Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T18:56:46Z No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T16:58:20Z (GMT) No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) / Made available in DSpace on 2018-02-22T17:05:19Z (GMT). No. of bitstreams: 1 Tese Doutorado - Tatiana Kurtz -FINAL (1).pdf: 3145488 bytes, checksum: 8d9c7ca2d5630d7d9e3c1060cce48762 (MD5) Previous issue date: 2017-08-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction and Objectives: Tuberculosis (TB) is a complex infectious disease that can occur in any age group. When the host comes in contact with Mycobacterium tuberculosis (MTB) the immune response of the organism may be sufficient to prevent the disease, resulting in total destruction of the bacteria or establishment of latency, termed latent tuberculosis (TBL). Due to the difficulty in demonstrating MTB in the clinical specimens of the child, the diagnosis of TB disease is based on the clinical, epidemiological and radiological bases associated with the interpretation of the cutaneous tuberculin (TT) test. In this context, Mycobacterium tuberculosis infection, mostly latent, represents an important reservoir for reactivation of the disease. This contingent is sufficient to continue generating new cases for many decades, even if the chain of transmission is interrupted. Thus, the precise definition of the criteria for diagnosis of latent TB has great relevance and TT is an important tool. The objective of the study is to evaluate the effect of the neonatal BCG vaccine on the tuberculin skin test cutaneous reaction and to define cutoff points to detect tuberculosis in the first two years of life. Methods: A cross-sectional study was carried out in children from the municipality of Santa Cruz do Sul, who met the inclusion criteria of the study: infants up to 2 years of age who received BCG vaccine during the neonatal period. Exclusion criteria were birth weight <2,000 grams, being the mother of HIV positive mother, or mother with persistence of tuberculosis in the perinatal period, or cases where there was evidence of primary immunodeficiency, absence of BCG vaccine scar after 6 months of life, in addition to TB research and TB contact. The children were identified and included through the authorization of the person responsible, explaining the project and accepting the consent term. The project was approved by the Research Ethics Committee of the Santa Cruz Hospital, where patient data were collected and the University of Santa Cruz do Sul (UNISC). The variable under study was the cutaneous induration reaction of the tuberculin test, in the first two years post-vaccination, using different cutoff points. A descriptive analysis of the variables was performed. Numerical variables were represented by mean and standard deviation and categorical variables by means of absolute number and percentage. To describe the data, we used means and standard deviation, or median and interquartile range for the quantitative variables; percentage for qualitative variables. For analysis of the Tuberculin Test, the sample was submitted to the analysis of variance (ANOVA), with significance level of p?0.05. The data analyzed in the SPSS Program 17.0. Results: Potentially eligible participants totaled 808, of which 90 were selected from the inclusion / exclusion criteria. Data collected included demographic characteristics, nutritional indexes, vaccination status and previous exposure to TB. TTs were administered and induration measured after 48-72 hours. The selected ones were of both sexes, with ages varying between 3 and 24 months. Of these, eleven were excluded because they did not attend the reading of the tuberculin test (TT), resulting in a sample of 79 patients. The median age was 9.5 months for boys and 11 months for girls. It was divided into 3 groups according to the age range: between 3-9 months (group 1), 10-18 months (group 2) and 19-24 months of age (group 3). We found that, when comparing the 3 groups, we showed a decrease in the mean response to tuberculin as the age group progresses, presenting statistical significance (p = 0.041). Considering the probable absence of Mycobacterium tuberculosis infection in the sample of patients included in the study, we observed that the tuberculin test with the highest reaction occurs in group 1. From the age of 10 months, no patient shows a reaction to the tuberculin test above 5 mm. The finding shows the decline in the tuberculin reaction curve in the first year of life. A complementary analysis was performed excluding patients who did not present an induration reaction (TT = 0 mm), and 28 patients were excluded from the interpretation. The 51 patients with Test Tuberculin reactor were divided into the same 3 groups according to age group. Between 3-9 months (group 1), 10-18 months (group 2) and 19-24 months of age (group 3), we found that when comparing the 3 groups, again we showed a decrease in the reaction to tuberculin according to age progresses, presenting significance (p = 0.031). We found that there were no adverse effects, described in the literature, in patients who underwent the Tuberculin Test. Conclusions: Based on the data from the study, we demonstrated that the induration reaction occurs in the tuberculin test in the first 12 months of age in previously healthy and BCG-vaccinated patients in the neonatal period. Therefore our results suggest that the cutoff point could be modified from 10mm to 5mm of induration after 12 months of age, improving the specificity of the TT diagnostic test to identify cases of TB infection. This reevaluation of the lowest cutoff point in the first two years of life may prevent inappropriate management in patients with tuberculosis. / Introdu??o e Objetivos: A tuberculose (TB) ? uma doen?a infecciosa complexa, podendo ocorrer em qualquer faixa et?ria. Quando o hospedeiro entra em contato com o Mycobacterium tuberculosis (MTB) a resposta imunit?ria do organismo pode ser suficiente para evitar a doen?a, ocorrendo destrui??o total das bact?rias ou estabelecimento de um estado de lat?ncia, denominado de tuberculose latente (TBL). Devido ? dificuldade em demonstrar MTB nos esp?cimes cl?nicos da crian?a, o diagn?stico da TB doen?a ? fundamentado em bases cl?nicas, epidemiol?gicas e radiol?gicas associados ? interpreta??o do teste tubercul?nico (TT) cut?neo. Neste contexto, verifica-se que a infec??o pelo Mycobacterium tuberculosis, na sua maioria forma latente, representa um importante reservat?rio de reativa??o da doen?a. Este contingente ? suficiente para continuar gerando novos casos por muitas d?cadas, mesmo que a cadeia de transmiss?o seja interrompida. Dessa forma, a defini??o precisa dos crit?rios para diagn?stico de TB latente tem grande relev?ncia e o TT ? uma importante ferramenta. O objetivo do estudo ? avaliar o efeito da vacina BCG neonatal na rea??o de endura??o cut?nea do Teste Tubercul?nico e definir pontos de corte para detectar tuberculose nos dois primeiros anos de vida. M?todos: Estudo transversal, em crian?as do munic?pio de Santa Cruz do Sul, que se adequaram aos crit?rios de inclus?o do estudo: lactentes at? 2 anos de idade que receberam vacina BCG durante o per?odo neonatal. Crit?rios de exclus?o foram: peso ao nascimento <2,000 gramas, ser filho de m?e HIV positiva, ou m?e com vig?ncia de tuberculose no per?odo perinatal, ou ainda os casos em que houve evid?ncia de imunodefici?ncia prim?ria, aus?ncia de cicatriz vacinal de BCG ap?s 6 meses de vida, al?m de investiga??o de TB e contato de TB. As crian?as foram identificadas e inclu?das atrav?s de autoriza??o do respons?vel, mediante explica??o do projeto e aceita??o do termo de consentimento. O projeto foi aprovado pela Comiss?o de ?tica em Pesquisa do Hospital Santa Cruz, onde foi realizada a coleta de dados dos pacientes e Universidade de Santa Cruz do Sul (UNISC). A vari?vel em estudo foi a rea??o de endura??o cut?nea do teste tubercul?nico, nos dois primeiros anos p?s-vacina??o, utilizando diferentes pontos de corte. Realizada uma an?lise descritiva das vari?veis. As vari?veis num?ricas foram representadas por meio de m?dia e desvio padr?o e as categ?ricas por meio de n?mero absoluto e porcentagem. Para descri??o dos dados, foram utilizados m?dias e desvio padr?o, ou mediana e intervalo interquartil para as vari?veis quantitativas; porcentagem para as vari?veis qualitativas. Para an?lise do Teste Tubercul?nico a amostra foi submetida ao teste de an?lise de vari?ncia (ANOVA), com n?vel de signific?ncia de p?0,05. Os dados analisados no Programa SPSS 17.0. Resultados: Os participantes potencialmente eleg?veis totalizaram 808, desses 90 foram selecionados a partir dos crit?rios de inclus?o/exclus?o. Dados coletados inclu?ram caracter?sticas demogr?ficas, ?ndices nutricionais, estado de vacina??o e exposi??o pr?via ? TB. TTs foram administrados e a endura??o medida ap?s 48-72 horas. Os selecionados foram de ambos os sexos, com idade variando entre 3 e 24 meses. Destes, onze foram exclu?das, pois n?o compareceram a leitura do teste tubercul?nico (TT), resultando em amostra final de 79 pacientes. A mediana das idades foi de 9,5 meses, entre os meninos, e 11 meses entre as meninas. Realizada divis?o em 3 grupos conforme faixa et?ria: entre 3-9 meses (grupo 1), 10-18 meses (grupo 2) e 19-24 meses de idade (grupo 3). Constatamos que, quando comparados os 3 grupos, evidenciamos queda na m?dia de rea??o ? tuberculina conforme a faixa et?ria progride, apresentando signific?ncia estat?stica (p= 0.041). Considerando a prov?vel aus?ncia de infec??o por Mycobacterium tuberculosis na amostra de pacientes inclu?dos no estudo, observamos que o teste tubercul?nico com rea??o mais elevada ocorre no grupo 1. A partir dos 10 meses de idade nenhum paciente demonstra rea??o ao teste tubercul?nico acima de 5 mm. O achado evidencia o decl?nio na curva de rea??o ? tuberculina j? no primeiro ano de vida. Realizada an?lise complementar excluindo os pacientes que n?o apresentaram rea??o de endura??o (TT= 0 mm), sendo exclu?dos da interpreta??o 28 pacientes. Os 51 pacientes com Teste Tubercul?nico reator foram divididos nos mesmos 3 grupos conforme faixa et?ria. Entre 3-9 meses (grupo 1), 10-18 meses (grupo 2) e 19-24 meses de idade (grupo 3), onde constatamos que quando comparados os 3 grupos, novamente evidenciamos queda na rea??o ? tuberculina conforme a faixa et?ria progride, apresentando signific?ncia (p= 0.031). Constatamos que n?o ocorreram efeitos adversos, descritos em literatura, nos pacientes que se submeteram a aplica??o do Teste Tubercul?nico. Conclus?es: A partir dos dados do estudo demonstramos que ocorre queda da rea??o de endura??o no teste tubercul?nico nos primeiros 12 meses de idade em pacientes previamente h?gidos e vacinados com BCG no per?odo neonatal. Portanto nossos resultados sugerem que o ponto de corte poderia ser modificado de 10mm para 5mm de endura??o ap?s os 12 meses de idade, melhorando a especificidade do teste diagn?stico TT para identifica??o dos casos de TB infec??o. Esta reavalia??o do ponto de corte menor nos dois primeiros anos de vida pode evitar manejos inadequados nos pacientes com contato com tuberculose.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which 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, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

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