Afhandling--Copenhagen. / Summary in Danish. Bibliography: p. [99]-110.

Thesis (M.S.)--University of Wisconsin--Madison, 1964. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 47-52.

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 94-108.

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 94-108.

Made available in DSpace on 2014-06-11T19:24:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-30Bitstream added on 2014-06-13T18:52:00Z : No. of bitstreams: 1 rosa_lc_me_botfm.pdf: 636625 bytes, checksum: 4e80c747550326696f8ac42a9a3aed5a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / O diabetes tipo I é uma doença causada pela destruição autoimune das células do pâncreas que resulta em deficiência na produção de insulina. O objetivo deste projeto foi, inicialmente, avaliar o potencial profilático de seis estratégias vacinais no controle do diabetes experimental induzido por estreptozotocina (STZ) em camundongos C57BL/6. As três primeiras são específicas e baseadas na imunização com insulina em condições tolerogênicas que incluem associação com alúmen, dexametasona ou Adjuvante Incompleto de Freund (AIF). As outras três são não-específicas e baseadas em estratégias do tipo prime-boost. Neste caso foram testadas as seguintes combinações: BCG/DNAhsp65, BCG/hsp65 e DNAhsp65/BCG. Nos três protocolos iniciais, camundongos C57BL/6 machos foram imunizados e posteriormente inoculados com múltiplas doses de streptozotocina para desencadeamento do diabetes tipo I. Para avaliação inicial do efeito protetor foram determinados os seguintes parâmetros: peso, glicemia, análise histopatológica do pâncreas e perfil de citocinas produzidas por células esplênicas. Dentre todos os protocolos testados com o modelo STZ, os resultados observados no grupo imunizado por priming com BCG seguido de boost com DNAhsp65 forneceu os resultados mais promissores. Neste caso, a hiperglicemia foi menor, ocorreu proteção parcial contra a insulite e produção significativa de IL-10 por células estimuladas in vitro com hsp65. Por último, avaliamos o efeito da estratégia BCG/DNAhsp65 na evolução do diabetes no modelo NOD. Esta imunização determinou maior ganho de peso, proteção contra a hiperglicemia, aumento na produção de citocinas e diminuição na porcentagem de ilhotas inflamadas quando comparado ao grupo de camundongos NOD não imunizados. Esta proteção se mostra promissora e deverá ser confirmada em estudos futuros / Diabetes type I is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this project was, initially, to evaluate the prophylactic potential of six different vaccine strategies on diabetes type 1 induced by streptozotocin (STZ) in C57BL/6 mice. The first three strategies were specific for diabetes and based on immunization with insulin in tolerogenic conditions including insulin associated with Incomplete Freund Adjuvant (IFA), alum or dexamethasone. The other three were based on prime-boost strategies involving BCG and the DNAhsp65 vaccine: BCG/DNAhsp65, BCG/hsp65 e DNAhsp65/BCG. In the first three experimental protocols, male C57BL/6 mice were immunized with one of the vaccine strategies and later diabetes was induced with multiple low doses of STZ. The following parameters were analyzed to evaluate the prophylactic potential: body weight, glycemic levels, pancreatic islets histopathological analysis and cytokine production by spleen cells. From all strategies tested in the STZ model, the one with the most promising results was BCG as a primer followed by a booster with DNAhsp65. In this case, we observed protection against weight loss and against hyperglycemia, a decreased islet inflammation and significantly increased production of IL-10 induced by in vitro stimulation with hsp65. As a final point, we evaluated the effect of BCG/DNAhsp65 in the evolution of diabetes in NOD mice. This immunization determined weight gain, protection against hyperglycemia, decreased islet inflammation and higher levels of cytokine production when compared with non-immunized NOD mice. These findings are very interesting and deserve further investigations

Orientador: Alexandrina Sartori / Banca: Maria Terezinha Serrão Peraçoli / Banca: Denise Morais da Fonseca / Resumo: O diabetes tipo I é uma doença causada pela destruição autoimune das células do pâncreas que resulta em deficiência na produção de insulina. O objetivo deste projeto foi, inicialmente, avaliar o potencial profilático de seis estratégias vacinais no controle do diabetes experimental induzido por estreptozotocina (STZ) em camundongos C57BL/6. As três primeiras são específicas e baseadas na imunização com insulina em condições tolerogênicas que incluem associação com alúmen, dexametasona ou Adjuvante Incompleto de Freund (AIF). As outras três são não-específicas e baseadas em estratégias do tipo prime-boost. Neste caso foram testadas as seguintes combinações: BCG/DNAhsp65, BCG/hsp65 e DNAhsp65/BCG. Nos três protocolos iniciais, camundongos C57BL/6 machos foram imunizados e posteriormente inoculados com múltiplas doses de streptozotocina para desencadeamento do diabetes tipo I. Para avaliação inicial do efeito protetor foram determinados os seguintes parâmetros: peso, glicemia, análise histopatológica do pâncreas e perfil de citocinas produzidas por células esplênicas. Dentre todos os protocolos testados com o modelo STZ, os resultados observados no grupo imunizado por priming com BCG seguido de boost com DNAhsp65 forneceu os resultados mais promissores. Neste caso, a hiperglicemia foi menor, ocorreu proteção parcial contra a insulite e produção significativa de IL-10 por células estimuladas in vitro com hsp65. Por último, avaliamos o efeito da estratégia BCG/DNAhsp65 na evolução do diabetes no modelo NOD. Esta imunização determinou maior ganho de peso, proteção contra a hiperglicemia, aumento na produção de citocinas e diminuição na porcentagem de ilhotas inflamadas quando comparado ao grupo de camundongos NOD não imunizados. Esta proteção se mostra promissora e deverá ser confirmada em estudos futuros / Abstract: Diabetes type I is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this project was, initially, to evaluate the prophylactic potential of six different vaccine strategies on diabetes type 1 induced by streptozotocin (STZ) in C57BL/6 mice. The first three strategies were specific for diabetes and based on immunization with insulin in tolerogenic conditions including insulin associated with Incomplete Freund Adjuvant (IFA), alum or dexamethasone. The other three were based on prime-boost strategies involving BCG and the DNAhsp65 vaccine: BCG/DNAhsp65, BCG/hsp65 e DNAhsp65/BCG. In the first three experimental protocols, male C57BL/6 mice were immunized with one of the vaccine strategies and later diabetes was induced with multiple low doses of STZ. The following parameters were analyzed to evaluate the prophylactic potential: body weight, glycemic levels, pancreatic islets histopathological analysis and cytokine production by spleen cells. From all strategies tested in the STZ model, the one with the most promising results was BCG as a primer followed by a booster with DNAhsp65. In this case, we observed protection against weight loss and against hyperglycemia, a decreased islet inflammation and significantly increased production of IL-10 induced by in vitro stimulation with hsp65. As a final point, we evaluated the effect of BCG/DNAhsp65 in the evolution of diabetes in NOD mice. This immunization determined weight gain, protection against hyperglycemia, decreased islet inflammation and higher levels of cytokine production when compared with non-immunized NOD mice. These findings are very interesting and deserve further investigations / Mestre

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Estudos em diferentes populaÃÃes tÃm mostrado que a vacinaÃÃo com a BCG concede proteÃÃo parcial contra a hansenÃase. Entretanto, a necessidade da revacinaÃÃo e seu impacto na resposta imune contra antÃgenos do Mycobacterium leprae à pouco compreendida, principalmente nas crianÃas que sÃo contatos destes pacientes com hansenÃase, e constituem uma populaÃÃo de risco para o adoecimento. Neste estudo, investigamos o papel da revacinaÃÃo com a BCG na modulaÃÃo da resposta imune em contatos menores de quinze anos de pacientes com hansenÃase. Amostras de sangue perifÃrico de vinte e cinco crianÃas, vacinadas com a BCG ao nascer, foram coletas antes e dois meses apÃs a revacinaÃÃo com a BCG. Suas cÃlulas mononucleares (PBMC) foram estimuladas com proteÃnas e peptÃdeos do M. leprae, dosando-se as quantidades de IFN-γ e IL-10 por ELISA. Anteriormente a revacinaÃÃo, foram coletas amostras para dosagem de IgM e IgG anti-PGL-1 e realizado o teste tuberculÃnico (PPD) para avaliaÃÃo da eficÃcia vacinal apÃs a revacinaÃÃo. Identificamos o potencial papel da BCG em estimular a produÃÃo de IFN-γ e/ou IL-10 dependendo do antÃgeno e da idade do contato, sem relaÃÃo com o PPD e sorologia anti-PGL-1. Estes resultados sugerem que a BCG modula a resposta imune dos contatos frente aos antÃgenos do M. leprae, com o frequente aumento nos nÃveis de IFN-γ, frente ao MLT, nas crianÃas com idade entre 1 a 4 anos (4.203 + 539,2 pg/mL prÃ-BCG x 9.141 + 860,9 pg/mL pÃs-BCG, p=0,001). Jà os contatos entre 5 a 9 anos mostram aumento na produÃÃo de IL-10 (210 +39,4 pg/mL prÃ-BCG x 680 + 76,74 pg/mL pÃs-BCG, p=0,001) e IFN-γ (4.912 + 1.065 pg/mL x 9.249 + 1.171 pg/mL, p=0,024). Estes achados sÃo acompanhados com o aumento dos casos de hansenÃase em crianÃas entre 5 a 9 anos na comunidade. Entretanto, nos contatos entre 10 a 15 anos a revacinaÃÃo falha em induzir o aumento de IFN-γ e IL-10. Os contatos de MB produziram, simultaneamente, altos nÃveis de IFN-γ e IL-10 em resposta ao MLT, enquanto que os contatos de PB somente produziram altos nÃveis de IFN-γ apÃs a revacinaÃÃo. A produÃÃo de IFN-γ e IL-10 frente aos peptÃdeos sintÃticos p38 e p69 indica que estes antÃgenos podem ser possÃveis marcadores de infecÃÃo. Assim, a BCG pode ser protetora, sendo eficiente em induzir o aumento da resposta por IFN-γ nestes contatos, frente aos antÃgenos do M. leprae. / Studies in different populations have shown that vaccination with BCG provides partial protection against leprosy. However, need of boost BCG vaccination and your impact in response immune against antigens of Mycobacterium leprae is poorly understood, mainly in children who are contacts of these patients with leprosy, and constitute population of risk for illness. This study, we investigated the paper a second dose of BCG given to contacts of leprosy patients under the age of fifteen years in modulate the response imune. Blood samples of twenty five children, who received a first dose of BCG at birth, were collected immediately before and two months after BCG revaccination. Your peripheral blood mononuclear cells (PBMC) stimulated by proteins and peptides of M. leprae, were measured IFN-γ and IL-10 by an ELISA assay. Before revaccination the serum anti-PGL1 IgG and IgM isotypes were measured by ELISA assays performed with native PGL1- coated microplates, and PPD-skin reaction was measured 2 to 3 months after revaccination. The study was approved by the local Ethic Committee, number 006-08, May 7th, 2008. BCG revaccination can induces IFN-gamma and/or IL-10 production related to antigen and age, but did not correlated with PPD-skin reaction or anti-PGL1 levels. The PBMCâs production of IFN-gamma against MLT (total M. leprae antigen) after BCG was higher on children under 4 years old (N= 8, 4203,0  539,2 pg/mL before BCG x 9141,0  860,9 pg/mL after BCG, p=0,015, Mann-Whitneyâs test). Children between 5 and 9 years old showed an increase either of IFN-gama levels (N=11, 4912  1065 pg/mL x 9249,0  1171 pg/mL, p=0,024, Mann-Whitneyâs test) or IL-10 levels (N=11, 210,6  39,4 pg/mL x 680,3  76,74 pg/mL, p=0, 0,001, Mann-Whitneyâs test). However children between 10 and 15 years old failed to increase the cytokines levels after BCG booster. Contacts of multibacillary patients produced higher IFN-gamma levels (N= 13, 4536,0  543,1 pg/mL x 9263,0  989,0 pg/mL, p=0,0012, Mann-Whitneyâs test) and IL-10 levels (N= 13, 235,9  46,5 pg/mL x 743,5  87,8, p=0,0012, Mann-Whitneyâs test) against MLT after BCG, but only IFN-gamma levels (N=12, 4975,0  995,8 pg/mL x 8126,0  788,6, p=0,0342, Mann-Whitneyâs test) increased after BCG on contacts of paucibacillary patients. The production of IFN-gamma and IL-10 against two synthetic peptides (p38 and p67), before and after BCG vaccine, were similar as seen with MLT antigen, but the absence of cytokine production against p69 help to identify this peptide as a effective diagnostic marker.

BCG is the only licensed human vaccine currently available against TB. Derived from a virulent strain of M. bovis, the vaccine was thought to have struck a balance between reduced virulence and preserved immunogenicity. Nowadays, BCG vaccine strains used in different countries and vaccination programs show clear variations in their genomes and immune protective properties. The aim of this study was to characterize the proteomic profile on Mycobacterium bovis and five BCG strains Pasteur, Tokyo, Danish, Phipps and Birkhaug by Tandem Mass Tag® (TMT®)-labeling quantitative proteomic approach. In total, 420 proteins were identified and 377 of them were quantitated for their relative abundance. We reported the number and relationship of differential expressed proteins in BCG strains compared to M. bovis and investigated their functions by bioinformatics analysis. Several interesting up-regulated and down-regulated protein targets were found. The identified proteins and their quantitative expression profiles provide a basis for further understanding of the cellular biology of M. bovis and BCG vaccine strains, and hopefully would assist in the design of better anti-TB vaccine and drugs.

No description available.

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. 141-163. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.