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Master's Dissertation
DOI
https://doi.org/10.11606/D.9.2020.tde-05072021-171431
Document
Author
Full name
Luiz Adriano Damasceno de Queiroz
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Martins, Joilson de Oliveira (President)
Cruz, José Walber Miranda Costa
Almeida, Sandro Rogerio de
Vinolo, Marco Aurelio Ramirez
Title in Portuguese
Caracterização fenotípica e funcional de linfócitos T em camundongos diabéticos induzidos por aloxana e estreptozotocina
Keywords in Portuguese
ALX
DM1
linfócitos
STZ
Abstract in Portuguese
A aloxana (ALX) e a estreptozotocina (STZ) são os agentes diabetogênicos mais utilizados na indução do diabetes mellitus tipo 1 (DM1) em animais, com vários estudos associando seu uso a efeitos tóxicos na resposta imune. O presente estudo tem como objetivo comparar o fenótipo e a funcionalidade dos linfócitos T em tecidos linfoides e não linfoides de camundongos C57BL/6J normais e diabéticos. Os animais diabéticos (inoculados com ALX ou STZ) e controles não diabéticos (CT) os seguintes parâmetros avaliados: (a) contagem total e diferencial de células da medula óssea e sangue periférico; (b) caracterização da composição de linfócitos em timo, baço e pâncreas, com marcadores de superfície (CD11b, CD3, CD4, CD8, CD19 e CD25); (c) determinação das citocinas fator de necrose tumoral (TNF)-α, interferon (IFN)-γ, interleucina (IL)-1β, IL-2, IL-4, IL-6, IL-10, IL12p70 e IL-17 no homogenato de baço e pâncreas; (d) análise morfológica de timo, baço e pâncreas; (e) ativação da resposta adaptativa e produção de imunoglobulinas (IgG)1 e IgG2a em animais imunizados e desafiados com ovalbumina; (f) ativação dos linfócitos T do baço estimulados com concanavalina (ConA). Em relação ao grupo CT, o grupo STZ exibiu aumento nos neutrófilos e redução nos linfócitos na medula óssea. Tanto o grupo ALX quanto o STZ mostraram diminuição nos leucócitos, e aumento nos granulócitos no sangue total. O grupo STZ apresentou diminuição nos linfócitos, T CD4-CD8+ e CD4+CD8+ no timo e linfócitos CD19+ no pâncreas e no baço. O grupo ALX apresentou aumento do número de linfócitos CD4-CD8+, CD4+CD25+ e CD19+ no timo. As citocinas IL-1β do baço e IL-6 do pâncreas, do grupo STZ, diminuíram em comparação ao grupo CT. O timo apresentou atrofia da região medular, nos grupos ALX e STZ, com diminuição nas células reticuloepiteliais; morte celular de linfócitos/timócitos; e aumento nos corpúsculos Hassall, no grupo ALX. O pâncreas de ambos os grupos exibiu atrofia das ilhotas de Langerhans e infiltração inflamatória e hiperemia com dilatação vascular. A ativação in vitro de linfócitos do grupo STZ foi comprometida. As análises in vivo do teste de hipersensibilidade mostraram resposta mais intensa no grupo STZ, com produção semelhante de anticorpos IgG1 e IgG2a nos grupos diabéticos e controle. Embora ambos os agentes diabetogênicos ALX e STZ tenham afetado os órgãos linfoides e suas populações celulares, o impacto disso só se refletiu em alterações no comportamento da resposta imune in vivo e in vitro de animais tratados com STZ.
Title in English
Phenotypic and functional characterization of T lymphocytes of alloxan and streptozotocin induced diabetes
Keywords in English
ALX
Diabetes mellitus
lymphocytes
STZ
Abstract in English
Alloxan (ALX) and streptozotocin (STZ) are the most commonly used diabetogenic agents to induce type 1 diabetes mellitus (DM1) in animals, with several studies associating their use with toxic effects on the immune response. The present study aims to compare the phenotype and functionality of T lymphocytes in lymphoid and non-lymphoid tissues of normal and diabetic C57BL / 6J mice. Diabetic animals (inoculated with ALX or STZ) and non-diabetic controls (CT) evaluated the following parameters: (a) total and differential count of bone marrow cells and peripheral blood; (b) characterization of the lymphocyte composition in the thymus, spleen and pancreas, with surface markers (CD11b, CD3, CD4, CD8, CD19 and CD25); (c) determination of cytokines tumor necrosis factor (TNF) -α, interferon (IFN) -γ, interleukin (IL) -1β, IL-2, IL-4, IL-6, IL-10, IL12p70 and IL- 17 in the spleen and pancreas homogenate; (d) morphological analysis of the thymus, spleen and pancreas; (e) activation of the adaptive response and production of immunoglobulins (IgG) 1 and IgG2a in animals immunized and challenged with ovalbumin; (f) activation of spleen T lymphocytes stimulated with concanavalin (ConA). In relation to the CT group, the STZ group exhibited an increase in neutrophils and a reduction in lymphocytes in the bone marrow. Both the ALX and STZ groups showed a decrease in leukocytes and an increase in granulocytes in whole blood. The STZ group showed a decrease in T CD4-CD8+ and CD4+CD8+ lymphocytes in the thymus and CD19+ lymphocytes in the pancreas and spleen. The ALX group showed an increase in the number of CD4-CD8+ , CD4+CD25+ and CD19+ lymphocytes in the thymus. The cytokines IL1β of the spleen and IL-6 of the pancreas, in the STZ group, decreased compared to the CT group. The thymus presented atrophy of the medullary region, in the ALX and STZ groups, with a decrease in reticuloepithelial cells; cell death of lymphocytes / thymocytes; and increase in Hassall corpuscles, in the ALX group. The pancreas of both groups exhibited atrophy of the islets of Langerhans and inflammatory infiltration and hyperemia with vascular dilation. The in vitro activation of STZ group lymphocytes was compromised. In vivo analyzes of the hypersensitivity test showed a more intense response in the STZ group, with similar production of IgG1 and IgG2a antibodies in the diabetic and control groups. Although both the diabetogenic agents ALX and STZ affected the lymphoid organs and their cell populations, the impact of this was only reflected in changes in the behavior of the immune response in vivo and in vitro of animals treated with STZ.
 
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Publishing Date
2021-07-26
 
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