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.

Dengue Virus Induces NK Cell Activation through TRAIL Expression during Infection.

Dengue is an acute febrile illness with a wide spectrum of signs and symptoms ranging from mild to severe forms characterized by plasma leakage that can be fatal. NK cells are one of the main effectors in early infection and may play an important role in dengue pathogenesis. We investigated NK cell involvement during dengue infection. A higher frequency of NK cell subsets and TRAIL+NK cells was found in mild DF cases when compared to that in severe cases or healthy donors. NK activation markers such as CD107a and TLR3 were upregulated in patients' cells compared to those in healthy donors. In addition, IL12 related to NK cell activation were upregulated in mild DF cases. In vitro PBMC culture models show that DENV-stimulated and IFNα-stimulated NK cells were able to express TRAIL, suggesting an indirect activation of cells, regarding TRAIL expression. Type I IFN receptor blockage on DENV-stimulated PBMCs showed TRAIL expression on NK cells is partially IFNα dependent. In addition, during PBMC stimulation, TRAIL expression on NK cells was inversely correlated with DENV-positive monocytes. Therefore, we observed DENV-induced activation of NK cell populations. A higher activation of NK cells would promote limited viral spread, resulting in decreased inflammatory response, contributing to protection against dengue severity.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7.

BACKGROUND: Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS: Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS: The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN. CONCLUSIONS: The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Increased circulating procoagulant and anticoagulant factors as TF and TFPI according to severity or infecting serotypes in human dengue infection.

Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders.

Cissampelos sympodialis has anti-viral effect inhibiting dengue non-structural viral protein-1 and pro-inflammatory mediators

ABSTRACT Dengue is the most important viral infection transmitted among humans by arthropod-borne. There are currently no vaccines or specific therapeutical treatment. Therefore, immunomodulatory compounds from plants have been widely examined for their antiviral effects. Cissampelos sympodialis Eichler, Menispermaceae, has scientifically proven to present immunomodulatory activities. Here we assessed the antiviral activity of leaf hydroalcoholic extract, warifteine or methylwarifteine from C. sympodialis in an in vitro dengue virus infection model. The results demonstrated that leaf hydroalcoholic extract or warifteine/methylwarifteine treatment did not reduce dengue virus-Ag+ hepatocyte (Huh-7 cell) rates in present experimental conditions. However, we assessed the potential antiviral effect of leaf hydroalcoholic extract or warifteine/methylwarifteine on dengue virus-infection by the production of inflammatory molecules, TNF-α, MIF, IL-8 and PGE2. Dengue virus infection enhanced TNF-α, MIF, IL-8 and PGE2 production in infected Huh-7 cells and leaf hydroalcoholic extract but not warifteine/methylwarifteine treatments, significantly reduced these molecules in infected cells. In dengue virus-infected Huh-7 cells, non-structural protein-1 is produced and leaf hydroalcoholic extract significantly inhibited it independently of alkaloids. Our findings imply that leaf hydroalcoholic extract may attenuate dengue virus infection in Huh-7 cells by inhibiting the enhanced of pro-inflammatory mediators and non-structural protein-1 production induce by dengue virus independently of warifteine/methywarifteine its major compound.

Characterization of clinical and immunological features in patients coinfected with dengue virus and HIV.

The pathogenesis of dengue in subjects coinfected with HIV remains largely unknown. We investigate clinical and immunological parameters in coinfected DENV/HIV patients. According to the new dengue classification, most coinfected DENV/HIV patients presented mild clinical manifestations of dengue infection. Herein, we show that DENV/HIV coinfected patients had higher CD8 T cells percentages reflected as a lower CD4/CD8 ratio. Furthermore, CCR5 expression on CD4 T cells and CD107a expression on both T subsets were significantly higher in coinfected patients when compared with monoinfected DENV and HIV individuals respectively. Increased inflammatory response was observed in treated HAART coinfected patients despite undetectable HIV load. These data indicate that DENV infection may influence the clinical profile and immune response in individuals concomitantly infected with HIV.

Influência do sistema complemento na infecção, ativação celular e alteração da permeabilidade endotelial na dengue / Influence of complement system in infection, cellular activation and alteration on endothelial cell permeability on dengue

O Sistema Complemento (SC) desempenha papel importante no controle de infecções atuando na eliminação do patógeno e na regulação da resposta imune. Contudo, uma ativação desregulada do SC gera efeitos deletérios ao hospedeiro, contribuindo para a patogênese de diversas doenças, como na Dengue. Entretanto, o envolvimento do SCna infecção pelo vírus Dengue (DENV) ainda tem vários aspectos a serem investigados. Assim, avaliamos a contribuição do SC na infecção in vitro de monócitos pelo DENV; o perfil de expressão dos Receptores de Complemento (CR) CR1, CR2,CR3, CR4, CD46, CD55 e CD59, e de moléculas de ativação nos monócitos e linfócitos T circulantes de pacientes infectados pelos DENV-1,-2 ou -4 por citometria de fluxo.Dosamos os níveis de SC5b-9 e citocinas em pacientes por ELISA. Avaliamos ainda, a contribuição da ativação do SC na permeabilidade e viabilidade endotelial, utilizando modelo in vitro de células endoteliais (CEs) pela medida da resistência elétrica transendotelial (TEER) e liberação de LDH sobrenadante de culturas. Por fim investigamos a interação DENV-2 com componentes purificados do SC por eletroforesedas proteínas. Como achados principais, observamos diminuição na frequência de monócitos CD14+ expressando CR3, CR4 e CD59 em pacientes comparado aos controles saudáveis. De forma interessante, o bloqueio do CR3 levou à diminuição em cerca de 30 por cento da infecção in vitro pelo DENV-2 em monócitos, sem alterar o fenótipo de ativação ou a ativação da caspase-1 destas células. No entanto, com o bloqueio deCR3, detectamos diminuição na produção de TNF-alfa e IFN-alfa. Não observamos diferença significativa na frequência de linfócitos T expressando CR3, CD46, CD55 eCD59 de em pacientes-Dengue-4...

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

Regulation of T lymphocyte apoptotic markers is associated to cell activation during the acute phase of dengue.

Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a+) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4+ and CD8+ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed.

Apoptotic mediators in patients with severe and non-severe dengue from Brazil.

Despite being the most significant arboviral disease worldwide, dengue has no antiviral treatment or reliable severity predictors. It has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. However, very little is known about the interplay between proapoptotic and antiapoptotic mediators in this disease. Therefore, plasma levels of the three proapoptotic mediators Fas ligand (FasL), tumor necrosis factor-α (TNF-α), and TNF-related apoptosis-inducing ligand (TRAIL) were measured in dengue patients. Patients were classified according to the World Health Organization classification of dengue revised in 2009. Additionally, inhibitors of apoptosis protein (IAPs) were determined in plasma (Survivin) and peripheral blood mononuclear cells (PBMCs) lysates (cIAP-1, cIAP-2, XIAP). Levels of apoptotic proteins in plasma were correlated with counts of blood cells. FasL and TRAIL levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. Dengue patients with warning signs showed decreased levels of Survivin compared to patients with severe dengue and controls. TRAIL was inversely correlated with counts of lymphocyte subsets. In contrast, Survivin was positively correlated with leukocyte counts. There was a trend of elevated IAPs levels in PBMCs of patients with severe dengue. The results suggest a likely antiviral effect of TRAIL in dengue. It appears that TRAIL might be involved with apoptosis induction of lymphocytes, whereas IAPs might participate in protecting leukocytes from apoptosis. Further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue pathogenesis.

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever.

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1ß but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1ß and CCL2/MCP-1 in dengue fever and parvovirosis.

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1ß, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Influência do sistema complemento na imunopatogênese da infecção pelo vírus dengue / Influence of complement system in the immunopathogenesis of dengue virus infection

O Sistema Complemento (SC) consiste numa rede de proteínas altamente regulada, podendo ser ativado pelas vias alternativa, clássica e da lectina e que convergem na clivagem de C3 e formação do complexo de ataque à membrana (C5b-9). Receptores do complemento (RC), envolvidos na regulação do SC, induzem mecanismos para eliminação de patógenos e a interação das imunidades inata e adaptativa. Estudos vem indicando que uma ativação desregulada do SC contribuiria à gravidade da dengue. A infecção humana pelo vírus Dengue (DENV) é caracterizada por um amplo espectro de sintomas clínicos, variando desde uma febre branda até distúrbios hemodinâmicos, podendo evoluir para choque hipovolêmico e morte. O objetivo principal do trabalho foi investigar o envolvimento do SC na infecção natural pelo DENV, através: (i) da avaliação quanto a expressão dos RC CR1/CD35, CR2/CD21, CR4/CD11c e CD59 nos monócitos CD14+ e linfócitos T CD4+ e TCD8+ por citometria de fluxo, (ii) dosagem de quantidades plasmáticas de SC5b-9 e das citocinas/quimiocinas TNF-alfa, IFN-gamma, CCL5, CCL2 por ELISA e por fim, (iii) avaliação da influência dos componentes do SC na alteração da permeabilidade de células endoteliais (CEs) pelo ensaio de citotoxidade pela enzima LDH. Neste estudo foram incluídos 66 pacientes com infecção pelo DENV-1 ou -2 e 12 indivíduos saudáveis. Os pacientes-DENV foram agrupados segundo a nova classificação da OMS: febre do dengue sem sinais de alarme (FD Sem SA), FD com sinais de alarme (FD Com SA) e dengue grave (G). Nossos resultados demonstraram diminuição significativa na frequência de monócitos CD14+ expressando CR1/CD35, CR4/CD11c e CD59 nos pacientes-DENV comparado aos controles. Além disso, diminuição na frequência de células T CD4+ e CD8+ expressando CR1/CD35 e CR2/CD21 nos pacientes-DENV comparado aos controles. Não observamos diferenças quanto a frequência de nonócitos CD14+ expressando CR2/CD21 ou de linfócitos T CD4+ e CD8+ expressando CR4/CD11c ou CD59 entre os grupos. A quantificação plasmática das citocinas/quimiocinas revelou: (i) maiores quantidades de TNF-alfa nos pacientes FD Sem SA e FD Com SA, (ii) quantidades aumentadas de CCL2 e (iii) diminuídas de CCL5 comparados aos controles saudáveis. Por fim, (iv) a quantidade de IFN-gamma não foi diferente entre grupos de pacientes e controles. Análises de correlação entre as quantidades de citocinas e expressão dos RC revelaram que TNF-alfa foi diretamente correlacionada com a frequência de monócitos CD14+ expressando de CD59 e CR4/CD11c, enquanto que CCL2 foi inversamente correlacionada com a frequência de linfócitos TCD8+ expressando CD59Quanto a dosagem do SC5b-9, quantidades plasmáticas desta molécula foram mais elevadas nos pacientes graves e que apresentaram manifestações hemorrágicas e extravasamento plasmático comparado aqueles sem estes sintomas. A adição de plasma de pacientes com quantidades elevadas de SC5b-9 promoveu maior lise de CEs in vitro, comparado aos plasmas de pacientes com quantidades menores de SC5b-9. Desta forma, nós concluímos que na infecção pelo DENV ocorre uma modulação dos RC nos monócitos e linfócitos T de pacientes, o que poderia levar a uma alteração funcional destas células. Além disso, TNF-alfa e CCL2 poderiam estar modulando a expressão de alguns dos RC nas células. Por fim, quantidades elevadas de SC5b-9, associada a ativação do SC, estaria associada as manifestações clínicas graves e na citotoxidade de CEs. Como perspectivas, serão avaliadas as alterações funcionais das células imunes e o grau de contribuição dos componentes do SC na patogênese da dengue. Desta forma, nossos dados levam a concluir que componentes do SC contribuem à patogênese da doença.