Stroke is a neurovascular disease that frequently results in decreased motor and cognitive functioning. Obesity is a major risk factor associated with ischemic stroke and is thought to worsen the functional deficits observed after stroke. Previous findings from our laboratory suggest that worse motor deficits in obese animals may be a result from an exacerbated neuroinflammatory response. Most animal studies demonstrate an association between obesity and worse cognitive functioning after stroke. However, the mechanisms are not well studied. This study examines the neuroinflammatory response, ischemic brain tissue damage, and cognitive functioning in diet-induced obese mouse models during the chronic phase after ischemic stroke, defined as weeks after stroke. Our study found an earlier onset of cognitive deficits in obese mice after stroke compared to normal weight mice. We found no differences in the degree of brain damage in obese animals and normal weight animals 11 weeks after stroke, but observed higher levels of microgliosis in obese animals compared to normal weight animals. Due to the limitations of our study, additional studies should be done to assess the severity of cognitive deficits in obese animals compared to normal weight animals in the chronic phase after stroke. Further studies also need to be done to confirm our findings regarding the microglial response and degree of ischemic brain damage during the chronic phase.

Camundongos C3H/HePAS infectados com Trypanosoma cruzi Sylvio X10/4 desenvolvem cardiomiopatia chagásica crônica semelhante aquela observada em pacientes portadores da doença de Chagas. A análise das células que infiltram o coração na fase crônica mostra uma maior frequência de leucócitos e maior expressão de PD-1 e PD-L1, moléculas inibitórias do sistema imune. Visando restaurar a resposta, tratamos camundongos primeiramente com anticorpos anti-PD-L1 e posteriormente com anticorpos anti-PD-L1 e anti-PD-1 em diferentes experimentos, que falharam em exercer uma diferença significativa na patologia cardíaca, parasitemia do sangue ou do coração associado ao aumento da resposta imune. Para fornecer co-estímulo além de bloquear interações inibitórias, realizamos o tratamento com anti-PD-1 e anti-PD-L1 associado a T. cruzi irradiado, que aumentou a patologia cardíaca e a bradicardia, assim como diminuiu a parasitemia sanguínea, sem mudanças no perfil das células T. O bloqueio das interações inibitórias associado ao desafio e ao co-estímulo das células T, leva a uma piora no funcionamento cardíaco associado à diminuição da parasitemia nos camundongos crônicos. / C3H/HePAS mice infected with T. cruzi Sylvio X10/4 parasites develop a chronic cardiomyopathy like the observed in human chagasic patients. Flow cytometry analysis of heart-infiltrating cells in chronically infected mice revealed higher leukocyte frequency with increased PD-1 and PD-L1 expression. In order to restore the immune response, mice were treated with anti-PD-L1 or with anti-PD-L1 and anti-PD-1, but neither exerted effects at heart pathology and parasitemia nor restored T cell response. Hence, we evaluated the possibility of using irradiated T. cruzi challenge to provide parasite antigen and co-stimulatory signaling to exhausted cells. Thus, association of anti-PD-1 and anti-PD-L1 treatment with irradiated T. cruzi challenge increased heart pathology and bradycardia as well as reduced blood parasitemia. Additionally, the treatment does not change T cell phenotype. Blocking both inhibitory interactions and provide antigen with co-stimulation seems to cause a loss in heart function despite decreasing parasitemia.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Chagas disease, an American trypanosomiasis caused by Trypanosoma cruzi, was discovered by Carlos Chagas in 1909, and presents a broad spectrum of clinical manifestations, including the acute and chronic phases. The latter is divided into three main forms: digestive, cardiac and indeterminate. The purposes of this study were to evaluate the immunoreactivity profile of recombinant peptides selected against purified human IgG from chronic chagas disease patients by Phage Display, to assess differential recognition patterns in the immune response among disease clinical forms, and to select mimotopes with potential use in diagnostics. The enzyme-linked immunosorbent assay (ELISA) was conducted with all 50 clones obtained after four rounds of selection. The trials were performed with phages supernatant. In order to evaluate the specific antibody response, plaque reduction tests were performed with clones that showed better performance in ELISAs. ELISA tests showed that the immune profile of some clones suggested the tendency of patients with the indeterminate form to develop cardiac or digestive form. Phage display approach has generated specific mimotopes that present differential recognition patterns of the immune response between the cardiac and digestive clinical forms of Chagas disease, and these mimotopes may become potential targets for vaccine development and for differential diagnostics of clinical forms. / A doença de Chagas foi descoberta e descrita por Carlos Chagas em 1909, e representa uma importante doença parasitária crônica causada pelo protozoário flagelado Trypanosoma cruzi. Depois de adquirida, a tripanossomíase americana pode apresentar-se sob duas fases: aguda e crônica, sendo esta dividida em três formas principais: indeterminada, digestiva e cardíaca. Objetivamos avaliar o perfil imunorreativo de peptídeos recombinantes selecionados contra IgG humana purificada de pacientes com as formas cardíaca, digestiva e indeterminada da fase crônica da doença de Chagas por Phage Display, a fim de avaliar a possibilidade de um padrão diferenciado de resposta imunológica entre as três formas da doença e buscar clones com potencial diagnóstico. Os ELISAs foram realizados com todos os 50 clones distintos obtidos pelos quatro ciclos de seleção, utilizando-se sobrenadante dos fagos como antígenos no sistema de captura. Além disso, foi realizado teste de redução em placas com os clones com melhor desempenho de reatividade nos ELISAs, a fim de demonstrar a especificidade de ligação dos anticorpos com esses epítopos. Os testes realizados demonstraram que o perfil imunorreativo de alguns clones sugere a propensão de indivíduos portadores da forma indeterminada a evoluírem para a forma cardíaca ou digestiva da doença, além de demonstrarem clones potenciais para o diagnóstico diferencial entre as formas cardíaca e digestiva, bem como epítopos para detecção geral da doença de Chagas. / Mestre em Ciências da Saúde