SummaryDengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus-target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. ing that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL-10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte-activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in order to control both intense immunoactivation and virus replication.

Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumournecrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. MethodsThe expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using N G -methyl L-Arginine (N G MLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. ResultsINOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with N G MLA. In ConclusionThis study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production.

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-α, IFN-α, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-α, IFN-α and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.Key words: dengue -dexamethasone -monocytes -cytokines -therapeutics Dengue fever (DF) is an acute infectious viral disease that presents a broad severity spectrum from asymptomatic or oligosymptomatic to extremely severe clinical manifestations. It is believed that immunological mechanisms play a key role in pathogenesis (WHO 1997, Gubler 2002. Proinflammatory cytokines have been associated with haemodynamic and coagulation disorders that may lead to increased vascular permeability and leakage and to hypovolemic shock resulting eventually in death (Green & Rothman 2006). No specific treatment exists for dengue and research has been unsuccessful in finding improved conditions with steroids, antivirals or substances that decrease capillary permeability (Sumarmo et al. 1982, Tassniyom et al. 1993, Pea et al. 2001, Gibbons & Vaughn 2002, Ligon 2005. In patients, fluid-replacement therapy should be administrated according to the severity and paracetamol/metamizole has been prescribed for fever and analgesia.Glucocorticoids are widely adopted as anti-inflammatory drugs for several inflammatory diseases and are known to suppress of inflammatory mediators expression (Barnes & Adcock 1995, Joyce et al. 1997. Dexamethasone inhibits cytokine gene expression such as TNF-α by blocking transcription factors NF-κB and activator factor-1 (Steer et al. 2000). IL-1β, iNOS, ciclooxigenase-2 and monocyte chemoattractant protein-1 have also been described to destabilize mRNAs during this treatment (Amano et al. 1993, Ristimaki et al. 1996, Poon et al. 1999, Lasa et al. 2001, Korhonen et al. 2002.Dexamethasone is applied in the clinics to treat pathologies with inflammatory and autoimmune origins, such as rheumatoid arthritis, multiple melanomas and bacterial meningitis (Jimenez-Zepeda & DominguezMartinez 2006, van de Beek & de Gans 2006, Schutt et a...

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 monocytederived 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.Key words: cytokines -dendritic cells -dengue virus -yellow fever vaccine -flavivirus dendritic cell activation by flavivirus • Mariana Gandini et al.

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.