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Doctoral Thesis
DOI
10.11606/T.5.2005.tde-14082007-140450
Document
Author
Full name
Maria Roseli Monteiro Callado
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2005
Supervisor
Committee
Yoshinari, Natalino Hajime (President)
Bonfa, Eloisa Silva Dutra de Oliveira
Fernandes, Sandra Regina Muchinechi
Pereira, Rosa Maria Rodrigues
Provenza, José Roberto
Title in Portuguese
Caracterização da resposta imune em modelo experimental de esclerodermia induzida por colágeno tipo V
Keywords in Portuguese
Coelhos
Colágeno/efeitos adversos
Escleroderma difusa/induzido quimicamente
Esquema de imunização
Formação de anticorpos/ imunologia
Modelos animais de doenças
Abstract in Portuguese
Os modelos experimentais reproduzem doenças que acometem seres humanos, sendo de extrema importância porque possibilitam o estudo da patogênese e abordagem terapêutica dessas enfermidades. Nesse grupo inclui-se o modelo experimental de esclerodermia induzida pela imunização de coelhos com colágeno V humano provocando alterações histológicas (pele, pulmão e rim) similares àquelas observadas em humanos. As doenças auto-imunes têm sua etiologia desconhecida e são particularmente caracterizadas pela presença de auto-anticorpos no soro. Nesse aspecto, 90 a 95% dos pacientes com esclerodermia apresentam algum auto-anticorpo contra antígenos intracelulares (proteínas nucleolares RNA polimerase I, II e III, Scl-70, centriolares ou golginas) ou da matriz extracelular (colágeno). O presente estudo tem como objetivo avaliar a resposta imunológica nos animais do modelo experimental de esclerodermia. Para tanto, o soro dos animais foram testados quanto à presença de auto-anticorpos e de outros fatores imunológicos séricos que indicassem um processo imunológico ativo paralelo às lesões teciduais em desenvolvimento e incluiu: pesquisa de anticorpos anticolágenos V, III e I, imunocomplexos circulantes, fator reumatóide, níveis de complemento, fatores antinucleares (FAN) por imunofluorescência indireta em células HEp-2 e caracterização dos antígenos alvos por immunoblot. A análise da resposta imune revelou que as alterações histológicas do pulmão, rim e pele se desenvolviam com o aumento dos níveis séricos de anticorpos anti-colágeno V. Além disso, todos os animais imunizados com Col V apresentavam anticorpos para outros tipos de colágeno. Esses animais desenvolveram uma marcante resposta imunológica a antígenos intracelulares com 100% de positividade para o FAN e presença de imunocomplexos nos soros obtidos 30, 75 e 120 dias pós-imunização quando comparados a dois grupos controles (albumina e adjuvante completo de Freud). Todos os animais do grupo Col V apresentaram na IFI padrão citoplasmático Golgi símile e, em 10% deles, reatividade adicional aos centríolos. Ambos auto-anticorpos são raros, mas já descritos em pacientes com esclerodermia. A pré-absorção dos soros desses animais com Col V não interferiu no resultado do FAN. A caracterização dos antígenos alvos mostrou uma reatividade uniforme a proteínas de alto peso molecular de células epiteliais humanas (maior ou igual 175 kDa) que progredia com o tempo de imunização. Eluatos ácidos contendo os anticorpos anti-fração 175 kDa reproduziram o padrão de IFI igual ao soro original. As análises demonstram que a imunização com Col V humano em coelhos resulta numa resposta imunológica exuberante e que se caracteriza pela presença de auto-anticorpos a componentes intracelulares.
Title in English
Humoral immune response characterization of the type V collagen induced scleroderma experimental model
Keywords in English
Antibody formation/immunology
Collagen/adverse effects
Disease models animal
Immunization schedule
Rabbits
Scleroderma diffuse/chemically induced
Abstract in English
Experimental models for human diseases are of utmost importance, since they allow the study of their pathogenesis and therapeutic approach. In this group we can include the experimental model of collagen V-induced scleroderma, in rabbits, with histological alterations (skin, lung and kidney) similar to those observed in humans. Auto-immune diseases have an unknown etiology, and are characterized by the presence of auto-antibodies in serum. In this aspect, 90%-95% of the patients with scleroderma present some kind of auto-antibody against intracellular antigens (RNA polymerase I, II and III nucleoproteins, Scl-70, centriolar or golgins) or to components of the extracellular matrix (collagen). This study aims to assess the immune response of the animal subjects in a scleroderma experimental model. The animals sera were tested for auto-antibodies and other serum immunological factors that would point to an active immunological process, parallel to the developing tissue lesions, including anti-collagen V, III and I antibodies, circulating immune complexes, rheumatoid factor, complement levels, antinuclear antibodies (ANA) by indirect immunofluorescence in HEp-2 (IFI) cells, and characterization of target antigens with an immunoblot. The analysis of the immune response in the studied animals revealed that histological alterations in lungs, kidneys and skin developed with an increase of the serum levels of anti-collagen V antibodies. Furthermore, all the Col Vimmunized animals presented antibodies against other types of collagen as well. These animals also developed a strong immune response against intracellular antigens, being 100% positive for ANA, showing also immune complexes in sera obtained 30, 75 and 120 days after immunization with Col V, when compared to the control groups (albumin and Freunds complete adjuvant). All the animals from the Col V group showed a cytoplasmic pattern at the IFI, Golgi simile and, in 10% of the cases, additional reactivity to the centrioles. Both auto-antibodies are rare, yet were already described in patients with scleroderma. The pre-absorption of these animals sera with Col V did not interfere with the ANA reactivity. The characterization of the target antigens showed a uniform reactivity to high molecular weight proteins of human epithelial cells (> or = 175 kDa), which progressed with the immunization time. Acid eluats containing antibodies against the 175 kDa fraction reproduced the same IFI reactivity pattern of the original serum. The results demonstrate that immunization of rabbits with human Col V results in an exuberant immune response, characterized by the presence of autoantibodies against intracellular components.
 
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Publishing Date
2007-08-22
 
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