INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.

INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.

Orientador: Daniela Leal Zandim-Barcelos / Banca: Joni Augusto Cirelli / Banca: Flávia Aparecida Chaves Furlaneto Messora / Resumo: Os peptídeos antimicrobianos são componentes da resposta imune inata, que apresentam propriedades imunomodulatórias adjuntas às propriedades antimicrobianas e, portanto, podem ter um papel chave na susceptibilidade ou resistência a doenças na cavidade oral.O propósito deste estudo foi investigar a influência do tabagismo sobre os níveis dos peptídeos antimicrobianos, alfa-defensinas (HNP 1-3) e LL-37, no fluido crevicular gengival de pacientes com periodontite crônica, e avaliar a relação destes peptídeos com saúde e doença periodontal. Um total de 40 pacientes com periodontite crônica, sendo 20 fumantes e 20 não fumantes, e 20 pacientes periodontalmente saudáveis foram incluídos no estudo. Os parâmetros clínicos avaliados foram: profundidade de sondagem, sangramento à sondagem, nível clínico de inserção, Índice de placa visível e Índice de sangramento gengival. Amostras de fluido crevicular gengival foram coletadas com tiras de papel absorvente e o volume mensurado com Periotron. Nos pacientes com periodontite crônica, foram coletadas amostras de sítios sadios e doentes. A quantificação da LL-37 e HNP 1-3 foi realizada pela técnica ELISA sanduíche.Os níveis de LL-37 e HNP 1-3 foram menores tanto nos sítios sadios como nos sítios doentes de pacientes fumantes em comparação com os não fumantes, sendo essas diferenças significativas exceto para os níveis de LL-37 nos sítios sadios (p<0,05). Os sítios doentes dos pacientes fumantes e não fumantes com periodontite crônica mostrar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Antimicrobial peptides are components of the innate immune response that have immunomodulatory properties adjuncts to the antimicrobial properties and, therefore, may have a key role in susceptibility or resistance to diseases in the oral cavity. The purpose of this study was to investigate the influence of smoking on the levels of antimicrobial peptides, alpha-defensins (HNP 1-3) and LL-37 in the gingival crevicular fluid of patients with chronic periodontitis, and evaluate the relationship of these peptides with health and periodontal disease. A total of 40 patients with chronic periodontitis, 20 smokers and 20 non-smokers, and 20 periodontally healthy patients were included in the study. The clinical parameters evaluated were probing depth, bleeding on probing, clinical attachment level, visible plaque index and gingival bleeding index. Samples of gingival crevicular fluid were collected with absorbent paper strips and the volume measured on Periotron. In patients with chronic periodontitis, samples of healthy and diseased sites were collected. The quantification of LL-37 and HNP 1-3 was carried out by sandwich ELISA tecnique. The levels of LL-37 and HNP 1-3 were lower both in healthy sites such as diseased sites in smokers compared to non-smokers, those with significant differences except for the LL-37 levels in healthy sites (p<0.05). Diseased sites of smokers and non-smokers with chronic periodontitis patients showed higher levels of LL-37 and lower levels of HNP 1-3 th... (Complete abstract click electronic access below) / Mestre

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

La prévention du risque infectieux représente un enjeu de sécurité majeur pour l'Etablissement Français du Sang. A l'heure actuelle, le risque bactérien constitue le risque infectieux majoritaire dans les pays développés. Le risque bactérien ne se limite pas au domaine de la transfusion. La résistance aux antibiotiques constitue un problème majeur de santé publique. Les peptides antimicrobiens sont des armes importantes de l'immunité innée qui représentent une alternative intéressante aux antibiotiques classiques. Chez l'Homme, les α-défensines 1, 2 et 3 (HNPs 1-3) sont présentes dans les granules azurophiles des polynucléaires neutrophiles. Nous avons développé un procédé innovant de purification des HNPs 1-3 à partir des filtres de déleucocytation utilisés lors de la préparation des produits sanguins labiles. Ce procédé permet la production d'un cocktail pur d'HNPs 1-3 dont l'activité antibactérienne a été démontrée dans ce travail. Les défensines HNPs 1-3 ont par ailleurs été utilisées comme élément biorécepteur dans une approche innovante de détection bactérienne. Dans un premier temps, un immunocapteur électrochimique a été développé exploitant des microparticules magnétiques fonctionnalisées par des anticorps commerciaux. Dans un second temps, nous avons fonctionnalisé des microparticules magnétiques par les défensines HNPs 1-3 purifiées par notre protocole. Nous avons obtenu une première preuve de concept attestant de la capture des bactéries par cette approche innovante. La stabilité des peptides, associée aux performances des biocapteurs électrochimiques permettrait d'élaborer un test générique de détection de bactéries dans les produits sanguins labiles. / The prevention of the infectious risk is a major issue for the Etablissement Français du Sang. Currently, bacterial contamination is the most infectious risk in developed countries. The bacterial risk is not limited to blood transfusion safety. The antimicrobial resistance is a major public health problem. Antimicrobial peptides are important arm of the innate immune system which represents an interesting alternative to antibiotics. Human neutrophil peptides 1, 2 and 3 (HNPs 1-3) are found in the azurophilic granules of neutrophils. We have developed an original approach of HNPs 1-3 purification from leucodepletion filters used in blood processing. This process allows the production of a pure cocktail of HNPs 1-3 displaying high antibacterial activity as demonstrated by this work. HNPs 1-3 have also been used as bioreceptor in an innovative approach for bacterial detection. Initially, an electrochemical immunosensor was designed, exploiting magnetic microparticles coated with commercially available antibodies. In a second step, magnetic microparticles have been coated efficently with the HNPs 1-3 purified according our protocol. We have obtained a first proof of concept showing the bacterial capture by this innovative approach. The peptides stability combined with the electrochemical biosensors performances would allow the development of a generic bacteria detection assay in labile blood products.