Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-05-11T20:41:59Z No. of bitstreams: 2 Tese - Adeliane Castro da Costa - 2016.pdf: 6997901 bytes, checksum: a6d453a5d3acafaed991fe945aa59330 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-12T10:41:03Z (GMT) No. of bitstreams: 2 Tese - Adeliane Castro da Costa - 2016.pdf: 6997901 bytes, checksum: a6d453a5d3acafaed991fe945aa59330 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-05-12T10:41:03Z (GMT). No. of bitstreams: 2 Tese - Adeliane Castro da Costa - 2016.pdf: 6997901 bytes, checksum: a6d453a5d3acafaed991fe945aa59330 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2016-03-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In the first chapter of this thesis we demonstrate, in a review article, some of the successful strategies employed in the construction of Bacillus Calmette-Guérin (BCG) vaccines, among others being: overexpression of promising Mycobacterium tuberculosis (Mtb) immunodominant antigens already expressed by BCG introduction of Mtb immunodominant antigens not expressed by BCG, such as antigens in the regions of difference (RD) 1 thru 16; combination of overexpression and introduction of novel antigens to BCG; BCG modification to skew immune response toward TCD8+, as for example recombinant BCG (rBCG) expressing cytokines. In the second chapter, we demonstrate that the recombinant fusion protein CMX is capable of aggregating important immunogenic properties to vaccine vectors, by inducing an effective response for the control of Mtb infection in the mouse tuberculosis infection model. It is hypothesized that the introduction of the rCMX protein in the BCG vaccine could add immunological properties that are absent in BCG, thus leading to the induction of important cell populations for the control of Mtb infection. Our results demonstrate that the introduction of the rCMX in the BCG vaccine, resulting the recombinant BCG vaccine (rBCG-CMX) was an important factor for the observed Th1 and Th17 responses, as well as polyfunctional cells, that could be responsible for the reduced inflammatory lesions seen in the lungs of Mtb infected BALB/c mice, significantly reducing the bacillary load in comparison to in comparison to mice immunized with BCG Moreau vaccine. Lastly, in the third chapter of this thesis we propose that rCMX protein could be responsible for modulating the BCG vaccine to activate a more adequate and protective innate immunity. Our results show that the rBCG-CMX vaccine induces the activation of alveolar macrophages by means of expression of activation-associated molecules CD86 and CD206. The increase in the expression of those molecules are accompanied by the production of TGF-β e IL-1α which in turn could be responsible for the decreased necrosis and higher apoptosis induction promoted by rBCG-CMX vaccination. This phenomenon could be providing a higher cellular survival rate of the recombinant vaccine, leading to a better processing and presentation by MHC-II. As rCMX was shown to induce the production of IL-1α, IL-6 e TGF-β by a pathway that seems to involve the participation of TLR-4, we hypothesize that this recombinant protein could be modulating the BCG vaccine to induce a more appropriate and protectiveresponse for Mtb infection. / A Tuberculose (Tb) é uma doença infecto contagiosa, causada pelo Mycobacterium tuberculosis (Mtb). Apesar de ser uma doença antiga, a Tb continua sendo um dos principais problemas de saúde pública. A Organização Mundial de Saúde acredita que cerca de um terço da população mundial está infectado com Mtb, gerando milhões de mortes por ano. Uma das medidas que podem melhorar a prevenção e bloquear a transmissão do Mtb é o desenvolvimento de novas vacinas que previnam o estabelecimento e a progressão da TB em humanos. Embora exista a vacina BCG que é eficiente contra formas graves de TB na infância, existe a necessidade do desenvolvimento de novas vacinas para controlar a disseminação da TB, que sejam mais eficientes e seguras que a BCG. Com este intuito, o objetivo deste trabalho é avaliar a proteção e a modulação da resposta imune induzida por BCG recombinante expressando espítopos imunodominantes Ag85C, MPT-51 e HspX do Mycobacterium tuberculosis induzida em modelo murino. Nossos resultados demonstram que a inserção da proteína CMX na vacina BCG recombinante (rBCG-CMX) foi um fator determinante para indução de resposta Th1 e Th17, além de células polifuncionais que possivelmente foram responsáveis pela redução das lesões inflamatórias no pulmão de camundongos BALB/c, reduzindo significantemente a carga bacilar em comparação com imunização com BCG Moreau. Além disso mostramos neste trabalho que a proteína rCMX é capaz de modular a vacina BCG e ativar a imunidade inata para a indução de uma melhor resposta protetora. Nossos resultados demonstram que a vacina rBCG-CMX induz ativação de macrófagos pulmonares por meio da expressão de moléculas de ativação CD86 e CD206. O aumento da expressão dessas moléculas é acompanhada por produção de TGF-β e IL-1α, sendo prováveis responsáveis pela menor indução de necrose e maior indução de apoptose pela vacina rBCG-CMX. Este fenômeno pode estar proporcionando a esta vacina maior capacidade de sobrevivência celular, colaborando para um melhor processamento e apresentação por MHC-II. Devido a proteína rCMX ser capaz de induzir produção de IL-1α, IL-6 e TGF-β por uma via que parece haver a participação de TLR-4. In vivo demonstramos que a vacina rBCG-CMX depende de TLR-2 e TLR-4 para induzir respostas Th1 e Th17, após imunização de camundongos com esta vacina. Neste trabalho hipotetizamos que a proteína CMX pode modular a resposta imune inata e adaptativa, por uma via em que há a participação do TLR-4. Esta pode ser a via pela qual a CMX, quando expressa por BCG favorece uma boa resposta protetora em animais desafiados com Mtb.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-12-14T16:06:41Z No. of bitstreams: 2 Dissertação - Grazzielle Guimarães de Matos - 2016.pdf: 6175984 bytes, checksum: 49ee98e2341033b338755369c46b165a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-12-15T17:30:05Z (GMT) No. of bitstreams: 2 Dissertação - Grazzielle Guimarães de Matos - 2016.pdf: 6175984 bytes, checksum: 49ee98e2341033b338755369c46b165a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-15T17:30:05Z (GMT). No. of bitstreams: 2 Dissertação - Grazzielle Guimarães de Matos - 2016.pdf: 6175984 bytes, checksum: 49ee98e2341033b338755369c46b165a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In Brazil, L. (V.) braziliensis is specie responsible for many cases of American Tegumentary Leishmaniasis (ETL). To effective vaccine development, formulations contained immunogenic antigens associated with adjuvants that induce a T helpe 1 (Th1) immune response are been investigated, among of antigen have the “Thiol Specific Antioxidant” (rTSA), “Stress Inducible protein 1” (rSTI) and “Leishmania elongation initiation fator” (rLeIF) of L. major, which have been immunogenic and promising. The genes that corresponding to these antigens in L. (V.) braziliensis were sequenced and recombinants proteins were obtained and used in this study. Works that used BCG and β-glucan showed that these could provide protection against not related pathogens. The present study had as aim to evaluate the immunogenicity and the effectiveness of the vaccine using the rTSA, rSTI and rLeIF proteins (Triple), associated or not with β-glucan, with or without BCG stimulus. BALB/c mice were divided in 2 groups, one of the groups was stimulated with BCG and the other not. Posteriorly, the animals were divided in subgroups and vaccinated with triple, associated or not with β-glucan. Mice’s serum were collected to search of cytokines and antibodies by immunoenzymatic technique. Lymph node and spleen’s cells were cultivated and cytokines searched in supernatants. BCG stimulation induced a greater IFN-γ production. The vaccinated mice with triple produced IgG total, IgG1 and IgG2a specifics to L. (V.) braziliensis. BCG stimulation induced a high IgG total and IgG2a production in animals that received the triple without β-glucan. To evaluate the protection provided by vaccine, the immunized animals were infected with L. (V.) braziliensis and paw them measured along of 8 weeks. The subgroup don’t stimulated with BCG and vaccinated with triple, associated with β-glucan, has developed bigger lesions that the others subgroups. The subgroups that received only BCG stimulation or BCG stimulation and vaccine with triple plus β-glucan presented bigger lesions that animal don’t stimulated or vaccinated. After infection by L. (V.) braziliensis, (i) the animals stimulated with BCG and vaccinated with triple presents more IgG specific to L. (V.) braziliensis; (ii) the immunizations with triple maintained IFN-γ levels elevated; (iii) all animals presented lower IL-17 levels in serum; (iv) the subgroups produced similar IFN-γ levels, but with a high IL-17 production in subgroup vaccinated with triple plus β-glucan, don’t BCG stimulated, in spleen; (v) the animals that received only BCG produced more IFN-γ and IL-17 in lymph node. In conclusion: the immunizations with triple were immunogenic; the immunization scheme containing triple plus β- glucan, without BCG stimulation, provided partial protection against L. (V.) braziliensis infection; BCG stimulations induced biggest lesions in subgroup that received only saline or triple plus β-glucan; the vaccination scheme containing triple maintained high IFN-γ production after L. (V.) braziliensis infection; in infection site, the BCG stimulation induced a Th1/Th17 heterologous response. / No Brasil, L. (V.) braziliensis é a espécie responsável pela maioria dos casos de Leishmaniose Tegumentar Americana (LTA). Para o desenvolvimento de uma vacina eficaz, formulações contendo antígenos imunogênicos, associados com adjuvantes, indutores de uma resposta imune do perfil T auxiliar 1 (Th1), estão sendo investigados, entre eles têm-se o “Thiol Specific Antioxidant” (rTSA), a “Stress Inducible protein 1” (rSTI) e o “Leishmania elongation initiation fator” (rLeIF) de L. major, que se mostraram imunogênicos e promissores. Os genes destas proteínas, de L. (V.) braziliensis, foram sequenciados e as proteínas recombinantes obtidas e utilizadas neste estudo. Trabalhos utilizando BCG e β-glucana mostraram a capacidade destes em fornecer proteção inespecífica. O presente estudo teve como objetivo avaliar a imunogenicidade e a eficácia da vacina com as proteínas rTSA, rSTI e Leif (Tríplice) de L. (V.) braziliensis associadas com o adjuvante β-glucana, com ou sem estímulo prévio com BCG. Camundongos BALB/c foram divididos em 2 grupos, um deles inoculado com BCG e o outro não. Posteriormente, os animais foram divididos em subgrupos e vacinados com tríplice, associadas ou não com β-glucana. Foram coletados os soros dos animais para dosagem de citocinas e anticorpos pela técnica imunoenzimática. Células do baço e linfonodo drenante foram cultivadas para dosagem de citocinas no sobrenadante. O estimulo com BCG induziu uma maior produção de IFN-γ. Os camundongos vacinados com a tríplice produziram anticorpos IgG total, IgG1 e IgG2a específicos para L. (V.) braziliensis. O estimulo com BCG induziu uma maior produção de anticorpos IgG total e IgG2a, nos animais vacinados com a tríplice sem β-glucana. Para avaliar a proteção fornecida pela vacina, os animais imunizados foram infectados com L. (V.) braziliensis e as patas mensuradas durante 8 semanas. O subgrupo, não estimulado com BCG e vacinado com a tríplice, associada com β-glucana, desenvolveu lesões menores que os demais subgrupos (p<0,05). Os subgrupos que receberam apenas BCG ou foram vacinados com a tríplice mais β-glucana apresentaram lesões maiores que os animais não estimulados e imunizados (p<0,05). Após a infecção por L. (V.) braziliensis, (i) os animais estimulados e vacinados com a tríplice apresentaram níveis elevados de IgG específicos para L. (V.) braziliensis; (ii) as imunizações com a tríplice mantiveram os níveis de IFN-γ aumentados (p<0,05); (iii) todos os animais apresentaram menores concentrações de IL-17, no soro; (iv) houve detecção similar de IFN-γ entre os subgrupos, mas com uma maior produção de IL-17 no subgrupo vacinado com a tríplice mais β-glucana, não estimulados com BCG, no baço (p<0,05); (v) os animais que receberam apenas BCG produziram mais IFN-γ e IL-17, no linfonodo drenante. Conclui-se que: as imunizações com tríplice foram imunogênicas; o esquema de imunização com a tríplice mais β-glucana, sem estimulo com BCG, forneceu proteção parcial contra a infecção por L. (V.) braziliensis; o estimulo com BCG induziu lesões maiores nos subgrupos que receberam salina ou a tríplice mais β- glucana; o esquema de vacinação com a tríplice manteve a produção de IFN-γ elevada após a infecção por L. (V.) braziliensis; no sitio de infecção, o estimulo com a BCG levou a uma resposta heteróloga Th1/Th17.

Made available in DSpace on 2014-08-20T13:32:52Z (GMT). No. of bitstreams: 1 tese_sibele_borsuk.pdf: 16306468 bytes, checksum: 4743a54f442368d81ff802d8290c7cfc (MD5) Previous issue date: 2008-02-28 / Mycobacterium bovis BCG has the potential to be an effective live vector for multivalent vaccines. However, there are two problems regarding the utilization of recombinant BCG as vaccine. The first one is that most mycobacterial cloning vectors rely on antibiotic resistance gene as selectable marker, which is used for genetic transformation. The second one is the limited use of BCG in animals because it interferes in the tuberculosis diagnosis by tuberculin skin test, which elicits delayed type hypersensitivity to the purified protein derivative (PPD). In this work we developed and evaluated the use of auxotrophic complementation as a new selectable marker, characterized the proteins that are present in the bovine and avium PPD and developed a knockout BCG strain by homologous recombination. To test the auxotrophic complementation as selectable marker, an auxotrophic BCG strain for the amino acid leucine was constructed by knocking out the leuD gene by homologous recombination. Expression of leuD on a plasmid acted as a selectable marker in the auxotrophic M. bovis BCG leuD and M. smegmatis mc2144. The auxotrophic complementation selection was similar to selection by antibiotic resistance, but with the advantage of promoting stability of the plasmid. The new system was highly stable even during in vivo BCG growth. The identification of proteins from PPD was archived by LC-MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry). A total of 147 proteins among five PPD samples (2 bovine PPD and 3 avium PPD) were identified. The bovine PPD had a considerable higher number of proteins comparing to the avium PPD. We identifying a group of 28 proteins present only in bovine PPD and a group of five proteins deleted in M. bovis BCG vaccinal strain. These two groups are of special interest as they can be used in tests with improved specificity, and potentially able to differentiate vaccinated and infected individuals. A mutant BCG strain with the DPPD antigen deleted was constructed. The Mb0092 coding sequence was knocked out by homologous recombination. The 11 sequences flanking the target gene were cloned into a suicide vector. Double crossovers were selected using sacB. The knockout genotype was determined by PCR and by Southern blot. This mutant BCG strain can be useful in animal vaccination as it will not interfere in the tuberculosis diagnostic test, when performed using recombinant DPPD. The results show alternatives for the problems related to the use of M. bovis BCG as a recombinant vaccine. The auxotrophic complementation system was highly stable, efficient and it is suitable for expressing heterologous antigens in BCG. The identification of proteins present in PPD preparations and the mutant BCG obtained provide the possibility for the development of differential diagnostic test, thus allowing the use of BCG as vaccine also in animals. / Mycobacterium bovis BCG tem o potencial para ser um vetor efetivo para vacinas recombinantes multivalentes. No entanto, existem dois problemas quanto a sua utilização como vetor vacinal. O primeiro é a presença de genes que conferem resistência a antibióticos nos vetores utilizados para transformação genética. O segundo é a limitação de uso de BCG em animais, principalmente por comprometer o teste de tuberculina, utilizado como diagnóstico de tuberculose, o qual se baseia em reação de hipersensibilidade ao PPD (Derivado Protéico Purificado). Neste trabalho desenvolvemos e avaliamos a complementação auxotrófica como novo marcador de seleção, fizemos a caracterização das proteínas componentes de amostras de PPD aviário e bovino e desenvolvemos um mutante de BCG por recombinação homóloga. Para o uso de complementação auxotrófica como marcador de seleção, uma cepa de BCG auxotrófica para o aminoácido leucina foi construída por knockout do gene leuD por recombinação homóloga. A expressão do gene leuD em um plasmídio atuou como marcador de seleção nas cepas auxotróficas de M. bovis BCG leuD e M. smegmatis mc2144. A seleção por complementação de BCG auxotrófica se mostrou equivalente à seleção por resistência a antibiótico, com a vantagem adicional de proporcionar maior estabilidade do vetor plasmidial, já que a pressão seletiva é mantida mesmo durante multiplicação da bactéria in vivo. A identificação das proteínas que compõem o PPD foi feita por espectrometria de massa utilizando-se LCMS/ MS (cromatografia líquida associada à espectrometria de massa em tandem). Foram identificadas 147 proteínas entre 5 amostras de PPD (2 PPD bovino e 3 PPD aviário). O PPD bovino teve um número maior de proteínas comparado ao PPD aviário. Foi identificado um grupo de 28 proteínas presentes em PPD bovino, mas ausentes em PPD aviário. Além disso, 5 proteínas encontradas no PPD estão ausentes em M. bovis BCG. Estes são de 9 especial interesse, pois poderão vir a contribuir para o desenvolvimento de um teste de diagnóstico mais específico, e possivelmente capaz de diferenciar indivíduo vacinado com BCG e infectado com o bacilo da tuberculose. Um mutante de M. bovis BCG Pasteur foi construído. O gene Mb0092 (dppd) foi alvo de inativação gênica por recombinação homóloga. Seqüências que flanqueiam o gene alvo foram clonadas em um vetor suicida. Duplo crossover foi selecionado utilizando sacB. O genótipo mutante foi determinado por PCR e por Southern blot. Esta cepa poderá ser utilizada como vacina em animais, quando o diagnóstico for feito com DPPD recombinante. Os resultados obtidos apresentam alternativas para os problemas envolvidos quanto à utilização de M. bovis BCG como vacina recombinante. O sistema de seleção por complementação auxotrófica foi estável, e pode ser empregado na expressão de antígenos heterólogos em BCG. A identificação dos principais componentes protéicos do PPD e o desenvolvimento da cepa mutante de BCG possibilitam o desenvolvimento de testes diagnósticos diferencias, permitindo a utilização de BCG como vacina também em animas.

No description available.

Asma alérgica é uma inflamação pulmonar crônica mediada por células Th2. A Hipótese da Higiene é a teoria aceita para explicar o aumento das alergias nas últimas décadas e preconiza que a menor exposição dos indivíduos a componentes microbianos prejudica a geração mecanismos imunorregulatórios. Nosso estudo abordou a modulação da resposta alérgica pulmonar induzida por ovalbumina, por cepas de bacilo Calmette-Guérin recombinantes (rBCG) que expressam fragmentos de toxinas bacterianas. Observamos que dependendo do antígeno heterólogo expresso, a imunização intranasal com rBCG pode levar tanto a supressão como a exacerbação da resposta alérgica pulmonar. Demonstramos que tanto em um contexto profilático quanto terapêutico, rBCG é capaz de suprimir os parâmetros alérgicos, e a supressão não envolve o recrutamento de células T regulatórias, é um fenômeno local, está associada a maior produção de IFN-g do que IL-4 e é dependente de IL-12. Estes dados sugerem que a infecção pulmonar por rBCG gera um milieu capaz de bloquear a migração de células Th2 inflamatórias. / Allergic asthma is an atopic disorder mediated by Th2 cells. The Hygiene Hypothesis is the accepted theory to explain the increasing in allergy in recent deacades. It states that modern health care and hygiene practices have led to a reduced exposure to microorganisms components which impairs the generation of immunoregulatory mechanisms. The present study analysed how the intranasal infection with recombinant bacillus Calmette-Guérin (rBCG) strains expressing fragments of bacterial toxins could modulate an allergic pulmonary inflammation induced by ovalbumin. We demonstrated that the rBCG strains could supress or exacerbate the allergic inflammation depending on the expressed heterologous antigen. We analysed the effect of the mycobacterial infection in a prophylactic and in a therapeutical contexts, and we have identified that for both situations the of supression allergic features does not involve the recruitment of regulatory T cells to the lungs, is a local phenomena, is associated with an increased production of IFN-g, and is an IL-12 dependent mechanism. Taken togheter, this data suggest that the rBCG pulmonary infection generates a milieu capable to supress the chemotaxis for Th2 cells, which suppress the establishment of the allergic inflammation in the lungs.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-14T11:53:05Z No. of bitstreams: 1 danilo-parmera.pdf: 3155055 bytes, checksum: ac63cd2b7c2719955b83925c310aabbc (MD5) / Made available in DSpace on 2012-11-14T11:53:05Z (GMT). No. of bitstreams: 1 danilo-parmera.pdf: 3155055 bytes, checksum: ac63cd2b7c2719955b83925c310aabbc (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / As culturas de células vêm sendo utilizadas extensivamente no desenvolvimento e na produção de uma variedade de produtos terapêuticos e profiláticos, tornando-se uma ferramenta indispensável para geneticistas, imunologistas, vacinologistas e a indústria farmacêutica. A adoção de sistemas de ensaios celulares in vitrotem sido aplicada como um método alternativo para a substituição ou diminuição do uso de animais nas fases de desenvolvimento, produção e testes de vacinas, demonstrando resultados promissores. Da mesma forma, sistemas computacionais podem ampliar a utilização desta ferramenta para etapas do desenvolvimento de vacinas candidatas na fase pré-clínica. O presente trabalho teve como objetivo o desenvolvimento de umprotocolo in vitroutilizando culturas de células humanas - a linhagem de monócitos THP-1, para a seleção de um protótipo vacinal - a cepa Pasteur de Mycobacterium bovisBCG expressando o antígeno Sm14 de Schistossoma mansoni(BCG/sm14). Para isso foram empregadas duas metodologias – citometria de fluxo e imunocitoquímica, para a identificação do perfil da expressão das citocinas IL-10, IL-12 e TNF-α. Foram utilizados como controles a cepa Pasteur do M. bovisBCG e a construção da cepa Pasteur do M. bovisBCG contendo o vetor de expressão pAU5 (BCG/pAU5).Após padronização, a multiplicidade de infecção utilizada para os experimentos foi de 10 bacilos para 1 célula THP-1 (10MOI). A expressão daproteína Sm14 foi detectada em todos os protótipos vacinais. Para assegurar queo protótipo BCG/sm14 era capaz de diferenciar a linhagem de monócitos THP-1 em macrófagos, avaliamos a taxa de crescimento celular e foi possível observarque após a infecção estas células apresentavam o índice de proliferação diminuído em 2 logs em relação à célula não infectada. O protótipo vacinal BCG/sm14não mostrou diferenças quanto a capacidade de infecção, a taxa de persistência intracelular e a estabilidade da construção plasmidial. As duas metodologias empregadas mostraram resultados discrepantes em relação ao percentual de citocinas expressas após a infecção com o protótipo vacinal ou os BCG controles, mostrando um maior percentual de células positivas quando avaliados por citometria de fluxo.Contudo, mesmo com esta diferença observamos que o protótipo vacinal BCG/sm14 não alterou o perfil de expressão de IL-10, IL-12 e TNF-α. O fato do plasmídeo pAU5 ser um vetor de expressão citoplasmática, sugere que a proteína Sm14 não foi capaz de estimular a mudança de citocinas em monócitos humanos. Experimentos futuros devem investigar o papel do BCG/sm14em macrófagos maduros, quanto a indução de citocinas pró e anti-inflamatórias, TLR, bem como na indução da resposta imune adaptativa, como a apresentação antigênica, na tentativa de melhor entender a resposta imune a esta vacina candidata. / Cell cultures has been used extensively in the development of a broad range of therapeutic and prophylactic products, and are an important tool for geneticists, immunologists, vaccinologists and the pharmaceuticsindustry. In vitrocell assay has been applied as an alternative method to replace ordiminish the use of animal model in the developmental, production in vaccine test phases, with promising results. Otherwise, computational methods should amplify theuse of cell cultures tools to test candidate vaccines. The mean goal of this work was to develop an in vitro protocol using human cell line – monocytic cell THP-1, to select a vaccine prototype – strain Pasteur Mycobacterium bovisBCG expressing Schistosoma mansoniSm14-antigen (BCG/sm14). Two methodologies were employed – a flow cytometry and immunocytochemistry, to quantify the expression of IL-10, IL-12 e TNF-α. Pasteur M. bovisBCG and the strain Pasteur do M. bovisBCG containing the expression vector pAU5 (BCG/pAU5) were used as controls. Multiplicityof infection (MOI) was determined and showed a better 10 bacilli to 1 cells ratios, regarding the expression of intracellular cytokines. Sm14 protein expressionwas detected in all vaccine prototype before use. To assure that BCG/sm14was able to differentiate monocytic THP1 cell line in mature macrophage, we evaluated the proliferative ration after BCGs infection and all strain showed the ability toinduce monocytic THP1 cells maturation, diminishing in 2.0 logs the cell proliferation. No differences were seen in the uptake, intracellular persistence and plasmidial stability. Interestingly, we observe discrepant results regarding the amount of positivecells expressing cytokines detected by the two methods used, independent of which BCG was tested. The overall results obtained by the cytometric method was high than immunocytochemistry. However, beside these ambiguous results, no alteration in the IL-10, IL-12 and TNF-alfa profile was observed whenBCG/sm14was compared with BCG. These results point to the plamidial BCG construction pAU5 and its intracellular expression, suggesting no modification in the cytokine profile in human monocytic cell lines. Further experiments should be addressedto identify the role of BCG/sm14 in modulate mature macrophage and, the induction ofadaptive immune response, as antigen presentation, pro- and anti-inflammatory cytokines, TLR expression and activation, to better understating the vaccine prototype immune response.

Orientador: Flávio Ruas de Moraes / Coorientador: Maria Isabel Quiroga Berdeal / Banca: Eduardo Juan Gimeno / Banca: Rogério Salvador / Banca: Laura Satiko Okada Nakaghi / Banca: Marco Antonio de Andrade Belo / Resumo: A piscicultura está caracterizada pela alta densidade de estocagem, transporte e altos níveis de arraçoamento, alterando a qualidade de água e a homeostase dos peixes. Uma das principais características frente a estas alterações é a resposta geral de adaptação, aumentando a susceptibilidade dos peixes às doenças infecciosas e parasitárias ao mesmo tempo em que a má qualidade da água particularmente quando rica em matéria orgânica, favorece a proliferação de agentes com potencial. Um dos processos fisiopatológicos de grande importância para manter a saúde do hospedeiro é a resposta inflamatória aguda que se cronifica com a persistência do agente. O implante de lamínulas de vidro no tecido subcutâneo de peixes induz a resposta inflamatória crônica, apresentando acúmulo de macrófagos e formação de células gigantes multinucleadas. O inóculo do BCG induz intenso infiltrado mononuclear com desorganização do tecido causado por edema com dissociação das fibras musculares e necrose. O surgimento de células gigantes ocorre de forma progressiva e com o tempo estas aumentam de tamanho e número de núcleos formando células gigantes tipo corpo estranho. Com o avanço do processo a disposição dos macrófagos ao redor do inóculo é maior e surgem fibroblastos, linfócitos e na maioria dos casos presença de melanomacrófagos formando o granuloma. Neste trabalho objetivou-se caracterizar morfologicamente o processo inflamatório crônico por corpo estranho induzido pelo implante de lamínulas de vidro no tecido subcutâneo e o epitelióide induzido pelo inóculo de BCG em Piaractus mesopotamicus e Oreochromis niloticus. 180 peixes de cada gênero foram utilizados no estudo, divididos em quatro grupos de 45 cada. Os tratamentos foram: Inoculados, Implantados, Inoculado e Implantados e grupo controle. Nos grupos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fish farming is characterized by high stocking density, transport and high levels of feeding, changing water quality and fish homeostasis. A major characteristic of these changes is opposite the overall response adjustment, increasing the susceptibility of fish to infectious and parasitic diseases at the same time that poor water quality particularly when rich in organic matter, favors the proliferation of agents with potential. One of the pathophysiological processes of major importance for maintaining the health of the host is the acute inflammatory response that chronic the persistence of the agent. The implantation of glass coverslips in the subcutaneous tissue of fish induces chronic inflammatory response, with accumulation of macrophages and multinucleated giant cell formation. The inoculum of BCG induces intense mononuclear infiltrate with disruption caused by tissue dissociation with edema and necrosis of muscle fibers. The emergence of giant cells occurs gradually and over time they increase in size and number of cores forming giant cells of foreign body type. With the progress of the process layout of macrophages around the inoculum is larger and appear fibroblasts, lymphocytes and in most cases the presence of melanomacrophages forming the granuloma. This study aimed to characterize morphologically the chronic inflammatory process induced by foreign body implant glass slides in the subcutaneous tissue and epithelioid induced by inoculation of BCG in Piaractus mesopotamicus and Oreochromis niloticus. 180 fish in each sex were used in the study were divided into four groups of 45 each. The treatments were: inoculated, deployed, deployed and Inoculated and control group). In inoculated groups were given 20 μl of BCG vaccine (40 mg/mL) (n° bacilli than 2.0 x 106 CFU / mg of BCG strain Mureau Rio de Janeiro) in skeletal striated... (Complete abstract click electronic access below) / Doutor

<p>New efficient vaccines against infectious diseases are in demand. Some important factors impeding the vaccine development are the poor immunogenicity and the MHC restriction of the immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins, which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and studied the mechanisms behind the adjuvanticity.</p><p>The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the efficacy of various adjuvants like alum, HSP70 from <i>Trypanosoma cruzi</i>, and the adjuvant combination (HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and magnitude of the response.</p><p>HSP belongs to a family of conserved molecules and the maximum homology lies on the N-terminal region of the protein, therefore there is a risk that use of a complete molecule would give rise to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved portion of HSP70 derived from <i>Plasmodium falciparum</i> (Pf70C). We found that the Pf70C exhibited similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization efficiently primed the immune system. This was evident as the subsequent boosting with the corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody responses. In contrast, no such priming effect was observed for <i>ex vivo</i> IFN-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ <i>in vitro</i>.</p><p>During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis by priming mice with different microorganisms such as BCG, <i>Mycobacterium vaccae</i> or <i>Chlamydia pneumoniae</i> and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We found that BCG and <i>M. vaccae</i> but not <i>C. pneumoniae</i> could provide priming of the immune system to induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens. However, no deposits were observed in the kidneys of HSP treated animals.</p><p>Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile of the induced immune response. Also, they possessed different requirements for signaling through TLRs. While HSP70 from <i>T. cruzi</i> induced antigen-specific humoral responses in wild type as well as in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice when both the whole and C-terminal fragment of HSP70 from <i>Mycobacterium tuberculosis</i> was used. However, the C-terminal fragment of <i>P. falciparum</i> HSP70 elicited responses only in wild type mice but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates in future vaccines.</p>

New efficient vaccines against infectious diseases are in demand. Some important factors impeding the vaccine development are the poor immunogenicity and the MHC restriction of the immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins, which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and studied the mechanisms behind the adjuvanticity. The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the efficacy of various adjuvants like alum, HSP70 from Trypanosoma cruzi, and the adjuvant combination (HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and magnitude of the response. HSP belongs to a family of conserved molecules and the maximum homology lies on the N-terminal region of the protein, therefore there is a risk that use of a complete molecule would give rise to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved portion of HSP70 derived from Plasmodium falciparum (Pf70C). We found that the Pf70C exhibited similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization efficiently primed the immune system. This was evident as the subsequent boosting with the corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody responses. In contrast, no such priming effect was observed for ex vivo IFN-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ in vitro. During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis by priming mice with different microorganisms such as BCG, Mycobacterium vaccae or Chlamydia pneumoniae and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We found that BCG and M. vaccae but not C. pneumoniae could provide priming of the immune system to induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens. However, no deposits were observed in the kidneys of HSP treated animals. Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile of the induced immune response. Also, they possessed different requirements for signaling through TLRs. While HSP70 from T. cruzi induced antigen-specific humoral responses in wild type as well as in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice when both the whole and C-terminal fragment of HSP70 from Mycobacterium tuberculosis was used. However, the C-terminal fragment of P. falciparum HSP70 elicited responses only in wild type mice but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates in future vaccines.

In light of the limitations of cost and profit centers, this research attempts to explore how enterprise N integrates managerial information and thus transforms into the concept of an investment center. The BCG matrix (Boston Consulting Group¡A1970) is adopted as the major analytical tool in this study. With this tool we are able to acquire the market attractiveness and profitability of enterprise N, its business units, and its subsidiaries, so as to facilitate business resource allocation. The FCF analysis reveals the balance of funds between individual business units, subsidiaries, and the enterprise as a whole. Calculation of EVA reveals the contribution that each business unit and subsidiary makes to the enterprise. We furthermore interview executives in order to verify the preliminary result of the BCG model. We also adopt SWOT analysis to investigate the strengths, weaknesses, opportunities, and threats of each business unit located in the respective quadrant of the BCG model. Thus, we may be lead to understand the fund allocation and strategic intention of respective business units and subsidiaries. Finally, we conclude and make suggestions for enterprise N, both regarding financial and strategic aspects. This study aims to find out the key success factors regarding how enterprise N makes and executes business resource allocation and synergy development. We hope that this case study may provide valuable information and serve as a refined tool of business analysis for enterprise N.