A View of Platelets in Dengue

Platelets were mainly associated with coagulation and hemostasis; however, other biological effects have been attributed to platelets, including angiogenesis, extracellular matrix synthesis, inflammation, and immune response. Dengue virus infection causes 200 million cases of severe flu-like illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome. Some hypotheses are postulated for immunopathogenesis of dengue, including antibody enhancement theory, T-cell activation of cross-reactive memory, and original antigenic sin. All hypotheses, to some extent, induce an overproduction or a skewed profile of cytokine release, giving rise to the term cytokine storm/cytokine tsunami. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. In dengue, platelets are one of the major cell populations affected by direct and/or indirect mechanisms of infection. It is common to observe both thrombocytopenia and platelet dysfunction in dengue, both strongly related to the clinical outcome. Thus, platelets are frequently affected in dengue, either for alteration of their own functionality, for “silent transport” of virus, or as an anti-viral immune cell. In this way, we describe some of functional aspects of platelets on dengue, observing circulating mediators, intraplatelet proteins contents, morphology, activation markers, and ability to interact with dengue virus

Avaliação do perfil de mediadores séricos e proteínas intraplaquetárias em relação à plaquetopenia em pacientes infectados pelo vírus dengue

Made available in DSpace on 2015-11-04T13:29:11Z (GMT). No. of bitstreams: 2 tamiris_barros_ioc_mest_2015.pdf: 3974773 bytes, checksum: 582ff742d19972a2ff51fc3e5895c17f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilPlaquetas são fragmentos celulares derivados dos megacariócitos, que desempenham papel na hemostasia, coagulação, angiogênese, inflamação e resposta imune. Na infecção humana pelo Vírus Dengue (DENV), plaquetas constituem uma das populações celulares mais afetadas devido à plaquetopenia e disfunção plaquetária. O objetivo deste trabalho foi investigar a influência de citocinas, quimiocina e fatores de crescimento séricos e de proteínas intraplaquetárias relacionadas à angiogênese, coagulação, regulação da matriz extracelular e inflamação na plaquetopenia de pacientes infectados pelo DENV. Para tal, realizamos: (i) estudo populacional de pacientes e obtenção de soro e plaquetas em 2013, (ii) ensaios multiplex de micrarranjo líquido para quantificação dos níveis séricos de citocinas, quimiocina e fatores de crescimento e (iii) ensaio de determinação do perfil de expressão 55 proteínas intraplaquetárias. Quarenta e três pacientes DENV foram confirmados, com predominância do DENV-4. Independente da forma clínica, pacientes DENV apresentaram níveis séricos elevados de IL-10, TNF-\03B1, CXCL8/IL-8, mas não de IL-1\03B2 e IFN-\03B3 quando comparados aos controles sadios. Análises estatísticas demonstraram que níveis de IL-10 e IFN-\03B3 apresentaram correlação, respectivamente inversa e direta com a contagem de plaquetas. Ainda, IL-10 diretamente com leucócitos e linfócitos e TNF-\03B1 com linfócitos Vinte e cinco proteínas intraplaquetárias foram quantificadas, mas apenas cinco delas, PDGF-AA, TGF-\03B21, HGF, IGFBP-1 e Angiopoetina-1, apresentaram correlação direta com a contagem de plaquetas nos pacientes DENV. A quantificação sérica de PDGF e VEGF demostrou que ambos estavam diminuídos no grupo DENV mais trombocitopênico. Análise entre proteínas intraplaquetárias com funções biológicas antagônicas demonstraram que a razão anti- versus pró-inflamatórios TGF-\03B21/MIP-1\03B1 foi diminuída em pacientes DENV trombocitopênicos e as razões anti versus pró-angiogênica serpina F1/angiopoetina-1 e serpina F1/ PDGF-AB/BB apresentaram níveis aumentados em pacientes DENV trombocipênicos. Concluímos brevemente que a reintrodução do DENV-4 não resultou numa maior ocorrência de gravidade. Contudo esta reintrodução, induziu aumento dos níveis de TNF-\03B1 e CXCL8/IL-8 e da IL-10, influenciando de maneira direta ou indireta contagens de plaquetas e/ou demais células em resposta à infecção. Níveis IX intraplaquetários de PDGF, TGF-\03B21, IGFPB-1, Angiopoetina e HGF em pacientes mais trombocitopênicos poderiam prejudicar a ativação de mecanismos relacionados à angiogênese, coagulação, integridade do endotélio vascular e produção de mediadores inflamatórios. Assim, plaquetas poderiam ser consideradas células atuantes da resposta imunológica anti-DENV e, portanto, plaquetopenia é um fator prognóstico chave da imunopatogênese da denguePlatelets are cell fragments derived from megakaryo cytes, which play a role in hemostasis, coagulation, angiogenesis, inflammation and immune response. In human infection with dengue virus (DENV), platelets are one of the most affecte d cell populations due to thrombocytopenia and platelet dysfunction. The objective of this stu dy was to investigate the influence of serum cytokines, chemokines, intraplatelet growth factors and proteins related to angiogenesis, coagulation, regulation of extracellular matrix and inflammation in thrombocytopenia of patients infected with DENV. For this purpose, we conducted: (i) population study of patients and obtaining their serum and platelets in 2013, (ii) l iquid microarray multiplex assays for quantitation of serum levels of cytokines, chemokine, and growth factors, and (iii) assay for determining expression profile of 55 intraplapletelet proteins. Forty-three DENV patients were confirmed, with a predominance of DENV-4. Regardless of type o f DENV, levels of IL-10, TNF- α , CXCL8 / IL-8, but not IL-1 β and IFN- γ were higher on serum of patients compared to healt hy individuals. Statistical analyses showed that levels of IL-10 an d IFN- γ presented correlation, respectively, inverse and direct with platelet count. Furthermore , IL-10 was directly correlated with leukocytes, lymphocytes, TNF- α and with lymphocytes. Twenty-five intraplatelet pr oteins were quantified, but only five of them, PDGF-AA, TGF- β 1, HGF, angiopoietin-1 and IGFBP-1 were directly correlated with platelet count in DENV pat ients. Both levels of PDGF and VEGF were decreased in group of DENV thrombocytopenic. Analys es between intraplatelet proteins with antagonic biological functions have shown that rati os anti- versus pro-inflammatory TGF- β 1 / MIP-1 α were decreased in thrombocytopenic DENV patients a nd the ratios anti-versus pro- angiogenic serpin-F1 / angiopoietin-1 and serpin-F1 / PDGF-AB / BB showed increased levels in thrombocytopenic DENV patients. Briefly been con cluded that the reintroduction of the DENV- 4 did not result in a higher incidence of gravity. However, levels of TNF- α and CXCL8 / IL-8 and IL-10 were increased, influencing directly or indir ectly platelet counts and/or other cells in response to infection. Levels of PDGF, TGF- β 1, IGFPB-1, angiopoietin and HGF intraplatelet of patients high thrombocytopenic may impair activatio n of mechanisms related to angiogenesis, coagulation, vascular endothelial integrity and pro duction of inflammatory mediators. Thus, platelets might be considered active anti-DENV immu ne response cells and hence thrombocytopenia is a key prognostic fator in the i mmunopathogenesis of dengue

Evaluation of Two Adjuvant Formulations for an Inactivated Yellow Fever 17DD Vaccine Candidate in Mice

The attenuated yellow fever (YF) vaccine is one of the most successful vaccines ever developed. After a single dose administration YF vaccine can induce balanced Th1/Th2 immune responses and long-lasting neutralizing antibodies. These attributes endorsed it as a model of how to properly stimulate the innate response to target protective immune responses. Despite their longstanding success, attenuated YF vaccines can cause rare fatal adverse events and are contraindicated for persons with immunosuppression, egg allergy and age 60 years. These drawbacks have encouraged the development of a non-live vaccine. The aim of the present study is to characterize and compare the immunological profile of two adjuvant formulations of an inactivated YF 17DD vaccine candidate. Inactivated YF vaccine formulations based on alum (Al(OH)3) or squalene (AddaVax®) were investigated by immunization of C57BL/6 mice in 3-dose or 2-dose schedules, respectively, and compared with a single dose of attenuated YF virus 17DD. Sera were analyzed by ELISA and Plaque Reduction Neutralization Test (PRNT) for detection of total IgG and neutralizing antibodies against YF virus. In addition, splenocytes were collected to evaluate cellular responses by ELISpot. Both inactivated formulations were able to induce high titers of IgG against YF, although neutralizing antibodies levels were borderline on pre-challenge samples. Analysis of IgG subtypes revealed a predominance of IgG2a associated with improved neutralizing capacity in animals immunized with the attenuated YF vaccine, and a predominance of IgG1 in groups immunized with experimental non-live formulations (alum and AddaVax®). After intracerebral (IC) challenge, attenuated and inactivated vaccine formulations showed an increase in neutralizing antibodies. The AddaVax®-based inactivated vaccine and the attenuated vaccine achieved 100% protection, and alum-based equivalent formulation achieved 70% protection