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.

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.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation.

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 etiologies. 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-alpha, IFN-alpha, 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-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applied for dengue fever.

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.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

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-alpha, IFN-alpha, 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-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

Produçäo de óxido nítrico por macrófagos peritoneias de camundongos tratados in vitro e in vivo com extratos de Phyllanthus tenellus

Phyllanthus Spp. säo usadas popularmente para tratamento de diversas doenças virais, bacteriana e parasitárias. Por outro lado, o óxido nítrico (NO) exerce uma atividade antimicrobiana, inibindo por exemplo a replicaçäo intracelular de vários vírus. No presente estudo, investigamos o papel imunomodulador dos extratos aquosos de P. tenellus fresco (Ptaf) e seco (Pts) e do extrato acetona/água de P. tenellus seco (Ptl) na produçäo de NO por macrófagos peritoneais de camundongos, tanto in vivo como in vitro. Os resultados demonstram que: 1) in vitro, a concentraçäo de 100æg/ml de extrato aquoso de P. tenellus fresco (Ptaf) estimulou significantemente (p<0,05) a produçäo de NO em todos os ensaios, sendo 48h de incubaçäo o tempo ideal de produçäo; 2) in vitro, os extratos Ptas e Ptl näo apresentaram significância, no estímulo, em todos os ensaios; 3) in vivo, as doses de 50, 100 e 200mg/Kg duas doses näo foram capazes de pré-ativar os macrófagos peritoneais in vivo mesmo quando adicionado um segundo estímulo desencadeante de Ptaf 100æ/ml in vitro; 4) as doses de 10 e 50mg/Kg administradas ip duas vezes, foram capazes de induzir in vivo a produçäo de NO a níveis estatisticamente significantes, em pelo menos um experimento , sem a necessidde de estímulo desencadeante. Ambas as doses foram capazes de pré-ativar os macrófagos in vivo, detectado após um segundo estímulo in vitro nos dois experimentos realizados, sugerindo um pré-ativaçäo do extrato in vivo. Estudos adicionais poderäo melhor avaliar a relaçäo dose/resposta do extrato a fim de se obter um melhor sinergismo e condiçöes ideais para uma atividade antiviral ou contra outras infecçöes microbianas. (AU)

Produçäo de óxido nítrico por macrófagos peritoneias de camundongos tratados in vitro e in vivo com extratos de Phyllanthus tenellus

Phyllanthus Spp. säo usadas popularmente para tratamento de diversas doenças virais, bacteriana e parasitárias. Por outro lado, o óxido nítrico (NO) exerce uma atividade antimicrobiana, inibindo por exemplo a replicaçäo intracelular de vários vírus. No presente estudo, investigamos o papel imunomodulador dos extratos aquosos de P. tenellus fresco (Ptaf) e seco (Pts) e do extrato acetona/água de P. tenellus seco (Ptl) na produçäo de NO por macrófagos peritoneais de camundongos, tanto in vivo como in vitro. Os resultados demonstram que: 1) in vitro, a concentraçäo de 100æg/ml de extrato aquoso de P. tenellus fresco (Ptaf) estimulou significantemente (p<0,05) a produçäo de NO em todos os ensaios, sendo 48h de incubaçäo o tempo ideal de produçäo; 2) in vitro, os extratos Ptas e Ptl näo apresentaram significância, no estímulo, em todos os ensaios; 3) in vivo, as doses de 50, 100 e 200mg/Kg duas doses näo foram capazes de pré-ativar os macrófagos peritoneais in vivo mesmo quando adicionado um segundo estímulo desencadeante de Ptaf 100æ/ml in vitro; 4) as doses de 10 e 50mg/Kg administradas ip duas vezes, foram capazes de induzir in vivo a produçäo de NO a níveis estatisticamente significantes, em pelo menos um experimento , sem a necessidde de estímulo desencadeante. Ambas as doses foram capazes de pré-ativar os macrófagos in vivo, detectado após um segundo estímulo in vitro nos dois experimentos realizados, sugerindo um pré-ativaçäo do extrato in vivo. Estudos adicionais poderäo melhor avaliar a relaçäo dose/resposta do extrato a fim de se obter um melhor sinergismo e condiçöes ideais para uma atividade antiviral ou contra outras infecçöes microbianas. (AU)