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Doctoral Thesis
DOI
https://doi.org/10.11606/T.25.2016.tde-03022016-152747
Document
Author
Full name
Heliton Gustavo de Lima
Institute/School/College
Knowledge Area
Date of Defense
Published
Bauru, 2015
Supervisor
Committee
Lara, Vanessa Soares (President)
Buzalaf, Camila Peres
Furuse, Cristiane
Mayer, Marcia Pinto Alves
Oliveira, Rodrigo Cardoso de
Title in Portuguese
Mecanismos de defesa dos mastócitos contra o periodontopatógeno Aggregatibacter actinomycetemcomitans: atividade microbicida intracelular
Keywords in Portuguese
Aggregatibacter actinomycetemcomitans
Doença periodontal
Mastócitos
Óxido nítrico
Peróxido de Hidrogênio
Abstract in Portuguese
Os mastócitos (MCs) estão presentes tanto no periodonto normal quanto inflamado, em diferentes quantidades e em vários locais. Nos últimos anos, a eficácia e a contribuição dos MCs em eliminar bactérias, através de sua atividade microbicida intracelular, estão se tornando cada vez mais reconhecidas. Assim, a partir de MCs murinos desafiados in vitro com o periodontopatógeno Aggregatibacter actinomycetemcomitans (ATCC 29523) por 3, 5, 10 e 24 horas, o presente estudo teve como objetivo investigar a capacidade microbicida intracelular de MCs, e comparar com a capacidade microbicida de macrófagos peritoneais murinos (MPs), considerados fagócitos profissionais, por meio da contagem das unidades formadoras de colônias. Além disso, avaliou-se a produção e liberação de mediadores microbicidas, óxido nítrico (NO) e peróxido de hidrogênio (H2O2), por meio do método colorimétrico de Griess e pela degradação de substratos fluorescentes, respectivamente. Para a análise estatística, foram utilizados os testes estatísticos ANOVA Fatorial seguido do teste de Tukey e teste de correlação de Pearson (p<0.05). Nossos resultados revelaram que os MCs foram capazes de eliminar eficientemente o periodontopatógeno, principalmente após 10h de desafio intracelular. Comparando-se a atividade microbicida dos dois tipos celulares, verificou-se, nos períodos de 3h e 5h de desafio, um menor percentual de colônias viáveis no interior de MPs, em comparação aos MCs. Inversamente, nos períodos de 10h e 24h, observaram-se menores valores percentuais de colônias intracelulares nos MCs em relação aos MPs. Além disso, a produção/liberação de NO bem como, em menor proporção, de H2O2 pelos MCs foram concordantes com a sua capacidade microbicida. Este é o primeiro estudo que demonstra a eficiente ação microbicida intracelular de MCs murinos contra Aggregatibacter actinomycetemcomitans, com produção e liberação de substâncias potencialmente bactericidas, e de forma mais eficaz que os macrófagos. Esses resultados sugerem a importância dessas células nos mecanismos de defesa presentes na doença periodontal induzida por placa dentobacteriana.
Title in English
Defense mechanisms of mast cells against periodontopathogen Aggregatibacter actinomycetemcomitans: intracellular microbicidal activity
Keywords in English
Aggregatibacter actinomycetemcomitans
Hydrogen peroxide
Mast cells
Nitric oxide
Periodontal diseases
Abstract in English
Mast cells (MCs) are present in both normal and inflamed periodontal tissues, in varying amounts and locations. Recently, MCs contribution in eliminating bacteria and its effectiveness, through its intracellular microbicidal activity, have been increasingly recognized. Thus, this study aimed to investigate the intracellular microbicide capacity of MCs, and compare it with the microbicide capacity of murine peritoneal macrophages (MPs), considered professional phagocytes, by counting the colony forming units. Both cell types were challenged in vitro with periodontopathogen Aggregatibacter actinomycetemcomitans (ATCC 29523) by 3, 5, 10 and 24 hours. Additionally, the production and release of microbicidal agents, nitric oxide (NO) and hydrogen peroxide (H2O2) were evaluated by means of colorimetric Griess method and by the degradation of fluorescent substrates, respectively. Statistical analysis was performed by ANOVA Factorial test followed by Tukey and Pearson's correlation test (p <0.05). Our results revealed that MCs are able to efficiently eliminate periodontopathogen, mainly after 10 hours of intracellular challenge. The microbicidal activity of both cell types, in 3 and 5 hours of challenge showed a lower percentage of viable colonies inside MPs, compared to MCs. Contradictorily, in 10 and 24 hours a lower percentage of intracellular colonies in MCs was observed in relation to MPs. Moreover, the production/release of NO and, in minor proportion, of H2O2 by MCs was in agreement with its microbicidal capacity. Therefore, this is the first report to describe the intracellular microicidal activity of murine MCs against Aggregatibacter actinomycetemcomitans, concerning production and release of potentially bactericidal substances, which is more effective than macrophages. These results suggest the importance of these cells in pathogenesis and defense mechanisms of biofilm-associated periodontal disease.
 
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Publishing Date
2016-02-04
 
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