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Previous issue date: 2015-07-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction : Respiratory Syncytial Virus (RSV) is a major cause of viral lower respiratory tract infection in children under two years of age. Memory CD8 T cell response to VSR does not provide an efficient and long-lasting immune response, so there are recurrent infections throughout life. The role of mTOR (mammalian target of rapamycin) and the purinergic receptor P2X7 in the memory CD8 T cell response during RSV infection has not been investigated. Objectives : To analize the effect of rapamycin on dendritic cells (DCs) during RSV infection, as well as to evaluate the role of P2X7 purinergic receptor in CD8 memory T cells response during RSV infection. Methodology : Bone marrow derived dendritic cells (BMDCs) differentiated from C57BL/6 P2X7-/- and C57BL/6 mice were infected with VSR virus and used to activate T cells purified from C57BL/6 P2X7-/- and C57BL/6 mice in vitro for 96 hours. In addition, BMDCs differentiated from C57BL/6 mice received 20ng/ml rapamycin during 1h prior infection. The following parameters were evaluated: BMDC cell death by apoptosis, memory cells markers by flow cytometry and RNA viral quantitation was performed by real time PCR. Results : Rapamycin treatment in RSV infected BMDCs decreases the frequency of CD8+CD44high cells and increases the level of viral RNA in DCs, but the rapamycin does not affect the viability of the infected dendritic cells. Furthermore, when BMDCs were treated with rapamycin, an increase of BMDC survival occurred, depending on the contact with the T cells. The absence of purinergic receptor P2X7 in T cells leads to a decrease in the frequency of CD8+CD122+KLRG1-, however the absence of purinergic receptor in infected BMDCs increases the frequency of CD8+CD122+ KLRG1- T cells. Conclusion : Our study suggests that P2X7 receptor is involved in memory CD8 T cell response. In addition our data indicated that mTOR inhibition increases the survival of BMDCs in a mechanism dependent on T-cell contact and also suggests that rapamycin treatment on dendritic cells during VSR infection affect CD8 T cell differentiation. / Introdu??o : O V?rus Sincicial Respirat?rio (VSR) ? um dos principais causadores de infec??o viral do trato respirat?rio inferior em crian?as menores de dois anos de idade. A resposta de c?lulas T CD8 de mem?ria para VSR n?o apresenta uma resposta imune eficiente e duradoura, por isso s?o recorrentes as infec??es durante a vida. At? o momento n?o se tem o conhecimento sobre o envolvimento do mTOR (mammalian target of rapamycin) e do receptor purin?rgico P2X7 na resposta de c?lulas T CD8 de mem?ria durante a infec??o de VSR. Objetivos : Investigar o efeito da rapamicina nas c?lulas dendr?ticas (DCs) durante a infec??o viral, bem como, avaliar o efeito do receptor purin?rgico P2X7 nas c?lulas dendr?ticas (DCs) infectadas com o v?rus VSR na resposta de c?lulas T CD8 de mem?ria. Metodologia : C?lulas dendr?ticas diferenciadas de medula ?ssea (BMDCs) de camundongos C57BL/6 P2X7-/- e C57BL/6 infectadas com VSR foram utilizadas para ativar c?lulas T purificadas de camundongos C57BL/6 P2X7-/- e C57BL/6 durante 96 horas. Al?m disso, um grupo de BMDCs de camundongos C57BL/6 recebeu 20ng/mL de rapamicina durante 1h antes da infec??o. As DCs foram marcadas para investiga??o de morte celular por apoptose, as c?lulas T marcadas para an?lise c?lulas de mem?ria e a quantifica??o do RNA viral foi realizada atrav?s de PCR em tempo real. Resultados : O tratamento com o inibidor de mTOR nas BMDCs infectadas pelo VSR diminuiu a gera??o de c?lulas T CD8+ CD44high e aumentou os n?veis de RNA viral nas DCs, por?m a rapamicina n?o influenciou a viabilidade das c?lulas dendr?ticas infectadas. Quando as BMDCs foram tratadas com rapamicina, houve um aumento de sobreviv?ncia das c?lulas, dependente do contato com as c?lulas T. Na aus?ncia do receptor purin?rgico P2X7 nas c?lulas T, ocorreu uma diminui??o na frequ?ncia das c?lulas T CD8+CD122+KLRG1-, entretanto a aus?ncia do receptor purin?rgico nas BMDCs infectadas aumentou a frequ?ncia destas c?lulas T CD8+CD122+KLRG1-. Conclus?o : Nosso estudo sugere que o receptor P2X7 est? envolvido na resposta de c?lulas T CD8 de mem?ria durante a infec??o pelo VSR. Al?m disso, sugere-se que as c?lulas dendr?ticas tratadas com rapamicina durante a infec??o com VSR prejudica a diferencia??o de c?lulas T CD8.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/6375 |
| Date | 29 July 2015 |
| Creators | Freitas, Deise do Nascimento de |
| Contributors | Souza, Ana Paula Duarte de, Morrone, Fernanda Bueno |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Medicina/Pediatria e Sa?de da Crian?a, PUCRS, Brasil, Faculdade de Medicina |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 3098206005268432148, 600, 600, 600, 600, -8624664729441623247, -969369452308786627, 2075167498588264571 |
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