Abstract The characteristics of humoral immunity have been proposed to reflect the bias between two T helper (Th) lymphocyte subsets: Th1 cells, which activate cell-mediated immunity, and Th2 cells, which mediate humoral immunity. The present study aimed to characterize the humoral immunity to beta-cell autoantigens insulin and glutamic acid decarboxylase (GAD65) in preclinical and clinical type 1 diabetes. Insulin antibodies were analyzed in pregnant women with or without type 1 diabetes and their newborn infants and in prediabetic children. Epitope or/and isotype-specific GAD65 antibodies (GAD65Abs) were analyzed in prediabetic children, in children and adolescents diagnosed with type 1 diabetes, and in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome. Antibodies were determined by radioligand immunoassays. The humoral immune response to insulin and GAD65 was observed to be a highly dynamic process, comprising mainly the IgG1 subclass and, less frequently, other IgG subclasses. GAD65Abs were directed primarily to the middle region and secondarily to the C-terminal region of GAD65 as a consequence of epitope spreading. Young children who progressed to overt type 1 diabetes were characterized by a broad initial isotype response to insulin and GAD65 and by a strong IgG1 and IgG3 response to insulin. Children who did not progress to clinical type 1 diabetes were characterized by an emerging IgG4 response to GAD65. Rising levels of GAD65Abs targeted to the middle region of GAD65 were associated with high titers of islet cell antibodies and a decreased requirement for exogenous insulin, probably reflecting a persistent residual beta-cell mass, in patients with manifest type 1 diabetes. Non-immunoglobulin insulin-binding activity was observed to be induced by pregnancy. APECED-associated humoral autoimmunity to GAD65 did not differ markedly from that observed in subjects with type 1 diabetes alone. In conclusion, isotype-specific GAD65 and especially insulin antibodies are valuable markers of the risk of progression to type 1 diabetes in young children. The appearance of an initial IgG3 subclass response and a strong IgG3 response to insulin in children who progressed to overt type 1 diabetes may reflect the role of cytotoxic Th1-biased immunity in the disease process leading to clinical presentation of type 1 diabetes.

Orientador: Hélio José Montassier / Banca: Antonio Carlos Alessi / Banca: Ricardo Luiz Moro de Souza / Banca: Luis Francisco Prata / Banca: Antonio Carlos Paulillo / Resumo: O vírus da bronquite infecciosa aviária (VBIG) permanece como um dos principais problemas para a avicultura industrial. Para a imunoprofilaxia dessa virose existem vacinas "vivas" atenuadas ou inativadas, mas elas não são efetivas para o controle dessa doença infecciosa a longo prazo, especialmente contra estirpes variantes desse vírus. A função primordial das vacinas usuais contra o VBIG são estimular as respostas imunes sistêmicas e locais, mediadas por anticorpos ou por células T efetoras. Com a finalidade de investigar os mecanismos envolvidos na imunidade protetora, os níveis de anticorpos locais e sistêmicos e a expressão de genes associados a respostas de linfócitos T citotóxicos, tais como o CD8 e a granzima A foi avaliada na mucosa traqueal, após a imunização primária de pintinhos de 1 dia de idade com vacinas atenuadas contra o VBIG seguida do desafio 42 dias depois. Proteção ao desafio foi avaliada por meio da ciliostase traqueal, histopatologia e detecção de vírus pela técnica de RT-PCR em tempo real na traquéia. Os níveis de anticorpos no soro e na secreção lacrimal foram mensurados pelo Sandwich- Concanavalina A - ELISA. A expressão dos genes de CD8 e de granzima A foram avaliadas pela técnica de RT-PCR em tempo real. Os resultados demonstraram que a proteção contra a infecção pelo VBIG em aves vacinadas se correlacionou com os níveis de anticorpos anti-virais específicos dos isótipos IgG, IgM e IgA na secreção lacrimal e com os níveis de expressão de CD8 e de granzima A. Concluindo, os resultados possibilataram definir o perfil cinético do desenvolvimento das respostas imunes de memória contra o VBIG na mucosa que é o sítio primário de replicação viral e indicaram que os mecanismos de imunidade humoral e celular podem ser muito importantes para a proteção de hospedeiros naturais contra a infecção pelo VBIG. / Abstract: Avian infectious bronchitis (IBV) remains a major problem in the poultry industry. Live and inactivated vaccines are available, but they are not effective long term in controlling IBV infection, specially against variant strains. The essential function of existing IBV vaccines is to elicit, ideally, local and systemic specific antibodies, as well as cell-mediated immunity to this virus. To investigate the mechanisms of protective immunity, the levels of systemic and local specific antibodies and the expression of genes responsible for cytotoxic T cell killing such as CD8-marker and granzyme-A at tracheal mucosa was evaluated after the primary immunization of 1- day-old chicks with an attenuated avian infectious bronchitis virus (IBV) and challenge 42 days later. Challenge protection was evaluated by tracheal ciliostasis, histopathology and virus detection by real time RT-PCR. The serum and lachrymal anti-IBV antibody levels of IgG, IgM and IgM isotypes were measured with a Sandwich-ELISA Concanavalina-A method. The expression of CD8 and granzyme A genes were evaluated by real time RT-PCR. The results showed protection against challenge with the M-41. Lachrymal IgG, IgM and IgA anti-IBV specific antibodies and the levels of expression of CD8 and granzyme A genes correlated significantly with the protection to challenge. Overall, the results provided the kinetics on the development of memory mucosal immune responses against IBV at the primary replication site and indicate that tracheal humoral and cellular immune mechanisms may be very important in protecting natural hosts against IBV infection. / Mestre

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-14Bitstream added on 2014-06-13T20:36:40Z : No. of bitstreams: 1 santos_il_me_jabo.pdf: 445425 bytes, checksum: 8eabdcdd56ae9af7bcf160cad345bd86 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da bronquite infecciosa aviária (VBIG) permanece como um dos principais problemas para a avicultura industrial. Para a imunoprofilaxia dessa virose existem vacinas “vivas” atenuadas ou inativadas, mas elas não são efetivas para o controle dessa doença infecciosa a longo prazo, especialmente contra estirpes variantes desse vírus. A função primordial das vacinas usuais contra o VBIG são estimular as respostas imunes sistêmicas e locais, mediadas por anticorpos ou por células T efetoras. Com a finalidade de investigar os mecanismos envolvidos na imunidade protetora, os níveis de anticorpos locais e sistêmicos e a expressão de genes associados a respostas de linfócitos T citotóxicos, tais como o CD8 e a granzima A foi avaliada na mucosa traqueal, após a imunização primária de pintinhos de 1 dia de idade com vacinas atenuadas contra o VBIG seguida do desafio 42 dias depois. Proteção ao desafio foi avaliada por meio da ciliostase traqueal, histopatologia e detecção de vírus pela técnica de RT-PCR em tempo real na traquéia. Os níveis de anticorpos no soro e na secreção lacrimal foram mensurados pelo Sandwich- Concanavalina A – ELISA. A expressão dos genes de CD8 e de granzima A foram avaliadas pela técnica de RT-PCR em tempo real. Os resultados demonstraram que a proteção contra a infecção pelo VBIG em aves vacinadas se correlacionou com os níveis de anticorpos anti-virais específicos dos isótipos IgG, IgM e IgA na secreção lacrimal e com os níveis de expressão de CD8 e de granzima A. Concluindo, os resultados possibilataram definir o perfil cinético do desenvolvimento das respostas imunes de memória contra o VBIG na mucosa que é o sítio primário de replicação viral e indicaram que os mecanismos de imunidade humoral e celular podem ser muito importantes para a proteção de hospedeiros naturais contra a infecção pelo VBIG. / Avian infectious bronchitis (IBV) remains a major problem in the poultry industry. Live and inactivated vaccines are available, but they are not effective long term in controlling IBV infection, specially against variant strains. The essential function of existing IBV vaccines is to elicit, ideally, local and systemic specific antibodies, as well as cell-mediated immunity to this virus. To investigate the mechanisms of protective immunity, the levels of systemic and local specific antibodies and the expression of genes responsible for cytotoxic T cell killing such as CD8-marker and granzyme-A at tracheal mucosa was evaluated after the primary immunization of 1- day-old chicks with an attenuated avian infectious bronchitis virus (IBV) and challenge 42 days later. Challenge protection was evaluated by tracheal ciliostasis, histopathology and virus detection by real time RT-PCR. The serum and lachrymal anti-IBV antibody levels of IgG, IgM and IgM isotypes were measured with a Sandwich-ELISA Concanavalina-A method. The expression of CD8 and granzyme A genes were evaluated by real time RT-PCR. The results showed protection against challenge with the M-41. Lachrymal IgG, IgM and IgA anti-IBV specific antibodies and the levels of expression of CD8 and granzyme A genes correlated significantly with the protection to challenge. Overall, the results provided the kinetics on the development of memory mucosal immune responses against IBV at the primary replication site and indicate that tracheal humoral and cellular immune mechanisms may be very important in protecting natural hosts against IBV infection.