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.

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.

Antiviral and Immunomodulatory Effects of Norantea brasiliensis Choisy on Dengue Virus-2.

BACKGROUND/AIMS: Severe dengue fever is a result of exacerbated immune responses and no specific treatments are available. We evaluated the antiviral and immunomodulatory effects of Norantea brasiliensis Choisy. METHODS: Human adherent monocytes infected in vitro with dengue virus (DENV)-2 were incubated with the crude ethanol extract from leaves (NB1) or 3 derived fractions: dichloromethane (NB3), ethyl acetate (NB5), and butanolic (NB6) partitions. The antiviral and immunomodulatory activities were determined by intracellular detection of DENV antigen within monocytes and by secreted NS1 viral protein and cytokines. RESULTS: The crude extract alone exhibited both antiviral activities (intracellular and secreted antigens) and all fractions derived from this extract modulated NS1 production. Regarding the immunomodulatory effect, among the secreted factors, TNF-α was inhibited by NB3 and NB6; IL-6 was inhibited by NB1, NB3, and NB6; IL-10 by NB1 and NB3; and IFN-α by NB6. The crude extract (NB1) presented the best antiviral effect, whereas the dichloromethane fraction (NB3) presented an immunomodulatory effect in the inflammatory and anti-inflammatory cytokines. CONCLUSION: During in vitro DENV infection, N. brasiliensis Choisy exerts both antiviral and immunomodulatory effects that are likely associated, considering that less viral load may lead to less immunostimulation.

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.

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.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles.

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Predictive factors of dengue severity in hospitalized children and adolescents in Rio de Janeiro, Brazil.

INTRODUCTION: Dengue is one of the most important mosquito-borne infections. Severe cases are more frequently observed in adults. However, in 2008, the State of Rio de Janeiro, Brazil, experienced a severe dengue epidemic that primarily affected children and caused many cases of dengue hemorrhagic fever (DHF) and death. METHODS: A cross-sectional analytical study was conducted to examine laboratory diagnosis and clinical epidemiologic factors for confirmed dengue cases in patients aged less than 16 years, from January to June 2008, at a municipal hospital in the City of Rio de Janeiro, Brazil. Variables associated with severe outcomes and P values less than .05 were evaluated by means of a logistic regression model. RESULTS: Of the 419 dengue cases studied, 296 were classified as DHF and 123 as classical dengue. Six patients who had DHF died. In multivariate analysis, some laboratory and clinical variables were independently associated with DHF: age 5 years or older (odds ratio [OR], 4.94; 95% confidence interval [CI], 1.30-18.71), abdominal pain (OR, 8.59; 95% CI, 3.17-23.27), hepatomegaly (OR, 15.87; 95% CI, 5.38-46.85), and positive tourniquet test (OR, 10.84; 95% CI, 3.96-29.71). Hypoalbuminemia occurred more frequently than hemoconcentration in DHF cases, and high aminotransferase levels were associated with severity. CONCLUSIONS: Age greater than 5 years, abdominal pain, painful hepatomegaly, and positive tourniquet test were predictors of DHF. The high frequency of hepatic impairment suggests that acetaminophen should be avoided in severe cases 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.

Analysis of Clinical and Laboratory Alterations Related to Dengue Case Severity: Comparison between Serotypes 2 and 4 in Brazil.

The increase in severe dengue (SD) cases has caused great impact on public health and has concerned authorities of countries where the disease is endemic and epidemics reach high proportions. The recognition of progression signs of this severe disease during the initial febrile phase can be difficult, since the symptoms are often indistinguishable from other febrile diseases. The aim of this study was to evaluate the clinical manifestations and laboratory findings in patients from two dengue outbreaks and their association with the disease. The study was conducted in patients (n = 153) with signs and symptoms consistent with dengue occurred during two distinct epidemics, 2010 and 2013, in the city of Campos dos Goytacazes, Rio de Janeiro, Brazil. According to the 2009 World Health Organization criteria, patients were classified as dengue without warning signs ([DwoWS] 60.6%, 57/94), dengue with warning signs ([DwWS] 30.9%, 29/94), and SD (4.25%, 4/94). Patients with DwWS/SD presented lower platelet and leukocyte counts and higher transaminase levels when compared with the DwoWS ones. Interestingly, patients from the epidemic of 2010 caused by dengue virus 2 (DENV-2) had lower platelet counts than patients of the 2013 epidemic caused by DENV-4. Furthermore, plasma leakage, gastrointestinal bleeding, and pleural effusion, hallmarks for a more severe disease, were also more frequently observed in those cases. Although previous studies may have extensively reported the wide range of the clinical aspects of dengue, the characterization of DENV-4 is desirable considering the burden of the disease during epidemics, especially for the health units and hospitals performing patient's management.

Association of rs1285933 single nucleotide polymorphism in CLEC5A gene with dengue severity and its functional effects.

Outbreaks of the Zika, dengue, and chikungunya viruses, especially in the Americas, pose a global threat due to their rapid spread and difficulty controlling the vector. Extreme phenotypes are often observed, from asymptomatic to severe clinical manifestations, which are well-studied in dengue. Host variations are also important contributors to disease outcomes, and many case-control studies have associated single nucleotide polymorphisms (SNPs) with severe dengue. Here, we found that the TC genotype and T-carriers for SNP rs1285933 in the C-type lectin superfamily member 5 (CLEC5A) gene was associated with severe dengue in a Northern Brazilian population (OR=2.75 and p-value=0.01, OR=2.11 and p-value=0.04, respectively). We also tested the functional effect of the CLEC5A protein and found that it is upregulated on the surface of human monocytes after in vitro dengue infection. CLEC5A was correlated with viral load inside the monocytes (Spearman r=0.55, p=0.008) and TNF production in culture supernatants (Spearman r=0.72, p=0.03). Analysis of mRNA in blood samples from DENV4-infected patients exhibiting mild symptoms showed that CLEC5A mRNA expression is correlated with TNF (r=0.67, p=0.0001) and other immune mediators. Monocytes from rs1285933 TT/TC individuals showed lower CLEC5A expression compared to CC genotypes. However, in these cells, CLEC5A was not correlated with TNF production. In summary, we confirmed that CLEC5A is genetically associated with dengue severity outcome, playing a central role during the immune response triggered by a dengue viral infection, and rs1285933 is a relevant SNP that is able to regulate signaling pathways after interactions between the dengue virus and CLEC5A receptors.

Dengue epidemics in two distinct periods reveal distinct epidemiological, laboratorial and clinical aspects in a same scenario: analysis of the 2010 and 2013 epidemics in Mato Grosso do Sul, Brazil.

BACKGROUND: Dengue is a major problem in Brazil. Epidemiological and clinical aspects were characterized in patients from two epidemics which occurred in Mato Grosso do Sul, Brazil. METHODS: Dengue cases were classified according to the 2009 WHO criteria, tested by serological and molecular biology tests and analysed for nonstructural protein 1 (NS1) antigenemia. RESULTS: Dengue was confirmed in 78.7% (48/61) and 75.6% (118/156) of the cases studied in 2010 and 2013, respectively. DENV-1 and DENV-2 were the serotypes involved in the 2010 epidemic and DENV-4 in the 2013 one. Most of the cases were classified as dengue without warning; however, severe dengue was observed in 18.7% (9/48) of the cases in 2010 and less observed in DENV-4 cases. NS1 levels were higher in patients with dengue with warning signs and severe dengue in 2010. Circulating aspartate aminotransferase (AST) and alanine transferase (ALT) were altered in all groups, independently of the infecting serotype or epidemic. Patients with DENV-1 and DENV-2 presented significant lower monocyte counts when compared to patients with DENV-4. An inverse correlation was found between platelet count, leucocytes, monocytes and NS1 levels. CONCLUSIONS: Epidemics caused by the prevalence of distinct DENV serotypes had different impacts and clinical characteristics in a same scenario and, despite the occurrence of secondary infections, the DENV-4 emergence was not associated with severe cases.

Circulating cytokines and chemokines associated with plasma leakage and hepatic dysfunction in Brazilian children with dengue fever.

Dengue fever is usually a benign acute viral infection transmitted by arthropods but may evolve to severe clinical manifestations such as coagulation and/or hemodynamic disorders, caused mainly by an increase of vascular permeability. Deregulated circulating immunological factors have been associated with severity. In Brazil severe cases appeared in children only recently and we evaluated the profile of cytokine/chemokine kinetics in 134 hospitalized young patients during the epidemic in Rio de Janeiro in 2008. Inflammatory cytokines TNF and IFNγ were found elevated during the acute phase in children as well as the anti-inflammatory IL10 and chemokines MIF and CXCL10/IP10, all last three persisting longer during the recovery phase. Severe disease fitting the dengue hemorrhagic fever pattern (WHO, 1997) was associated with higher IL10 and CXCL10/IP10 circulating levels (peak levels at seven days with P<0.01 and P<0.001 respectively as compared to DF). These factors were higher in patients pulmonary effusion or ascites (P<0.05 for IL10 and P<0.01 for CXCL10/IP10). Both factors were also associated with liver changes such as AST increase correlated with CXCL10/IP10 (r=0.4300 with P<0.0001) and patients presenting painful hepatomegaly showed higher circulating levels of IL10 (P<0.01, at 7-9 days) and of CXCL10/IP10 (P<0.05, 4-6 days and P<0.001, 7-9 days) when compared to patients without apparent liver alterations. Most cases presented a history of prior infection (93%). This is the first study demonstrating cytokine and chemokine association with severity during dengue fever in Brazilian children. IL10 and CXCL10/IP10 play a role in the disease severity associated with induction of vascular leakage and a novel association with changes in liver dysfunction.

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.

TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

Uncaria tomentosa alkaloidal fraction reduces paracellular permeability, IL-8 and NS1 production on human microvascular endothelial cells infected with dengue virus.

Dengue is the major Arbovirus in the world, annually causing morbidity and death. Severe dengue is associated with changes in the endothelial barrier function due to the production of inflammatory mediators by immune cells and by the endothelium. Dengue virus (DENV) replicates efficiently in human endothelial cells in vitro and elicits immune responses resulting in endothelial permeability. Uncaria tomentosa (Willd.) DC.(Rubiaceae), known as cat's claw, has been used in folk medicine for the treatment of a wide-array of symptoms, and several scientific studies reported its antiviral, immunomodulatory, anti-inflammatory and antioxidant properties. Here we infected a human lineage of dermal microvascular endothelial cells (HMEC-1) with DENV-2 and treated it with an alkaloidal fraction from U. tomentosa bark (AFUT). We showed antiviral and immunomodulatory activities of U. tomentosa by determining the NS1 antigen and IL-8 in supernatant of DENV-2 infected HMEC-1. Furthermore, by measurement of transendothelial electrical resistance (TEER) we demonstrated, for the first time, that a plant derivative contributed to the reduction of paracellular permeability in DENV-2 infected HMEC-1. We also showed that IL-8 contributed significantly to the induction of permeability. Although further investigations should be conducted before a new drug can be suggested, our in vitro data support evidence that AFUT could be potentially useful in developing a treatment for severe dengue.

Dengue virus activates membrane TRAIL relocalization and IFN-α production by human plasmacytoid dendritic cells in vitro and in vivo.

BACKGROUND: Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. METHODS FINDINGS: Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production. CONCLUSIONS: This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.

Predictive factors of dengue severity in hospitalized children and adolescents in Rio de Janeiro, Brazil

Abstract INTRODUCTION: Dengue is one of the most important mosquito-borne infections. Severe cases are more frequently observed in adults. However, in 2008, the State of Rio de Janeiro, Brazil, experienced a severe dengue epidemic that primarily affected children and caused many cases of dengue hemorrhagic fever (DHF) and death. METHODS: A cross-sectional analytical study was conducted to examine laboratory diagnosis and clinical epidemiologic factors for confirmed dengue cases in patients aged less than 16 years, from January to June 2008, at a municipal hospital in the City of Rio de Janeiro, Brazil. Variables associated with severe outcomes and P values less than .05 were evaluated by means of a logistic regression model. RESULTS: Of the 419 dengue cases studied, 296 were classified as DHF and 123 as classical dengue. Six patients who had DHF died. In multivariate analysis, some laboratory and clinical variables were independently associated with DHF: age 5 years or older (odds ratio [OR], 4.94; 95% confidence interval [CI], 1.30-18.71), abdominal pain (OR, 8.59; 95% CI, 3.17-23.27), hepatomegaly (OR, 15.87; 95% CI, 5.38-46.85), and positive tourniquet test (OR, 10.84; 95% CI, 3.96-29.71). Hypoalbuminemia occurred more frequently than hemoconcentration in DHF cases, and high aminotransferase levels were associated with severity. CONCLUSIONS: Age greater than 5 years, abdominal pain, painful hepatomegaly, and positive tourniquet test were predictors of DHF. The high frequency of hepatic impairment suggests that acetaminophen should be avoided in severe cases of dengue.