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Master's Dissertation
DOI
10.11606/D.17.2010.tde-20072010-100607
Document
Author
Full name
Daiane Fernanda dos Santos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2010
Supervisor
Committee
Faccioli, Lucia Helena (President)
Marchetti, Juliana Maldonado
Martins, Vania Luiza Deperon Bonato
Title in Portuguese
Atividades in vitro e in vivo de microesferas biodegradáveis contendo leucotrieno B4 e/ou antígenos livres de células de Histoplasma capsulatum
Keywords in Portuguese
histoplasmose
imunomodulação
leucotrieno B4
microesferas biodegradáveis
Abstract in Portuguese
O Histoplasma capsulatum é um fungo dimórfico responsável por infecções pulmonares graves caracterizadas por reação granulomatosa. Sua incidência vem aumentando nos últimos anos devido principalmente às alterações imunológicas relacionadas ao comprometimento da imunidade celular. Anteriormente, nosso grupo de pesquisa demonstrou o envolvimento dos leucotrienos (LTs) nos mecanismos de defesa do hospedeiro durante a histoplasmose. Além disso, demonstrou que antígenos livres de células (CFAgs, do inglês cell-free antigens), derivados de H. capsulatum, quando empregados na imunização de animais, conferem proteção eficiente aos mesmos e controle da infecção, uma vez que ativam a imunidade celular, e aumento da produção de LTs nos pulmões dos animais imunizados. Assim, nosso grupo desenvolveu microesferas (MS) biodegradáveis, constituídas de ésteres derivados dos ácidos láctico e glicólico (PLGA), contendo LTB4. Estas MS foram avidamente fagocitadas por macrófagos in vitro e aumentaram o recrutamento de leucócitos para os pulmões, quando administradas intratraquealmente. Diante do papel dos LTs na histoplasmose e dos potenciais terapêutico e profilático dos CFAgs, o objetivo deste estudo foi avaliar as atividades biológicas in vitro e in vivo de MS contendo LTB4 e/ou CFAgs. Assim, foram desenvolvidas MS (PLGA) contendo LTB4 e/ou CFAgs através do processo de simples ou dupla emulsão seguido pela extração do solvente. Este método permitiu uma eficiente encapsulação tanto do mediador lipídico quanto dos antígenos protéicos e um perfil de liberação sustentada ao longo dos dias avaliados. O potencial zeta e a morfologia das MS não foram alterados com o processo de microencapsulação; da mesma forma, a integridade dos CFAgs não foi interferida. Para estudos in vitro, empregamos macrófagos diferenciados de medula óssea murina (BMDM). O tamanho adequado das MS contribuiu para sua eficiente fagocitose pelos BMDM e as MS contendo LTB4 e/ou CFAgs modularam a produção de TNF-, IL-1, IL-6 e IL-12, quimiocinas (KC, MCP-1 e RANTES), e nitrito, sendo que as MS-CFAgs mostraram-se mais potentes. O estímulo com as diferentes MS induziu discreto aumento na expressão de CD86 na superfície de BMDM. Neste contexto, verificamos o envolvimento do fator de transcrição NF-B durante a ativação de BMDM induzida pelas MS. Apesar da eficiente ativação dos BMDM induzida pelas MS, não foi possível evidenciar uma resposta imune celular em animais imunizados com as MS-LTB4+CFAgs ou MS-CFAgs. Portanto, futuros experimentos deverão ser realizados a fim de investigar o potencial profilático das MS contendo LTB4 e/ou CFAgs na histoplasmose.
Title in English
In vitro and in vivo activities of biodegradable microspheres containing leukotriene B4 and/or cell-free antigens from Histoplasma capsulatum
Keywords in English
biodegradable microspheres
histoplasmosis
immunomodulation
leukotriene B4
Abstract in English
Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a pulmonary disease characterized by chronic granulomatous reaction. In the last years, the incidence of histoplasmosis has increased, mainly as a result of the immunological alterations involved with deficiency of the cellular immunity. Previously, our research group demonstrated the involvement of leukotrienes (LTs) on host defense mechanisms during the histoplasmosis. Cell-free antigens (CFAgs) derived from H. capsulatum, when employed for animals´ immunization, can confer efficient protection and control of the infection, since they activate the cellular immunity. Furthermore, the protection of CFAgs-immunized mice was associated with increased LTB4 generation in the lungs. Based on these results, our group developed biodegradable microspheres (MS) based on PLGA containing LTB4. We showed that these MS were phagocytosed by macrophages in vitro and increased the leukocyte recruitment into the lungs, when administrated via intratracheal. Because the role of leukotrienes in the histoplasmosis and therapeutic and profilatic effects of CFAgs, the aim of this study was evaluate the in vitro and in vivo biological activities of MS containing LTB4 and/or CFAgs. Then, MS (PLGA) containing LTB4 and/or CFAgs were developed through simple or double emulsion/extraction process. This method allowed an efficient encapsulation of the lipid mediator and CFAgs, and a sustained release profile during the evaluated days. Zeta potential and morphology of MS were not altered with the microencapsulation process; CFAgs integrity was not interfered. For in vitro studies, we employed bone marrow-derived macrophages (BMDM). The appropriate size of MS contributed for efficient uptake by BMDM. MS containing LTB4 and/or CFAgs modulated the TNF-, IL-1, IL-6 and IL-12, chemokines (KC, MCP-1 and RANTES), and nitrite production by BMDM, since the MS-CFAgs showed potent immunostimulant effect. Moreover, the stimulus with different MS provoked a discreet increase in the CD86 cell expression. Also we verified an involvement of the transcription factor NF-B during the BMDM activation induced by MS. Even though the in vitro biological activities on BMDM, it was not possible to evidence a cellular immune response in immunized mice with the MS-LTB4+CFAgs or MS-CFAgs. Therefore, future experiments should be conducted in order to investigate the profilatic potential of MS containing LTB4 and/or CFAgs in the histoplasmosis.
 
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mestrado.pdf (18.49 Mbytes)
Publishing Date
2010-08-27
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • SANTOS, Daiane F. dos, et al. Biodegradable microspheres containing leukotriene B4 and cell-free antigens from Histoplasma capsulatum activate murine bone marrow-derived macrophages. European Journal of Pharmaceutical Sciences, 2011, vol. 44, n. 5, p. 580-588.
  • SANTOS, Daiane F. dos, et al. Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres. European Journal of Pharmaceutical Sciences, 2009, vol. 38, n. 5, p. 548-555.
  • SANTOS, Daiane F. dos, et al. Evaluation of cell activation by leukotriene B4 and cell-free antigens from Histoplasma capsulatum entrapped into PLGA microspheres. In XXXIV Congress of the Brazilian Society for Immunology and X International Symposium on Allergy and Clinical Immunology, Salvador, 2009.
  • SANTOS, Daiane F. dos, et al. Immunostimulant effect of biodegradable microspheres containing cell-free antigens from Histoplasma capsulatum on murine bone marrow-derived macrophages. In 37th Annual Meeting & Exposition of the Controlled Release Society, Portland, 2010.
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