMR, with K/H/D/A residues enabled polymerization. GTP binding and GTPase activities of the mutants were partially affected. The indispensable nature of C-terminal residues for MtFtsZ polymerization in vitro is contrary to the dispensability of the equivalent extreme C-terminal residues of Escherichi coli, Pseudomonas aeruginosa, and Bacillus subtilis FtsZ (Wang et. Al., 1997; Cordell et al., 2003; Singh et al., 2007) for in vitro polymerization. The essentiality of C-terminal extreme residues of BtFtsZ for polymerization offers direction to design anti MtFtsZ polymerization agents. Chapter 5. An attempt to find correlation between Biochemical properties of FtsZ and Generation Time of the Bacterium The clue that there might be a correlation between FtsZ polymeristion and generation time of the bacterium came from the observation mentioned in chapter 3. The presence of polymerization-aversive T172 in the FtsZ of extremely slow-growing M. leprae 913.5 days generation time, Levy, 1970) and polymerization-favouring A172 in the FtsZ of M. tuberculosis(18hrs generation time, Patterson and Youmans, 1970). For a bacterium, which has short generation time, it might be conducive to have an FtsZ that will also polymerise fast. Conversely, for a bacterium, which has long generation, it might be conducive to have an FtsZ molecule that will polymerise slow. In this respect, a preliminary comparative study was carried out between the generation time of bacterial species, E. coli, Mycobacterium smegmatis, Streptomyces coelicolor, M leprae, and M. tuhberculosis and their respective FtsZ (EcFtsZ, MsFtsZ, M1FtsZ and MtFtsZ). Detailed biochemical characterization of EcFtsZ and MtFtsZ has already been reported in the literature. In this thesis work, biochemical characterisation of M1FtsZ(Chapter 3), ScFtsZ and MsFtsZ (in this Chapter) were carried out. E. coli, which has a generation time of 18-55 min(labrum, 1953), possesses FtsZ (EcFtsZ) that reaches steady state of polymerization in about 10 sec under standard conditions in vitro (Beamhill and Thompson, 1994), using 90º light scattering assay (Mukherjee and Lukenhaus, 1999). On the other hand, M. tuberculosis, which has a generation time of 18hrs in vivo (Patterson and Youmans, 1970) and 24 hrs in vitro (Hiriyanna and Ramakrishnan, 1986) possesses FtsZ (MtFtsZ) that reaches steady state of polymerization in about 6 min post-addiction of GTP in vitro (White et al., 2000). Further, M. leprae, which takes 13.5 days tp divide once in vivo (levy, 1970), possesses an FtsZ (M1FtsZ) that does not even show polymerization under standard conditions in vitro (Chapter 3 of this thesis). The organisms Mycobacterium smegmatis and Streptomyces coelicolor have generation times that fall in between those of the other three organisms mentioned above. While M. smegmatis divides once in 2-3 hrs (Husson, 1998), S. coelicolor has a variable generation time depending on growth condition, which can be as fast as once in 2.31 hours, depending upon growth conditions (Cox, 2004). We found ScFtsZ and MsFtsZ takes around 4 min to reach polymerization saturation after addition of GTP, EcFtsZ( 10 sec), MtFtsZ (10 min) and M1FtsZ (dose not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation after addition of GTP, EcFtsZ (10sec), MtFtsZ (10 min) and M1FtsZ (does not polymerise in vitro) seem to indicate that there exists a correlation between polymerization saturation time and the generation time of the respective bacterium. But when we compared polymerization time of ScFtsZ and MsFtsZ (4 min both case) with MtFtsZ ( 6 min), we found that there is no linear correlation with generation time of these bacteria and the time taken by their FtsZ to reach steady state of polymerization. Many more bacterial FtsZ proteins need to be characterized to conclusively state wthether there exist a correlation between generation time of bacteria and the time taken for their FtsZ to reach steady state of polymeristion. Such correlation would simply reveal the fact that the primary structure of an FtsZ protein might have evolved to suit the generation time of the bacterium.

Made available in DSpace on 2016-12-23T13:56:10Z (GMT). No. of bitstreams: 1 Dissertacao de Cristina Paula do Nascimento.pdf: 967221 bytes, checksum: 66ef634f97ef2bec6f402d2a4a644c7f (MD5) Previous issue date: 2011-04-26 / Em ensaios clínicos para avaliação de novos fármacos contra a tuberculose a principal metodologia utilizada é a avaliação da atividade bactericida precoce (ABP), que consiste em quantificar a queda da carga bacilar presente na amostra de escarro de pacientes com tuberculose (TB), por meio de coletas noturnas com duração de 12 a 16 horas, durante os dois primeiros dias de tratamento. Contudo, o procedimento de coleta de escarro por um período de 12 a 16 horas apresenta vários inconvenientes. Em virtude desses, nos propusemos a desenvolver e avaliar um procedimento de coleta de escarro mais simples, com menor tempo, que não requeira a internação do paciente e que apresente a mesma eficiência de um procedimento por 12 horas. Para esse propósito foram realizadas culturas quantitativas de escarro, para mensurar a carga bacilar de diferentes procedimentos de coletas: 5 horas e 12 horas. Assim este trabalho foi dividido em duas etapas. Na primeira etapa ao comparar-se as cargas bacilares de escarro provenientes de coletas de 5 (6,17 log10UFC/mL) ou 12 horas (6,23 log10UFC/mL), não houve diferença estatística significante (p=0,27). Para a segunda etapa os pacientes foram submetidos a 4 procedimentos de coletas em dias consecutivos: 2 coletas por 5 horas e 2 coletas por um período de 12 horas para se analisar a variação de carga bacilar intra e interpaciente. A variação de carga bacilar intra-paciente dos procedimentos de 5 horas (0,037 Log10UFC/mL) e 12 horas (- 0,022 Log10UFC/mL), não apresentou diferença estatística significante entre os procedimentos (p=0,56). Além disso, a coleta matinal permitiu reduzir a variação inter-paciente e melhorar a precisão de estimativa da média de variação de carga bacilar de um dia de coleta para o outro (EP 5 horas =0,019 e EP 12 horas =0,028). Dessa forma, demonstrou-se que uma coleta de escarro por 5 horas apresenta uma população de Mycobacterium tuberculosis tão representativa quanto à proporcionada pela coleta com duração de 12 horas. Portanto, o procedimento de escarro por 5 horas pode ser utilizado em ensaios clínicos de avaliação de ABP em substituição a coleta de 12 horas / In clinical trials studies on new drugs against tuberculosis, the main methodology used is the evaluation of the early bactericidal activity (EBA), which quantifies the decrease of CFU of Mycobacterium tuberculosis in sputum samples during the first 2 days of treatment, through 12 to 16 hours of nocturnal sputum collections. However, the sputum collection for a period of 12 to 16 hours has several shortcomings. In this context, we proposed to develop and evaluate a simple, less time-consuming procedure for sputum collection that does not require hospitalization and presents the same efficiency as a 12-hour collection procedure. We performed quantitative cultures of sputum to measure the bacterial load after three different procedures: 5-hour and 12-hour sputum collections. So this study was divided into two stages. In the first step, the bacillary loads observed in sputum were compared to those obtained after a 5-hour (6,17 log10CFU/mL) and 12 hour (6,23 log10CFU/mL) collection, and there was no statistically significant difference (p =0,27). For the second stage, the patients underwent 4 procedures for sputum collection on consecutive days - two 5-hour collections and two 12-hour collections, in order to analyze the bacterial load variation intra and inter-patients. When the intra-patient variation in the bacillary load was compared between the procedures lasting 5 hours (0,037 Log10CFU/mL) and 12 hours (- 0,022 Log10CFU/mL), no statistically significant difference was observed (p = 0,56). Moreover, the morning collection has reduced the inter-patient variation and improved the accuracy for estimation of the average change in bacterial load from a collection of one day to the other (standard error for 5-hour =0,019 and standard error for 12-hour =0,028). These results show that a 5-hour morning sputum collection is as representative as that provided by a 12-hour overnight collection. In conclusion, the simplified procedure of sputum collection for a period of 5 hours proposed by this study could be used in clinical assessment of EBA as an alternative to 12-hour collections, with no impairment to the EBA evaluation of the study drug

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST &#8805; 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-&#947; e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-&#947; e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Depuis peu, un intérêt croissant est manifesté pour l’étude de la tuberculose pédiatrique ;les enfants constituent, en effet, une grande partie du réservoir des futurs cas de tuberculose active (TBA) maladie. Selon les estimations de l’Organisation Mondiale de la Santé (OMS), ce réservoir est constitué par 2 milliards de personnes porteuses d’une forme latente de tuberculose (LTBI). La LTBI est définie par l’existence d’une réponse immunitaire adaptative aux antigènes de Mycobacterium tuberculosis (M.tuberculosis) en l’absence de signes cliniques de maladie. A partir de ce réservoir, 8,7 millions de nouveaux cas de TBA et 1,4 millions de décès liés à ces TBA ont été déclarés par l’OMS en 2011. On estime que la TBA pédiatrique, dont l’ampleur réelle est probablement sous-estimée, représente 9 à 12 % de tous les cas incidents. <p>Une meilleure coordination des efforts et leur augmentation à l’échelle mondiale, au cours de ces dernières années, a permis de réduire l’incidence de la TB en diagnostiquant, traitant rapidement et plus efficacement les TBA. Cette stratégie est insuffisante si l’objectif, comme défini par l’OMS à l’horizon de 2050, est d’éradiquer la TB. Il faudrait, pour cela, développer de nouvelles stratégies vaccinales pour améliorer l’efficacité du bacille Calmette et Guérin (BCG), mais également s’attaquer au réservoir de M. tuberculosis. <p>Le seul vaccin disponible, le BCG, confère une protection variable et souvent faible contre la TB pulmonaire. La stratégie, dans le développement de vaccins contre la tuberculose, est de mimer les réponses immunitaires naturelles développées par les sujets avec une LTBI puisque 90% d’entre eux sont définitivement protégés contre la maladie. Il apparaît donc important de caractériser ces réponses de l’immunité adaptative qui apparaissent lors d’une primo-infection par M. tuberculosis (avant l’état de latence) chez l’homme. L’étude de ces réponses immunitaires primaires est difficilement réalisable chez les adultes car, chez la majorité d’entre eux, les cas de TBA résultent soit d’une réactivation, soit d’une réinfection. Dans un pays à faible prévalence comme la Belgique où, par ailleurs, la vaccination par le BCG n’est pas administrée en routine, l’étude d’un « groupe modèle » constitué d’enfants exposés à M. tuberculosis est une opportunité inespérée pour caractériser la réponse immunitaire primaire spécifique. La comparaison des réponses immunitaires de primo-infection aux réponses immunitaires secondaires (enfants exposés à M. tuberculosis et précédemment vaccinés par le BCG) présente également un intérêt pour l’établissement de nouvelles stratégies vaccinales telles que le « prime-boost ». De plus, les approches vaccinales actuelles incluent préférentiellement les antigènes de la phase réplicative alors que l’ajout d’un antigène de latence pourrait induire une réponse immunitaire plus protectrice et plus complète.<p>Les réponses de l’immunité adaptative primaires ou secondaires des jeunes enfants dirigées contre les antigènes de M. tuberculosis sont relativement peu caractérisées si bien que les facteurs responsables de la grande susceptibilité des enfants à développer une infection sévère sont loin d’être complétement identifiés. Par ailleurs, au vu des difficultés diagnostiques de l’infection par M. tuberculosis chez l’enfant et, en particulier, de différentier les TBA des formes latentes, de nombreuses recherches sont menées pour tenter d’identifier des biomarqueurs qui pourraient aider à séparer ces deux phénotypes. En effet, la TBA pédiatrique est pauci-bacillaire, ce qui rend les moyens diagnostiques classiques comme la microbiologie et/ou la biologie moléculaire, tous deux dépendant de la charge mycobactérienne, peu sensibles. De plus, le diagnostic de la LTBI par le test cutané à la tuberculine (TCT)et les tests in vitro aujourd’hui commercialisés mesurant les sécrétions d’interféron-gamma (IFN-γ), les IGRA, ne sont pas suffisamment sensibles et spécifiques, surtout chez les enfants de moins de 5 ans.<p>L’étude des réponses immunitaires induites par la « Heparin-Binding Haemagglutinin » (HBHA) a été menée dans l’objectif d’améliorer la performance des techniques de laboratoire. La HBHA est une adhésine exprimée par le complexe M. tuberculosis. Elle est impliquée dans la dissémination extrapulmonaire du bacille et constitue donc un facteur de virulence. Par ailleurs, dans un modèle murin, le caractère protecteur d’une vaccination avec la HBHA contre l’infection par M. tuberculosis a également pu être démontré ;l’efficacité était comparable à celle du vaccin BCG. Si l’effet d’une telle vaccination n’a pas encore été évalué chez l’homme, l’immunogénicité de cet antigène a, par contre, pu être établie en clinique humaine. En effet, les lymphocytes circulants, isolés chez des adultes infectés par M. tuberculosis de façon latente, sécrètent plus d’IFN-γ en réponse à la HBHA que les lymphocytes des patients présen-tant une TBA non traitée. La HBHA est donc un antigène de latence puisqu’il induit une réponse immunitaire principalement pendant cette phase. De plus, la HBHA est une protéine méthylée et la méthylation s’avère essentielle pour garantir ses propriétés immuno-protectrices. <p>Nos travaux ont été orientés selon deux axes :d’une part, l’analyse des réponses immunitaires primaires et secondaires dirigées contre la HBHA comparées à celles induites par des antigènes spécifiques de M.tuberculosis connus et, d’autre part, l’exploitation de la réponse immunitaire à la HBHA ainsi qu’à d’autres antigènes spécifiques de M. tuberculosis dans le but de définir des biomarqueurs de susceptibilité et proposer de nouveaux moyens immuno-diagnostiques. <p>L’évaluation de la réponse immunitaire primaire, chez les enfants de moins de 5 ans et non vaccinés par le BCG, aux antigènes mycobactériens spécifiques démontre que la réponse IFN-γ primaire induite par la HBHA est peu élevée quantitativement chez les enfants infectés par M. tuberculosis contrairement aux réponses IFN-γ induites par les antigènes de la phase réplicative, l’ESAT-6 et le CFP-10. Cette faiblesse de la réponse immunitaire, de type TH1, vis–à-vis de la HBHA, n’est pas due à une immaturité du système immunitaire des jeunes enfants ni à un shift des réponses vers un profil cytokinique de type TH2. Elle reflète pro-bablement le phénotype clinique de l’infection affiché par ces jeunes enfants récemment infectés par M. tuberculosis qui ne sont pas encore parvenus au stade de latence. Cette faible réponse pourrait, néanmoins, constituer un biomarqueur de susceptibilité accrue à la TB des jeunes enfants. De plus, si la réponse primaire HBHA-spécifique n’est quantitativement pas différente de celle des enfants présentent une TBA ou une LTBI, elle l’est cependant qualitativement si l’on compare ces deux phénotypes cliniques. En effet, seuls les enfants «protégés» (avec une LTBI ou une TBA traitée) présentent des réponses envers des épitopes protecteurs de la forme méthylée de la HBHA. La mise en évidence de réponses contre ces épitopes de la forme méthylée pourrait donc être évaluée en tant que biomarqueur de protection. Parallèlement, l’absence ou la présence d’une telle réponse dirigée contre les épitopes de cette forme méthylée de la HBHA pourrait également permettre de différentier un enfant avec une TBA d’un enfant avec une LTBI. <p>La caractérisation de la réponse immunitaire se-condaire à la HBHA, chez les enfants infectés par M. tuberculosis et préalablement vaccinés par le BCG, a permis de démontrer que cette réponse est quantitativement plus importante si on la compare aux réponses de type primaire. <p>Puisque le caractère protecteur du BCG adminis-tré à la naissance est bien reconnu, il semblerait logique d’utiliser en priorité la HBHA dans une approche vaccinale «prime-boost », c’est-à-dire une amplification par la HBHA d’une immunité «protectrice» déjà acquise lors de l’administration du vaccin BCG dans l’enfance.<p>L’exploitation de l’analyse des réponses immuni-taires induites par les antigènes spécifiques de M. tuberculosis, l’ESAT-6, le CFP-10 et la HBHA, a ensuite permis d’établir que la réponse IFN-γ induite par la HBHA est faible chez l’enfant et non discriminante entre une TBA et une LTBI, ce qui limite l’intérêt diagnostique de la HBHA chez l’enfant contrairement à ce qui a pu être observé chez les adultes. Par contre, les résultats des tests IGRA à longue durée d’incubation utilisant l’ESAT-6 et surtout le CFP-10 comme antigènes, peuvent apporter des arguments en faveur d’une infection par M. tuberculosis chez l’enfant et être ainsi intégrés dans la triade diagnostique utilisée par les pédiatres. En effet, la mise en évidence d’une réponse IFN-γ induite par le CFP-10 très élevée est plus souvent associée à une TBA et, en combinaison avec le TCT ou la PPD-IGRA, elle a permis d’identifier, au sein du groupe d’enfants que nous avons étudié, ceux qui présentaient une TBA. <p>Les tests immuno-diagnostiques in vitro et in vivo manquent de sensibilité pour le diagnostic des TBA extra-pulmonaires. Dans ces cas particuliers, l’analyse de la réponse IFN-γ par les lymphocytes isolés directement au niveau du site d’infection apparait prometteuse chez l’enfant. Parmi les TB extra-pulmonaires, l’adénite tuberculeuse est une présentation fréquente difficile à différentier cliniquement ou radiologiquement d’une lymphadénopathie causée par une autre mycobactérie non tuberculeuse comme M.avium. Souvent, le recours à une technique d’investigation plus invasive (biopsie) est nécessaire. Nous avons développé au sein du laboratoire des tests in vitro comparant la réponse IFN-γ induite par la PPD-tuberculosis et la PPD-avium. Le ratio entre ces deux réponses permet d’orienter le clinicien dans le diagnostic différentiel entre des infections induites par l’une ou l’autre de ces deux mycobactéries et permettra peut-être à l’avenir de ne pas imposer aux patients la pratique de manœuvres chirurgicales.<p><p>En conclusion, ce travail confirme que la suscepti-bilité des jeunes enfants aux formes sévères de tuberculose n’est pas la conséquence d’un « état d’immunodéficience ». Par ailleurs, ils sont ca-pables de développer une réponse immunitaire contre la HBHA, candidat pour le développement d’un nouveau vaccin ou d’une nouvelle stratégie vaccinale contre la tuberculose. Enfin, nous avons identifié de nouveaux biomarqueurs susceptibles d’aider les pédiatres à diagnostiquer et à différencier les différentes formes que peut prendre l’infection tuberculeuse chez leurs jeunes patients. <p> / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Realizou-se estudo comparando o uso da reação em cadeia da polimerase à evolução clínica e painel de exames tradicionais para diagnóstico em pacientes com suspeita clínica de tuberculose cutânea e micobacteriose atípica. Observou-se sensibilidade da reação em cadeia da polimerase de 88%, especificidade de 83%, valor preditivo positivo de 82%, valor preditivo negativo de 88% e acurácia de 85% com concordância pelo teste de McNemar (p= 0,655). Os exames do painel de maior acurácia, após a reação em cadeia da polimerase, foram o teste tuberculínico com acurácia de 79% e a presença de dermatite crônica granulomatosa com reação em cadeia da polimerase positiva com acurácia também de 79%, ambos com concordância pelo teste de McNemar (p= 0,179 e p= 0,655, respectivamente) / A study was performed comparing the polymerase chain reaction and the traditional panel of exams for the diagnosis in patients with a clinical suspicion of cutaneous tuberculosis and atypical mycobacteria infection to the clinical evaluation. It was observed that the sensitivity of the PCR was 88%, the specificity was 83%, the positive predictive value was 82%, the negative predictive value was 88% and the accuracy was 85% in agreement with the McNemar test (p=0.655). The panel exams of second highest accuracy, were the tuberculin test with an accuracy of 79% and the chronic granulomatous dermatitis with positive PCR, also with an accuracy of 79%, both in agreement with the McNemar test (p=0.179 and p=0.655, respectively)

A doença infecciosa que causa o maior número de mortes no mundo é a tuberculose, causada pelo bacilo Mycobacterium tuberculosis. Um dos fatores de risco que aumenta três vezes o desenvolvimento de tuberculose é a diabetes, sendo a obesidade associada com predisposição à diabetes. A obesidade gera inflamação de baixo grau que agrava a progressão de doenças crônicas. Estudos que avaliaram a associação da obesidade com tuberculose são controversos, e o tema merece maior investigação. No presente estudo, usamos um modelo experimental para determinar a interface da obesidade e da tuberculose. Camundongos C57BL/6 foram alimentados com dieta hiperlipídica (HFD - High Fat Diet) durante 60 dias, quando foram infectados com M. tuberculosis (HFD/Mtb) por via intra-traqueal. Como controles experimentais, animais foram alimentados com dieta padrão (LFD - Low Fat Diet) e infectados (LFD/Mtb). Paralelamente, um grupo recebeu HFD e outro LFD, e seguiram sem infecção. Após 30 dias de infecção, totalizando 90 dias de dieta, os diferentes grupos foram avaliados. Os animais obesos e infectados (HFD/Mtb) apresentaram aumento do peso corporal e do peso dos tecidos adiposos, aumento da expressão gênica de IL-1? no tecido adiposo, intolerância à glicose, deficiência na produção de insulina e aumento dos níveis séricos de IFN-? comparados aos animais LFD/Mtb. Além disso, o grupo HFD/Mtb foi mais suscetível e apresentou maior inflamação pulmonar comparado ao grupo LFD/Mtb. A inflamação foi caracterizada por aumento na expressão gênica para IL-17, IFN-?, TNF, IL-1?, IL-1?, NLRP3, caspase-1, IL-18, IL-6, aumento de células CD4+ produtoras de IFN-? e/ou IL-17 nos pulmões, e foi também acompanhada por aumento de células CD8+ e células CD4+Foxp3+ quando comparado ao grupo LFD/Mtb. Como NLRP3 é uma molécula chave na metainflamação induzida pela obesidade, mas seu papel ainda não está bem definido na tuberculose, animais deficientes de NLRP3 receberam HFD e foram infectados (NLRP3-/- HFD/Mtb). Esse grupo NLRP3-/- HFD/Mtb foi mais resistente e exibiu redução da inflamação pulmonar comparado ao grupo WT (Wild Type) HFD/Mtb. Sabendo que a obesidade está associada à disbiose e que produtos bacterianos derivados da dieta alimentar ou da microbiota podem estimular a liberação de IL-1? pela ativação de NLRP3, avaliamos o papel da microbiota na comorbidade obesidade e tuberculose. Encontramos disbiose intestinal, caracterizada por aumento do Filo Firmicutes e redução dos Filos Bacteroidetes e Proteobacteria, além do aumento de butirato e redução de acetato e propionato nos intestinos do grupo HFD/Mtb comparado ao grupo LFD/Mtb. O aumento na expressão de claudina-2 sugere alteração na permeabilidade intestinal e possível translocação bacteriana, caracterizada pela disbiose nos pulmões, nos quais foi detectado aumento de Firmicutes, Bacteroidetes e Actinobacteria, e redução de Proteobacteria no grupo HFD/Mtb. Em conclusão, a obesidade aumenta a magnitude da inflamação pulmonar e a suscetibilidade à infecção por M. tuberculosis por um mecanismo dependente de NLRP3. Ambos, aumento da suscetibilidade à infecção e da inflamação pulmonar estão associadas com disbiose intestinal e pulmonar, e aumento da permeabilidade intestinal. / The infectious disease that causes the largest number of deaths in the word is tuberculosis, caused by Mycobacterium tuberculosis bacilli. One of the risk factors that increases the devolpment of tuberculosis three times is diabetes. Obesity generates lowgrade inflammation that magnify the progression of chronic disease. Studies that have evaluated the association between obesity and tuberculosis are controversial, and the issue requires further investigation. In this study, we used an experimental model to determine the interface between obesity and tuberculosis. C57BL/6 mice were fed a highfat diet (HFD) for 60 days and infected by M. tuberculosis (HFD/Mtb) via intratracheal. As experimental control, animals were fed a light-fat diet (LFD) and were infected (LFD/Mtb). In parallel, one group was fed with HFD and another LFD, and they remained without infection. After 30 days of diet completing 90 days of feeding, the different groups were evaluated. Obese and infected animals (HFD/Mtb) showed increased body mass and adipose tissue weight, increased of IL-1? gene expression in adipose tissue, glucose intolerant, impaired insulin production and increased of serum levels of IFN-? compared to LFD/Mtb animals. In addition to, HFD/Mtb animals were more susceptible and exhibited higher lung inflammation compared to LFD/Mtb animals. The inflammation was characterized by increased of IL-17, IFN-?, TNF, IL-1?, IL-1?, NLRP3, caspase-1, IL-18, IL-6 gene expression and increase of IFN-? and/ or IL-17- producing CD4+ cells in the lungs, and was also accompanied by increased CD8+ and CD4+Foxp3+ cells compared to the LFD/Mtb group. As NLRP3 is a key molecule in obesity-induced meta-inflammation, but its role is still not well defined in tuberculosis, NLRP3 deficient animals fed with HFD and were infected (NLRP3-/- HFD/Mtb). This NLRP3-/- HFD/Mtb group was more resistant and exhibited reduction of lung inflammation compared to the WT (Wild Type) HFD/ Mtb group. Considerate that obesity-associated dysbiosis and that bacterial products derived from diet or microbiota can stimulate the release of IL-1? by the activation of NLRP3, we evaluated the microbiota role in obesity and tuberculosis comorbidity. We found intestinal dysbiosis characterized by increased Firmicutes phylum and reduction of Bacteroidetes and Proteobacteria phylum, as well as increased butyrate and diminished acetate and propionate in the intestine of the HFD/Mtb group compared to the LFD/Mtb group. An increase of claudin-2 expression suggests an alteration in intestinal permeability and a possible bacterial translocation characterized by dysbiosis in the lungs, with increased of Firmicutes, Bacteroidetes and Actinobacteria and diminished of Proteobacteria in the HFD/Mtb group. In conclusion, obesity increases the magnitude of pulmonary inflammation and susceptibility to M. tuberculosis infection by an NLRP3-depedent mechanism. Both increased susceptibility to infection and pulmonary inflammation are associated with intestinal and pulmonary dysbiosis, and increased intestinal permeability.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-13T18:08:23Z No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T12:58:18Z (GMT) No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) / Made available in DSpace on 2016-10-22T12:58:18Z (GMT). No. of bitstreams: 1 carolinedesouzaalmeida.pdf: 1716960 bytes, checksum: 4e2f5cb727250f9b08b6d1633c2a22dc (MD5) Previous issue date: 2008-04-11 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Um terço da população mundial está infectada com o Mycobacterium tuberculosis. Novos métodos de diagnóstico e a vacinação contra tuberculose (TB) são necessários para o controle da doença. A resposta imunológica contra o M. tuberculosis envolve a produção de várias citocinas e quimiocinas, porém suas interações durante a infecção são bastante complexas. O presente trabalho teve como objetivo o estudo da produção de citocinas e quimiocinas durante a tuberculose pulmonar, frente a estímulos de antígenos do M. tuberculosis. Através do método citométrico de esferas ordenadas (CBA-Cytometric Bead Array) foi possível avaliar os níveis plasmáticos das quimiocinas MIG, IP-10, IL-8, RANTES e MCP-1 em pacientes com tuberculose ativa não tratada (TBA), em pacientes sob tratamento (TBST), em pacientes tratados (TBT) e em indivíduos controles sadios (CS). Utilizando esse mesmo método também foi realizada a mensuração das citocinas IFN-γ, TNF-α, IL-6, IL-2, IL-10 e IL-4 e das quimiocinas MIG, IP-10, IL-8, RANTES e MCP-1, produzidas em cultura de células mononucleares do sangue periférico após estímulo com os antígenos ESAT-6/CFP-10 e 16kDa, nos grupos de pacientes com tuberculose ativa não tratada (TBA), de pacientes tratados (TBT), de indivíduos controles sadios (CS) e de indivíduos contatos de pacientes com tuberculose (CT). As quimiocinas plasmáticas MIG, IP-10 e IL-8 estavam elevadas no grupo TBA, em comparação aos demais grupos (CS, TBST e TBT); e ainda os níveis das quimiocinas analisadas no plasma diminuíram após o início do tratamento. Nos ensaios “in vitro” com PBMC humanas, as citocinas IFN-γ, TNF-α, IL-6, IL-2 e as quimiocinas MIG, IP-10, IL-8 e RANTES foram estimuladas em níveis maiores, pelo ESAT-6/CFP-10, nos grupos TBA e CT em relação aos controles e pacientes tratados, enquanto que o estímulo com antígeno 16kDa não permitiu diferenciar entre os grupos estudados quanto a produção dessas citocinas e quimiocinas. Já as citocinas IL-10 e IL-4 e a quimiocina MCP-1 apresentaram níveis elevados nas culturas de PBMC de indivíduos controles e contatos quando estimuladas com os antígenos do M. tuberculosis estudados em relação aos pacientes com tuberculose. O antígeno ESAT-6/CFP-10 diferenciou a tuberculose ativa quanto à produção das citocinas IFN-γ, TNF-α, IL-6 e IL-2, e das quimiocinas MIG, IP-10, IL-8 e RANTES, as quais podem contribuir para uma resposta protetora na tuberculose. O antígeno 16kDa se mostrou um forte estimulador de IL-10 e MCP1, moléculas que podem atuar como reguladoras ou inibidoras da resposta imunológica contra a infecção por M. tuberculosis, sugerindo o papel importante deste antígeno em favorecer a persistência do bacilo no hospedeiro. Os níveis de quimiocinas plasmáticas podem ser eficazes em diferenciar os pacientes com tuberculose ativa de indivíduos controles e ainda pode ser útil em monitorar o efeito do tratamento quimioterápico anti-tuberculose. O entendimento da dinâmica resposta imunológica contra antígenos do M. tuberculosis pode auxiliar no desenvolvimento de novas estratégias de combate à tuberculose. / One-third of the world's population is currently infected with Mycobacterium tuberculosis. The development of new methods of diagnosis and vaccines against tuberculosis (TB) are necessary for disease control. The immune response against M. tuberculosis involves the production of many cytokines and chemokines, but their interaction during the infection is complex. The objective of this work was to study the production of cytokines and chemokines in pulmonary TB under stimulation of M. tuberculosis antigens. The method of Cytometric Bead Array (CBA) was used to evaluate plasma levels of chemokines MIG, IP-10, IL-8, MCP-1 and RANTES in untreated patients with active TB (ATB), in patients during treatment (DTB) and in treated patients (TTB) and healthy controls individuals (HC). We also evaluated, by CBA, the levels of cytokines IFN-γ, TNF-α, IL-6, IL-2, IL-10 and IL-4, and chemokines MIG, IP-10, IL-8, MCP-1 and RANTES, in culture of peripheral blood mononuclear cells (PBMC) after stimulation with the antigens ESAT-6/CFP-10 and 16kDa, in groups of untreated patients with active TB (ATB), in treated patients (TTB), healthy controls individuals (HC) and in contact individuals of patients with TB (CT). The production of plasma chemokines, the levels of MIG, IP-10 and IL-8 were higher in the group ATB, compared to the other (HC, DTB and TTB), and the levels of chemokines analyzed in plasma decreased after the beginning of the treatment. In assays with human PBMC, the cytokine IFN-γ, TNF-α, IL-6, IL-2 and chemokines MIG, IP-10, IL-8 and RANTES were stimulated at higher levels, by the fusion protein ESAT-6 / CFP-10, in groups ATB and CT in relation to the controls and treated patients. The stimulation with antigen 16kDa did not allow for differentiation of these cytokines and chemokines between the groups. IL-10, IL-4 and MCP-1 showed high levels in the cultures of PBMC from healthy control individuals and contacts when stimulated with the studied M. tuberculosis antigens compared to patients with TB. The antigen ESAT-6/CFP-10 differentiated active TB with regard to the production of cytokines IFN-γ, TNF-α, IL-6 and IL-2 and chemokines MIG, IP-10, IL-8 and RANTES, which can contribute to a protective response in TB. The antigen 16kDa was a strong stimulator of IL-4, IL-10 and MCP-1 molecules that can regulate or inhibit the immune response against M. tuberculosis infection, demonstrating the important role of this antigen in promoting the persistence of the bacillus in the host. The plasma levels of chemokines can be effective in differentiating patients with active TB from the healthy control individuals, and can also be useful in monitoring the effect of anti-TB chemotherapy. The comprehension of the dynamics of the immune response against M. tuberculosis antigens can aid the development of new strategies to combat tuberculosis.