Subsets of Cytokines and Chemokines from DENV-4-Infected Patients Could Regulate the Endothelial Integrity of Cultured Microvascular Endothelial Cells.

Introduction: It is a consensus that inflammatory mediators produced by immune cells contribute to changes in endothelial permeability in dengue. We propose to relate inflammatory mediators seen in dengue patients with the in vitro alteration of endothelial cells (ECs) cultured with serum from these patients. Methods: Patients with mild (DF) to moderate and severe dengue (DFWS/Sev) were selected. ELISA quantified inflammatory mediators. Expression of adhesion molecules and CD147 were evaluated in the ECs cultured with the patient's serum by flow cytometry. We assessed endothelial permeability by measuring transendothelial electrical resistance in cocultures of ECs with patient serum. Results: Dengue infection led to an increase in inflammatory mediators-the IL-10 distinguished DF from DFWS/Sev. There were no changes in CD31, CD54, and CD106 but decreased CD147 expression in ECs. DFWS/Sev sera induced a greater difference in endothelial permeability than DF sera. Correlation statistical test indicated that low IL-10 and IFN-γ and high CCL5 maintain the integrity of ECs in DF patients. In contrast, increased TNF, IFN-γ, CXCL8, and CCL2 maintain EC integrity in DFWS/Sev patients. Conclusions: Our preliminary data suggest that a subset of inflammatory mediators may be related to the maintenance or loss of endothelial integrity, reflecting the clinical prognosis.

Different Profiles of Cytokines, Chemokines and Coagulation Mediators Associated with Severity in Brazilian Patients Infected with Dengue Virus.

The incidence of dengue in Latin America has increased dramatically during the last decade. Understanding the pathogenic mechanisms in dengue is crucial for the identification of biomarkers for the triage of patients. We aimed to characterize the profile of cytokines (IFN-γ, TNF-α, IL-1ß, IL-6, IL-18 and IL-10), chemokines (CXCL8/IL-8, CCL2/MCP-1 and CXCL10/IP-10) and coagulation mediators (Fibrinogen, D-dimer, Tissue factor-TF, Tissue factor pathway inhibitor-TFPI and Thrombomodulin) during the dengue-4 epidemic in Brazil. Laboratory-confirmed dengue cases had higher levels of TNF-α (p < 0.001), IL-6 (p = 0.005), IL-10 (p < 0.001), IL-18 (p = 0.001), CXCL8/IL-8 (p < 0.001), CCL2/MCP-1 (p < 0.001), CXCL10/IP-10 (p = 0.001), fibrinogen (p = 0.037), D-dimer (p = 0.01) and TFPI (p = 0.042) and lower levels of TF (p = 0.042) compared to healthy controls. A principal component analysis (PCA) distinguished between two profiles of mediators of inflammation and coagulation: protective (TNF-α, IL-1ß and CXCL8/IL-8) and pathological (IL-6, TF and TFPI). Lastly, multivariate logistic regression analysis identified high aspartate aminotransferase-to-platelet ratio index (APRI) as independent risk factors associated with severity (adjusted OR: 1.33; 95% CI 1.03-1.71; p = 0.027), the area under the receiver operating characteristics curve (AUC) was 0.775 (95% CI 0.681-0.869) and an optimal cutoff value was 1.4 (sensitivity: 76%; specificity: 79%), so it could be a useful marker for the triage of patients attending primary care centers.

SARS-CoV-2 diagnostic diary: from rumors to the first case. Early reports of molecular tests from the military research and diagnostic institute of Rio de Janeiro.

Corona virus disease (COVID-19) presents a serious threat to global health. A historical timeline of early molecular diagnostics from government alert (January 22) (D) was presented. After in silico analysis, Brazilian Army Institute of Biology (IBEx-RJ) tested samples in house using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (fast mode) based on Centers for Disease Control and Prevention (CDC) recommendations. First cases from Brazil, Rio de Janeiro, IBEx, and diagnosis team were reported in D36, D44, D66, and D74 respectively. Therefore, after 1300 tests, we recommend N1/N2 primer sets (CDC) for preliminary and Charité protocol confirmation in case of positive results. Moreover, every professional should be tested before starting work, in addition to weekly tests for everyone involved.

SARS-CoV-2 diagnostic diary: from rumors to the first case. Early reports of molecular tests from the military research and diagnostic institute of Rio de Janeiro

Corona virus disease (COVID-19) presents a serious threat to global health. A historical timeline of early molecular diagnostics from government alert (January 22) (D) was presented. After in silico analysis, Brazilian Army Institute of Biology (IBEx-RJ) tested samples in house using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (fast mode) based on Centers for Disease Control and Prevention (CDC) recommendations. First cases from Brazil, Rio de Janeiro, IBEx, and diagnosis team were reported in D36, D44, D66, and D74 respectively. Therefore, after 1300 tests, we recommend N1/N2 primer sets (CDC) for preliminary and Charité protocol confirmation in case of positive results. Moreover, every professional should be tested before starting work, in addition to weekly tests for everyone involved.

Influence of chemokines on the endothelial permeability and cellular transmigration during dengue.

During a pathogenic infection, an inflammatory process is triggered in which several inflammatory mediators, such as cytokines, chemokines, growth factors, complement system components, nitric oxide, and others induce integrity alteration on the endothelial barrier. Chemokines are responsible for regulating leukocyte trafficking under homeostatic conditions as well as activating immune system cells under inflammatory conditions. They are crucial molecules in the early stages of infection, leading to the recruitment of immune cells, namely neutrophils, monocytes, natural killer (NK) cells, natural killer T cells (NKT), dendritic cells (DC), T lymphocytes and all cells expressing chemokine receptors for inflammatory sites. Other functions, such as collagen production, tissue repair, a proliferation of hematopoietic precursors and angiogenesis, are also performed by these molecules. Chemokines, amongst inflammatory mediators, play a key role in dengue immunopathogenesis. Dengue fever is a disease caused by the dengue virus (DENV). It is characterized by a broad spectrum of clinical manifestations ranging from asymptomatic cases to mild and severe symptomatic ones. As for the latter, the appearance of hemorrhagic manifestations and changes in vascular permeability may lead the patient to develop cavitary effusions, organ involvement, and even death. As chemokines exert an influence on various homeostatic and inflammatory processes, acting vigorously on vascular endothelial activation and cell migration, the main purpose of this chapter is to discuss the influence of chemokines on the alteration of endothelial permeability and migration of T lymphocytes in DENV infection.

Influência das Quimiocinas na migração dos linfócitos T e na permeabilidade de células endoteliais na infecção pelo vírus Dengue / Influence of chemokines on the migration of T lymphocytes and on the permeability of endothelial cells in the infection by the Dengue virus

Dengue é uma doença causada pelo vírus dengue (DENV) com amplo espectro clínico, variando desde uma infecção assintomática a casos graves, com manifestações hemorrágicas e/ou extravasamento plasmático, que podem levar o paciente ao óbito. Durante a infecção, mediadores pró-inflamatórios, como citocinas e quimiocinas, apresentam papel chave na imunopatogênese da dengue. Dentre esses mediadores, as quimiocinas exercem influência em diversos processos homeostáticos e inflamatórios, atuando na ativação do endotélio vascular e na migração celular. O objetivo desse trabalho foi avaliar a influência das quimiocinas CCL5, CX3CL1 e CXCL10 na alteração da permeabilidade endotelial e na migração de linfócitos T em pacientes infectados com DENV. Soro e células mononucleares do sangue periférico (do inglês PBMC) dos pacientes foram utilizados neste estudo, e obtidas durante as epidemias de 2013 e 2016.

Após a confirmação do diagnóstico laboratorial para dengue, os pacientes foram classificados clinicamente segundo critérios da OMS 2009. Níveis séricos das citocinas TNF-α, IL-1ß e IL-10 e quimiocinas CCL2, CCL5, CXCL8, CXCL10 e CX3CL1 de pacientes foram quantificadas por ELISA e LUMINEX. Para avaliação da permeabilidade endotelial in vitro foi realizado medida da resistência elétrica transendotelial (TEER) em HMVEC-d, através do equipamento Milicell-ERS acoplada a câmara Endohm 6, na presença de soro pré-tratados ou não com anticorpos neutralizantes anti-CCL5, anti-CXCL10 ou anti-CX3CL1. A caracterização do perfil de expressão dos receptores de quimiocinas CCR4, CCR5, CCR7 e CX3CR1 em linfócitos T de pacientes e controles sadios foram realizados por citometria de fluxo. Para avaliação da capacidade in vitro de migração de linfócitos T em câmara transwell foram utilizados os recombinantes TNF+CXCL8/IL-8, CCL5 ou CX3CL1.

Os resultados demonstraram a influência de constituintes presentes no soro de pacientes na alteração da permeabilidade da monocamada de células endoteliais. Essa alteração foi observada com mais frequência nos casos FDSA/Grave comparado aos casos FD e controles sadios. Dentre os mediadores próinflamatórios quantificados, os níveis de CCL5 foram menores em pacientes cujo soro foi capaz de induzir alteração da permeabilidade das HMVEC-d, comparado aos pacientes cujo soro não induziu. O pré-tratamento do soro de pacientes que promoveram a redução do TEER relativo com os anticorpos bloqueadores CCL5 e CX3CL1, levou ao aumento do valor TEER relativo das células HMVEC-d, indicando que, neste grupo de pacientes, existe uma influência das quimiocinas CCL5 e CX3CL1 na manutenção da integridade endotelial. Pacientes FDSA/Grave apresentam maior frequência de linfócitos T CD4 e CD8 expressando CCR5 e CX3CR1 em comparação aos linfócitos T de pacientes FD. Estudo sobre a influência das quimiocinas CCL5 e CX3CL1 na migração de linfócitos T de pacientes indicou que tanto os linfócitos T CD4 como os CD8 de pacientes FDSA/Grave migram mais, independente da presença do estímulo quimiotático, do que os linfócitos T de pacientes FD, provavelmente porque as células dos FDSA/Grave expressam mais a molécula CD49d. Em condições nas quais foram utilizados os recombinantes CCL5 e CX3CL1 como estímulos quimiotáticos, os linfócitos T CD4 e CD8 de pacientes FDSA/Grave foram mais responsivos ao CCL5 e, portanto, migraram mais na presença de CCL5, comparado aos linfócitos T dos pacientes FD. O conjunto desses dados demonstra a influência das quimiocinas CCL5 e CX3CL1 na regulação da integridade endotelial e de CCL5 na migração dos linfócitos T, mais evidenciado nos pacientes FDSA/Grave. (AU)

Following in the Footsteps of the Chikungunya Virus in Brazil: The First Autochthonous Cases in Amapá in 2014 and Its Emergence in Rio de Janeiro during 2016.

Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amapá (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV (n = 8) and by CHIKV/ZIKV (n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.

CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role, whereas CD8(+)Pfn(+) may play a detrimental role in T. cruzi-elicited heart injury.