Myeloid-derived suppressor cells (MDSCs) constitute of monocytic (M-MDSCs) and granulocytic cell subsets (G-MDSCs)and were initially described as suppressors of T-cell function in tumor microenvironments. Recent studies have shown the involvement of MDSCs in a number of infectious diseases including Mycobacterium tuberculosis (Mtb) infection. MDSCs are tremendously accumulated in patients with Mtb infection and exert a suppressive effect on T cell responses against mycobacteria. Mycobacterium bovis BCG, the only available vaccine against Mtb fails to protect against the adult pulmonary tuberculosis (TB). Understanding the mechanisms of MDSC suppression for immunity against mycobacterial infection will provide a rational basis to improve anti- TB vaccination and host-directed therapies against TB. In this study, we investigated the role of three lipid-rich components of the plasma membrane, Caveolin-1(Cav-1), Acid Sphingomyelinase (ASM) and asialo-GM1 on BCG-activated MDSCs. Cav-1 is one of the vital components of caveolae (plasma membrane invaginations) which regulates apoptosis and lipid metabolism. In this work, we found that MDSCs upregulated Cav-1, TLR4 and TLR2 expression after BCG infection on the cell surface. However, Cav-1 deficiency resulted in a selective defect in the intracellular TLR2 accumulation in the M-MDSC, but not G-MDSC subset. Further analysis indicated no difference in the phagocytosis of BCG by M-MDSCs from WT and Cav1-/- mice but a reduced capacity to up-regulate surface markers, to secrete various cytokines, induce iNOS and NO production. These defects correlated with deficits of Cav1-/- MDSCs in the suppression of T cell proliferation. Among the signaling pathways that were affected by Cav-1 deficiency, we found lower phosphorylation of NF-kB and p38 mitogen-activated protein kinase (MAPK) in BCG - activated MDSCs. ASM is an enzyme present in lysosomes and is translocated to the cell surface where it hydrolyzes sphingomyelin into ceramide. Flow cytometric studies revealed that MDSCs phagocytosed BCG independent of inhibiting ASMase using pharmacological inhibitors (amitryptiline or desipramine) or MDSCs from WT and ASM-/-. Suppression of ASMase or using ASM-/- MDSCs resulted in reduced NO production and decreased cytokine secretion by MDSCs in response to BCG. Furthermore, MDSCs inhibited by amitryptiline had impaired AKT phosphorylation upon BCG infection. Asialo-GM1 is a ganglioside expressed on the cell surface of MDSCs reported to cooperate with TLR2 for activating ERK signaling. Here, in this study, we found that asialo-GM1 expression was upregulated specifically upon mycobacterial infection and not upon any other stimulus. We noted that the soluble form of asialo-GM1 bound to BCG. Flow cytometric studies revealed that blocking 81 asialo-GM1 did not affect the phagocytosis of BCG into MDSCs. Furthermore, blocking of asialo- GM1 had no effect on the cytokine and NO secretion or AKT signaling. Collectively, the data presented in this work implicated that Cav-1, ASM, asialo-GM1 are dispensable for the internalization of BCG. Rather, Cav-1 and ASM are required for the functional activation of MDSCs. Although asialo-GM1 binds to BCG, we did not find any difference in the functional activation of MDSCs after blocking asialo-GM1. This study provides insights into the role of lipid raft components of the MDSC cell membrane during mycobacterial infection. / Myeloide Suppressorzellen (engl, myeloid-derived suppressor cells MDSCs) bestehen aus monozytischen (M-MDSCs) und granulozytären Subtypen (G-MDSCs) und wurden anfangs als Suppressoren der T-Zellfunktion in Tumormikroumgebungen beschrieben. Kürzlich durchgeführte Studien haben gezeigt, dass MDSCs an einer Reihe von Infektionskrankheiten beteiligt sind, einschließlich einer Infektion mit Mycobacterium tuberculosis (Mtb). MDSCs sind bei der Patienten Mtb-Infektion enorm akkumuliert und üben eine supprimierende Wirkung auf die T-Zell-Antworten gegen Mykobakterien aus. Mycobacterium bovis BCG, der einzige verfügbare Impfstoff gegen Mtb, schützt nicht gegen die Lungentuberkulose bei Erwachsenen (TB). Das Verständnis der Mechanismen über welche MDSCs eine der Immunsuppression bei mykobakteriellen Infektionen vermitteln, bilden eine rationale Grundlage für die Verbesserung der Anti-TB-Impfung und Therapien gegen TB. In dieser Studie wurden die Rolle der drei lipidreichen Komponenten der Plasmamembran, Caveolin-1 (Cav-1), Saure Sphingomyelinase (ASM) und Asialo-GM1 bei BCGaktivierten MDSCs untersucht. Cav-1 ist eine der Komponenten von Caveolae (Plasmamembran-Invagination), die die Apoptose und den Fettstoffwechsel regulieren. Diese Arbeit zeigte, dass MDSCs die Expression von Cav-1, TLR4 und TLR2 nach BCG-Infektion auf der Zelloberfläche hochregulierten. Eine Cav-1 Defizienz führte jedoch zu einem selektiven Defekt in der intrazellulären TLR2-Akkumulation bei MMDSCs, jedoch nicht bei G-MDSCs. Weitere Analysen zeigten keinen Unterschied in der Phagozytose von BCG durch M-MDSCs von WT- und Cav1-/- Mäusen, jedoch eine verringerte Fähigkeit, Oberflächenmarker hoch zu regulieren, verschiedene Zytokine zu sekretieren und die Produktion von iNOS und NO zu induzieren. Diese Defekte korrelierten mit Defiziten von Cav1-/- MDSCs bei der Unterdrückung der T-Zell-Proliferation. Unter den von Cav-1-Mangel betroffenen Signalwegen fanden wir eine geringere Phosphorylierung der NF-KB- und p38- Mitogen-aktivierten Proteinkinase (MAPK) in BCG-aktivierten MDSCs. ASM ist ein in Lysosomen vorhandenes Enzym, das an die Zelloberfläche transloziert wird, wo es Sphingomyelin zu Ceramid hydrolysiert. Durchflusszytometrische Studien ergaben, dass MDSCs BCG unabhängig von der Hemmung von ASMase mit pharmakologischen Inhibitoren (Amitryptilin oder Desipramin) oder MDSCs von ASM-/- Mäusen BCG phagozytierten. Die Suppression von ASMase oder die Verwendung von ASM-/- MDSCs führte zu einer verringerten NO Produktion und einer verringerten Zytokinsekretion durch MDSCs als Antwort auf BCG. Darüber hinaus hatten MDSCs, die durch Amitryptilin inhibiert wurden, die AKT-Phosphorylierung bei einer BCG-Infektion beeinträchtigt. Asialo-GM1 ist ein Gangliosid, das auf der Zelloberfläche von MDSCs exprimiert wird, von dem berichtet wurde, dass es mit TLR2 kooperiert, um ERK-Signale zu aktivieren. Hier in dieser Studie haben wir festgestellt, dass die Expression von Asialo-GM1 spezifisch bei mycobakterieller Infektion und nicht bei einem anderen Stimulus hochreguliert wurde. Wir haben festgestellt, dass die lösliche Form von Asialo-GM1 an BCG binden kann. Durchflusszytometrische Studien ergaben, dass die Blockade von Asialo-GM1 die Phagozytose von BCG in MDSCs nicht beeinflusst. Darüber hinaus hatte die Blockierung von Asialo-GM1 keinen Einfluss auf die Zytokin- und NO-Sekretion oder das AKT-Signal. Zusammenfassend ergaben die in dieser Arbeit präsentierten Daten, dass Cav-1, ASM, asialoGM1 für die Internalisierung von BCG entbehrlich sind. Dagegen sind Cav-1 und ASM für die funktionale Aktivierung von MDSCs erforderlich. Obwohl Asialo-GM1 an BCG bindet, konnten wir nach der Blockierung von Asialo-GM1 keinen Unterschied in der funktionellen Aktivierung von MDSCs feststellen. Diese Studie liefert Einblicke in die Rolle einiger Komponenten der lipid-reicher Areale der MDSC-Zellmembran bei mykobakteriellen Infektionen.

O presente trabalho tem como finalidade estudar a resposta imunológica em escolares do sexo masculino, de 14 a 20 anos de idade, vacinados há 2 e 12 meses com BCG intradérmico, pertencentes a três Escolas do SENAI, nos municípios de Osasco, São Bernardo do Campo e São Paulo, Brasil, no período de agosto a dezembro de 1979. A população de estudo, constituída de 900 escolares, foi distribuída em três grupos: vacinados há 2 meses, vacinados há 12 meses e não vacinados, segundo as variáveis: idade, cor, sintomático respiratório, comunicante e presença de cicatriz pós-vacinal. Foram feitos 270 testes imunológicos, de transformação blástica de linfócitos para avaliar a resposta celular, e teste de hemaglutinação passiva para verificar a resposta humoral, pela pesquisa de anticorpos específicos. Simultaneamente foi realizado o teste tuberculínico com PPD Rt23 2UT,para avaliar a conversão tuberculínica pós-vacinal e a correlação da mesma com as respostas imunológicas. A resposta imunológica celular apresentou maior porcentagem de positividade do que a resposta humoral e se manteve praticamente inalterada no decurso de 2 para 12 meses. Não houve associação estatisticamente significativa entre a resposta imunológica celular e reatividade tuberculínica, nas pessoas vacinadas há 2 e 12 meses. Não houve relação entre reatividade tuberculínica, presença de cicatriz pós-vacinal e resposta imunológica. / This survey is to study the immune response of male students, between 14 and 20 years old, who had received intradermal BCG 2 and 12 months ago. They studied at SENAI schools in Osasco, São Bernardo do Campo and São Paulo, Brazil, during august to december of 1979. The population of this study was 900 students separated in three groups: one who had received vaccine 2 months ago; other after 12 months and the last one who did not receive vaccine. The variables age, color, respiratory symptom, contact and presence of post vaccinal cicatrix were analysed. They were realised 270 immunologic tests of blastic transformation of lymphocyte in order to evaluate the cellular response and the hemagglutination test to find out the specific antibody to verify the humoral response. The tuberculin test was realized with PPD Rt23 2UT to evaluate the post-vaccinal tuberculin conversion and the correlation with the immune response. The cellular immune response demonstrated that has major positive percentage than the humoral response and was unaltered between 2 and 12 months groups. There were no statistical significant correlation between cellular immune response and tuberculin reativity in boys who received vaccine 2 and 12 months ago. There was not relation among tuberculin reativity, post vaccinal cicatrix and immune response.

Dissertation is about the problems with vaccination of children, whose part is also the inoculation against tuberculosis. The objective is the analysis of opinions and approaches of parents toward the problems with vaccination of children in Czech Republic and to discover the level of their knowledge about tuberculosis disease and about vaccination against it. To get the information needed, the anonymous questionary has been used. In the first part we will familiarize ourselves with the main terms from the area of vaccination and also about tuberculosis's basic data. The second part includes presentation of data collected, which are sorted into the bars and graphs. It includes also the calculation of coefficient of benefit for area vaccination against tuberculosis. In the conclusion of work is the summary of collected data and their rating

Made available in DSpace on 2012-09-06T01:12:12Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 249.pdf: 2200380 bytes, checksum: 3d3205b0b06a23c66e31518e6c25237b (MD5) Previous issue date: 1998 / Foi realizado com a finalidade de avaliar o papel protetor do BCG-ID, recebido nos primeiros anos de vida na proteçäo contra a hanseníase. Foram incluídos 620 comunicantes de pacientes com as formas multibacilares da hanseníase e acompanhados por um período de 5 anos. Os comunicantes foram recrutados no período de junho de 1987 a dezembro de 1992. Desses, 468 apresentavam cicatriz de BCG e 152 näo apresentavam cicatriz de BCG. A faixa etária variou de 0 a 28 anos e 72 (11,6 por cento) dos contatos estavam doentes ou adoeceram em algum momento do acompanhamento. O percentual de adoecimento foi de 8,33 por cento (39) com cicatriz de BCG e 21,7 por cento (33) sem cicatriz vacinal. Entre aqueles com cicatriz de BCG, 35 (7,5 por cento) foram classificados como PB e 4 (0,85 por cento) foram classificados MB. Para aqueles sem cicatriz de BCG, 24 (15,8 por cento) foram PB e 9 (5,9 por cento) foram MB. A proteçäo conferida pelo BCG para as formas multibacilares (BB/BL/LL) foi de 86 por cento e de 53 por cento para as formas paucibacilares (TT/BT/HI/NI). Entre as 59 contatos que adoeceram apresentando a forma paucibacilar 12 (20 por cento) tinham idade entre 0 a 9 anos e desenvolveram a forma Nodular Infantil (NI) e desses 10 (83,3 por cento) apresentaram cura espontânea, sugerindo que o BCG pode polarizar para a forma tuberculóide, crianças submetidas precocemente à infecçäo pelo M.leprae. A taxa de adoecimento considerando-se somente os casos co-prevalentes, foi 2 vezes maior nos pacientes sem cicatriz de BCG enquanto que, entre os casos incidentes esta taxa foi 7 vezes maior nos contatos sem cicatriz de BCG. Considerando todos os 72 contatos que adoeceram, a taxa de adoecimento foi 3 vezes maior para aqueles sem cicatriz de BCG quando comparados àqueles com cicatriz vacinal. Os dados incidentes se concentraram no primeiro ano após o diagnóstico do caso índice, sendo praticamente ausente após o quarto ano de seguimento. O risco de adoecer variou com a carga bacilar do caso índice, número de pessoas por cômodo da casa, mais de um doente multibacilar na família e tipo de convivência intradomiciliar com o caso índice. A proteçäo conferida pelo BCG variou de acordo com a faixa etária do contato, sendo de 55 por cento para aqueles com idade de 0 a 9 anos, de 91 por cento para 10 a 18 anos e nenhuma para aqueles com 19 a 28 anos, sugerindo um decréscimo no efeito protetor do BCG-ID provável influência da idade em que o contato recebeu a vacina. / The present study aimed to assess the role of the BCG-ID as a protector, received in the first years of life as a protection against leprosy. A total of 620 contacts of patients with the multibacillary forms of leprosy have been included and followed for period of five years. Such contacts were recruited from June 1987 to December 1992. These, 468 presented BCG scars and 152 did not present BCG scars. The age group varied from 0 to 28 years old and 72 (11.6%) of the contacts were sick or got sick sometime during the follow-up. The percentage of sickening was of 8.33% (39) with BCG scars and 21.7% (33) without vaccinal scars. Among those with BCG scars 35 (7.5%) were classified as PB and 4 (0.85%) were classified as MB. For those without BCG scars, 24 (15.8%) were PB and 9 (5.9%) were MB. Thus, the protection given by the BCG to the multibacillary forms (BB/BL/LL) was of 86% and of 53% to the paucibacillary forms (TT/BT/HI/INL). It was observed that among the 59 contacts who got sick presenting the paucibacillary form 12 (20%) were between 0 and 9 years old and developed the Infantum Nodular Leprosy (INL) form and out of these 10 (83.3%) presented spontaneous healing, suggesting that the BCG may polarize to the tuberculoid form, children that were early submitted to infection by the M.leprae. The sickening rate considering only the co-prevailing cases, was twice higher in those patient who did not present the BCG scar whereas, among the incident cases this rate was 7 times higher in the contacts without BCG scars. Considering all the 72 contacts who got sick, the sickening rate was 3 times higher for those without the BCG scars than when compared to those with the vaccinal scar. Another finding of this study was that the incident cases concentrated themselves in a meaningful way in the first year after the diagnosis of the rate case, being practically absent after the fourth year of follow-up, suggesting that the main source of infection in these contacts concentrated itself in the family environment. The risk of getting sick varied with the bacillus load of the rate case, number of people per room in the house, more than one multibacillary patient in the family and the kind of introdomicile living with the rate case. The protection given by the BCG varied according to the age group of the contact, being 55% for those with age from 0 to 9 years old, of 91% for 10 to 18 years old and none for those of 19 to 28 years old, suggesting a decrease in the protector effect of the BCG-ID and a probable influence of the age in which the contact received the vaccine.

Made available in DSpace on 2016-04-15T12:59:56Z (GMT). No. of bitstreams: 2 melissa_pereira_ioc_dout_2009.pdf: 8311387 bytes, checksum: fc500887f8ee4dfdef6441b8f9d7d150 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A tuberculose (TB) ocupa o segundo lugar em causa de morte por doença infecciosa de notificação compulsória. Uma das estratégias para seu controle é a vacinação, e a única vacina disponível é o BCG (Bacilo de Calmette-Guèrin). Embora utilizada mundialmente, a vacina BCG apresenta diversas limitações. O Brasil utiliza a cepa Moreau para produção da vacina, sendo o único país do mundo autilizar, desde os anos 1930, esta cepa para vacinação. Como observado para outros patógenos intracelulares, a imunidade celular desempenha papel importante na proteção do hospedeiro contra a TB. O IFNy, secretado por células T, é descrito como uma citocina particularmente importante participando na resposta imune protetora, e é empregado como correlato de proteção, embora sua utilidade seja ainda questionável, sobretudo em populações adultas. Isto evidencia a necessidade de buscar biomarcadores adicionais que permitam uma correlação de proteção mais confiável. As proteínas extracelulares micobacterianas estão entre as primeiras moléculas a interagirem com o sistema imune do hospedeiro, e uma melhor compreensão de seu papel imunomodulatório poderá contribuir para o desenvolvimento de novos alvos para o controle e erradicação da TB. Uma estratégia para a identificação de proteínas imunologicamente relevantes é a análise do imunoproteoma, que combina a abordagem proteômica com uma apresentação imunológica dos dados. A caracterização de soros, provenientes de indivíduos vacinados, muitas vezes revela diferentes padrões de reconhecimento que podem ser indicativos dos diferentes graus de proteção conferidos pela vacina em uma população heterogênea Empregamos assim esta tecnologia, combinando a separação por eletroforese bi-dimensional (2DE) de antígenos protéicos extracelulares da cepa vacinal brasileira seguida de western-blot, para avaliar o perfil imunoproteômico da resposta humoral de indivíduos saudáveis, PPD negativos, revacinados com BVG Moreau e classificados como alto e baixo produtores de IFNy. Nossos resultados revelam um padrão de reconhecimento claramente distinto entre os grupos avaliados, correlacionado com o perfil de produção de IFNy. Dentre as proteínas imunoreativas identificadas, um grupo (antígenos do complexo 85) foi reconhecido por todos os soros incluídos no estudo, inclusive controles. Alguns antígenos foram reconhecidos exclusivamente pelos indivíduos classificados como alto produtores de IFNy e outros somente pelos indivíduos enquadrados no grupo de baixo produtores. Algumas proteínas foram selecionadas e produzidas sob a forma recombinante em Escherichia coli possibilitando sua avaliação mais detalhada frente aos soros individualizados. Entre os antígenos diferencialmente reconhecidos, as proteínas Mpb70 e Mpb83 se destacaram como potenciais correlatos da proteção oferecida pela vacinação com BCG Moreau, tendo sido reconhecidos somente pelos indivíduos produtores de elevados níveis de IFNy. Estudos adicionais são necessários para determinar a aplicabilidade efetiva de tais antígenos como potenciais correlatos da proteção conferida pela vacinação com BCG Moreau. Este é o primeiro relato que detalha o perfil de reconhecimento sorológico por indivíduos revacinados com BCG Moreau frente a antígenos extracelulares da cepa homóloga, e contribui para uma melhor compreensão da cepa vacinal brasileira contra a TB / Tuberculosis (TB) holds the second place as cause o f death by an infectious disease of obligatory notification. One of the stra tegies for its control is vaccination, and the only vaccine available is BCG (Bacillus Cal mette-Guerin). Although used worldwide, BCG shows various limitations. Brazil us es the Moreau strain for vaccine production, being the only country in the world to use, since the 1930s, this strain for vaccination. As observed for other intracellular pa thogens, cellular immunity plays an important role in host protection against TB. IFN γ secreted by T cells is described as a particularly important cytokine participating in protective immune responses, being used as a correlate of protection, although its uti lity remains questionable, especially in adult populations. This highlights the need to s eek additional biomarkers yielding better surrogates of protection. Mycobacterial extr acellular proteins are among the first molecules to interact with the host immune sy stem and a better understanding of their immunomodulatory role may contribute to the d evelopment of new targets for the control and eradication of TB. A strategy for t he identification of immunologically relevant proteins is the analysis of the immunoprot eome, combining a proteomic approach with an immunological presentation of the data. The characterization of sera from vaccinated individuals often reveals diff erent patterns of recognition that may be indicative of the different degrees of prote ction conferred by the vaccine in a heterogeneous population. We have thus employed thi s technology, combining the separation by two-dimensional electrophoresis (2DE) of extracellular proteins from the Brazilian vaccine strain, followed by Western b lot to assess the immunoproteomic profile of the humoral response in healthy, PPD negative subjects, re-vaccinated with BCG Moreau and classified as hig h or low producers of IFN γ . Our results reveal a clearly distinct recognition patte rn between the evaluated groups, correlating with the profile of IFN γ production. Among the immunoreactive proteins identified, one group (85 complex antigens) was rec ognized by all sera included in the study, including controls. Some antigens were r ecognized only by individuals classified as high producers of IFN γ and others only by the individuals included in the group of low producers. Some proteins were selected and produced in recombinant form in Escherichia coli allowing for their further evaluation against the individual sera. Among the antigens differentially recognized, Mpb70 and Mpb83 proteins stood out as potential correlates of protection offered b y vaccination with BCG Moreau, and vi were only recognized by individuals producing high levels of IFN γ . Additional studies are needed to determine the effective applicability of these antigens as potential correlates of protection provided by vaccination wi th BCG Moreau. This is the first report detailing the serologic recognition profile of individuals re-vaccinated with BCG Moreau against extracellular antigens of the homolo gous strain, and contributes to a better understanding of the Brazilian vaccine strai n against TB.

O presente trabalho tem como finalidade estudar a resposta imunológica em escolares do sexo masculino, de 14 a 20 anos de idade, vacinados há 2 e 12 meses com BCG intradérmico, pertencentes a três Escolas do SENAI, nos municípios de Osasco, São Bernardo do Campo e São Paulo, Brasil, no período de agosto a dezembro de 1979. A população de estudo, constituída de 900 escolares, foi distribuída em três grupos: vacinados há 2 meses, vacinados há 12 meses e não vacinados, segundo as variáveis: idade, cor, sintomático respiratório, comunicante e presença de cicatriz pós-vacinal. Foram feitos 270 testes imunológicos, de transformação blástica de linfócitos para avaliar a resposta celular, e teste de hemaglutinação passiva para verificar a resposta humoral, pela pesquisa de anticorpos específicos. Simultaneamente foi realizado o teste tuberculínico com PPD Rt23 2UT,para avaliar a conversão tuberculínica pós-vacinal e a correlação da mesma com as respostas imunológicas. A resposta imunológica celular apresentou maior porcentagem de positividade do que a resposta humoral e se manteve praticamente inalterada no decurso de 2 para 12 meses. Não houve associação estatisticamente significativa entre a resposta imunológica celular e reatividade tuberculínica, nas pessoas vacinadas há 2 e 12 meses. Não houve relação entre reatividade tuberculínica, presença de cicatriz pós-vacinal e resposta imunológica. / This survey is to study the immune response of male students, between 14 and 20 years old, who had received intradermal BCG 2 and 12 months ago. They studied at SENAI schools in Osasco, São Bernardo do Campo and São Paulo, Brazil, during august to december of 1979. The population of this study was 900 students separated in three groups: one who had received vaccine 2 months ago; other after 12 months and the last one who did not receive vaccine. The variables age, color, respiratory symptom, contact and presence of post vaccinal cicatrix were analysed. They were realised 270 immunologic tests of blastic transformation of lymphocyte in order to evaluate the cellular response and the hemagglutination test to find out the specific antibody to verify the humoral response. The tuberculin test was realized with PPD Rt23 2UT to evaluate the post-vaccinal tuberculin conversion and the correlation with the immune response. The cellular immune response demonstrated that has major positive percentage than the humoral response and was unaltered between 2 and 12 months groups. There were no statistical significant correlation between cellular immune response and tuberculin reativity in boys who received vaccine 2 and 12 months ago. There was not relation among tuberculin reativity, post vaccinal cicatrix and immune response.

Orientadores: Ubirajara Ferreira, Valéria Cagnon Quitete / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T22:24:14Z (GMT). No. of bitstreams: 1 Reis_LeonardoOliveira_D.pdf: 2126558 bytes, checksum: 304094622429151dac9d10d151c9e58b (MD5) Previous issue date: 2011 / Resumo: O tratamento do câncer de bexiga urotelial não músculo invasivo (CBNMI) com Bacilo Calmette-Guerin (BCG) tem efeito comprovado na redução de recidiva tumoral, embora ocorram efeitos colaterais de intensidades variadas, desde sintomas irritativos leves até reação sistêmica grave e o impacto na progressão tumoral seja controverso. Neste cenário a enterotoxina B do Staphylococcus aureus (EBS) destaca-se como alternativa promissora na terapêutica do CBNMI. Assim, os objetivos principais do presente estudo foram comparar e caracterizar os efeitos morfológicos, antiangiogênicos e o balanço entre proliferação e apoptose das imunoterapias com BCG, EBS e BCG-EBS no tratamento do CBNMI de ratos induzidos quimicamente. Um total de 75 ratas da linhagem Fisher 344 foram utilizadas. Para a indução do câncer, 60 animais foram anestesiados e receberam uma dose intravesical de 1,5mg/kg de N-metil-N-nitrosouréia (MNU) a cada 15 dias, nas semanas 0, 2, 4 e 6 (4 doses). Os outros 15 animais receberam 0,3ml de solução fisiológica intravesical e constituíram o grupo Controle (CT). Após o período de indução com MNU, os animais foram divididos em 5 grupos (15 animais cada): grupo MNU: recebeu o mesmo tratamento que o grupo CT - 0,3 ml de solução fisiológica intravesical; grupo BCG: recebeu dose intravesical de 106 UFC (2x106 UFC/mg) de BCG; grupo EBS: recebeu dose intravesical de 10?g/ml de EBS; grupo BCG-EBS: recebeu tratamento simultâneo com BCG e EBS nas mesmas concentrações que nos grupos BCG e EBS; todos por 6 semanas consecutivas. Após 15 semanas de protocolo, os animais foram sacrificados e as bexigas urinárias coletadas e submetidas às análises histopatológica, imunohistoquímica e Western Blotting. Os resultados demonstraram que as alterações histopatológicas mais frequentes em cada grupo foram carcinoma papilífero e carcinoma in situ no grupo MNU, hiperplasia papilífera nos grupos BCG e EBS e hiperplasia plana no grupo BCG-EBS. Não houve toxicidade relevante. Os índices apoptótico e proliferativo foram aumentados em todos os grupos de tratamento. Contudo, esses índices foram menores nos grupos BCG, EBS e BCG-EBS em relação ao grupo MNU. Intensas imunorreatividades para VEGF, MMP-9 e IGFR-1 foram verificadas no grupo MNU; moderadas nos grupos BCG e EBS; e fracas nos grupos BCG-EBS e Controle. Em contraste, intensa imunorreatividade para endostatina foi verificada nos grupos Controle e BCG-EBS em relação aos demais grupos experimentais. Assim, pode-se concluir que o tratamento associado BCG-EBS foi mais eficaz em restaurar o equilíbrio entre os processos de apoptose e proliferação celular e as alterações histopatológicas do que os tratamentos isolados com BCG e EBS. Além disso, a diminuição da proliferação celular e das alterações histopatológicas correlacionaram-se com o aumento da reatividade para endostatina e diminuição das reatividades para VEGF, IGFR-1 e MMP-9. A associação entre BCG e EBS abre uma nova perspectiva terapêutica no CBNMI / Abstract: PURPOSE: Compare and characterize effects of intravesical Bacillus Calmette-Guerin (BCG), Staphylococcal Enterotoxin B (SEB) and BCG plus SEB treatments in the non-muscle invasive bladder cancer (NMIBC). METHODS: Seventy-five female Fisher 344 rats were anesthetized and 15 of them received 0.3 ml of saline (Control group), 60 animals received 1.5 mg/kg dose of N-methyl-N-nitrosourea (MNU), all intravesically every other week for 6 weeks. The rats were divided into 5 groups: MNU and Control groups received 0.3 ml of saline; BCG group received 106 CFU dose of BCG; SEB group received 10 ?g/ml dose of SEB and BCG-SEB group received simultaneous BCG and SEB, all intravesically for 6 weeks. After 15 weeks, all urinary bladders were collected for histopathological, immunological and Western Blotting analyses. RESULTS: Papillary carcinoma (pTa) and high-grade intraepithelial neoplasia (carcinoma in situ) were more often in MNU group, papillary hyperplasia in BCG and SEB groups and flat hyperplasia in BCG-SEB group. No significant toxicity was observed. Apoptosis and cellular proliferation indexes decreased in BCG, SEB and BCG-SEB groups compared to MNU group. Intensified VEGF, MMP-9, Ki-67 and IGFR-1 immunoreactivities were verified in the MNU group; moderate in the BCG and SEB groups; and weak in the BCG-SEB and Control groups. In contrast, intense endostatin immunoreactivity was verified in the Control and BCG-SEB groups. CONCLUSIONS: BCG and SEB presented similar anti-angiogenic effects. BCG-SEB treatment showed an additional activity compared to monotherapy, being more effective in restoring apoptosis and cellular proliferation balance, correlating with increased endostatin and decreased VEGF, MMP-9, Ki-67 and IGFR-1 reactivities / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

No description available.

Reproduction de : Thèse d'exercice : Médecine. Médecine générale : Nantes : 2008. / Bibliogr.

No description available.