RESUMEN
Dengue is an arboviral disease caused by dengue virus (DENV), which is transmitted to humans by Aedes aegypti mosquitoes. Infection by DENV most commonly results in a mild flu-like illness; however, the disease has been increasingly associated with neurological symptomatology. This association draws attention to further investigations on the impact of DENV infection in the host's central nervous system. Here, we analyzed brain samples of three fatal dengue cases that occurred in 2002 during an outbreak in Rio de Janeiro, Brazil. Brain tissues of these cases were marked by histopathological alterations, such as degenerated neurons, demyelination, hemorrhage, edema, and increased numbers of astrocytes and microglial cells. Samples were also characterized by lymphocytic infiltrates mainly composed of CD8 T cells. DENV replication was evidenced in neurons, microglia and endothelial cells through immunohistochemistry and in situ hybridization techniques. Pro-inflammatory cytokines, such as TNF-α and IFN-γ were detected in microglia, while endothelial cells were marked by the expression of RANTES/CCL5. Cytoplasmic HMGB1 and the production of nitric oxide were also found in neurons and microglial cells. This work highlights the possible participation of several local pro-inflammatory mediators in the establishment of dengue neuropathogenesis.
Asunto(s)
Encéfalo/virología , Dengue/patología , Replicación Viral , Adulto , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Quimiocina CCL5/metabolismo , Dengue/mortalidad , Dengue/virología , Femenino , Humanos , Interferón gamma/metabolismo , Microglía/patología , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
Zika virus (ZIKV) is an emerging virus involved in recent outbreaks in Brazil. The association between the virus and Guillain-Barré syndrome (GBS) or congenital disorders has raised a worldwide concern. In this work, we investigated a rare Zika case, which was associated with GBS and spontaneous retained abortion. Using specific anti-ZIKV staining, the virus was identified in placenta (mainly in Hofbauer cells) and in several fetal tissues, such as brain, lungs, kidneys, skin and liver. Histological analyses of the placenta and fetal organs revealed different types of tissue abnormalities, which included inflammation, hemorrhage, edema and necrosis in placenta, as well as tissue disorganization in the fetus. Increased cellularity (Hofbauer cells and TCD8+ lymphocytes), expression of local pro-inflammatory cytokines such as IFN-γ and TNF-α, and other markers, such as RANTES/CCL5 and VEGFR2, supported placental inflammation and dysfunction. The commitment of the maternal-fetal link in association with fetal damage gave rise to a discussion regarding the influence of the maternal immunity toward the fetal development. Findings presented in this work may help understanding the ZIKV immunopathogenesis under the rare contexts of spontaneous abortions in association with GBS.
RESUMEN
Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.
Asunto(s)
Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Virus del Dengue/patogenicidad , Adulto , Animales , Encéfalo/patología , Encéfalo/virología , Línea Celular , Cerebelo/patología , Cerebelo/virología , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Dengue is an important infectious disease that presents high incidence and yields a relevant number of fatal cases (about 20,000) every year worldwide. Despite its epidemiological relevance, there are many knowledge gaps concerning dengue pathogenesis, especially with regards to the circumstances that drive a mild clinical course to a severe disease. In this work, we investigated the participation of high mobility group box 1 (HMGB1), an important modulator of inflammation, in dengue fatal cases. Histopathological and ultrastructural analyses revealed that liver, lung and heart post-mortem samples were marked by tissue abnormalities, such as necrosis and apoptotic cell death. These observations go in line with an HMGB1-mediated response and raised concerns regarding the participation of this cytokine in promoting/perpetuating inflammation in severe dengue. Further experiments of immunohistochemistry (IHC) showed increased expression of cytoplasmic HMGB1 in dengue-extracted tissues when compared to non-dengue controls. Co-staining of DENV RNA and HMGB1 in the host cell cytoplasm, as found by in situ hybridization and IHC, confirmed the virus specific induction of the HMGB1-mediated response in these peripheral tissues. This report brings the first in-situ evidence of the participation of HMGB1 in severe dengue and highlights novel considerations in the development of dengue immunopathogenesis.
Asunto(s)
Virus del Dengue/patogenicidad , Dengue/metabolismo , Dengue/patología , Proteína HMGB1/metabolismo , Adulto , Citocinas/metabolismo , Femenino , Proteína HMGB1/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Inflamación/metabolismo , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Adulto JovenRESUMEN
Diseases caused by flaviviruses, such as dengue and zika, are globally recognized as major threats. During infection, a critical point in their replicative cycle is the maturation step, which occurs throughout the cellular exocytic pathway. This step is a pH-dependent process that involves the modification of the viral envelope by converting prM (pre-membrane) into M (membrane) proteins with the release of a "pr peptide". After this reaction, the pr peptides remain bound to the viral envelope while the virions cross the acidic trans-Golgi network, and are released only at neutral pH after secretion of the virus particles. Despite this current knowledge, the molecular basis of the flavivirus maturation step is largely unknown. Here, based on the crystal structure of the dengue pr-E complex ("pr peptide" bound to virus envelope protein) and using molecular dynamics simulations, we found that the pH shift from acidic to neutral yields considerable structural changes in the system. Dynamic cross correlation maps and root mean square deviation analyses revealed that the pr-E junction is clearly unstable under neutral pH. Secondary structure analysis also revealed that the fusion loop region, present in the E protein, is sensitive to pH and tends to unstructure at a neutral environment. Moreover, we found that five residues present in the E protein, Gly102, His244, Thr70, Thr68 and Asn67 are critical to confer stability to the pr-E complex while inside the Golgi apparatus. This work brings details about the dynamical behavior of the pr-E system, helps to better understand the flavivirus biology and may also be of use in the development of novel antiviral strategies.
Asunto(s)
Virus del Dengue/metabolismo , Simulación de Dinámica Molecular , Proteínas del Envoltorio Viral/química , Virus Zika/metabolismo , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Estructura Secundaria de Proteína , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer-dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel antichemotactic agents.
Asunto(s)
Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Receptores CCR2/química , Receptores CCR2/metabolismo , Sitios de Unión , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
Dengue disease is an acute viral illness caused by dengue virus (DENV) that can progress to hemorrhagic stages leading to about 20000 deaths every year worldwide. Despite many clinical investigations regarding dengue, the immunopathogenic process by which infected patients evolve to the severe forms is not fully understood. Apart from differences in virulence and the antibody cross reactivity that can potentially augment virus replication, imbalanced cellular immunity is also seen as a major concern in the establishment of severe dengue. In this context, the investigation of cellular immunity and its products in dengue fatal cases may provide valuable data to help revealing dengue immunopathogenesis. Here, based in four dengue fatal cases infected by the serotype 3 in Brazil, different peripheral organs (livers, lungs and kidneys) were studied to evaluate the presence of cell infiltrates and the patterns of local cytokine response. The overall scenario of the studied cases revealed a considerable systemic involvement of infection with mononuclear cells targeted to all of the evaluated organs, as measured by immunohistochemistry (IHC). Quantification of cytokine-expressing cells in peripheral tissues was also performed to characterize the ongoing inflammatory process by the severe stage of the disease. Increased levels of IFN-γ- and TNF-α-expressing cells in liver, lung and kidney samples of post-mortem subjects evidenced a strong pro-inflammatory induction in these tissues. The presence of increased RANTES-producing cell numbers in all analyzed organs suggested a possible link between the clinical status and altered vascular permeability. Co-staining of DENV RNA and IFN-γ or TNF-α using in situ hibridization and IHC confirmed the virus-specific trigger of the pro-inflammatory response. Taken together, this work provided additional evidences that corroborated with the traditional theories regarding the "cytokine storm" and the occurrence of uneven cellular immunity in response to DENV as major reasons for progress to severe disease.
Asunto(s)
Quimiocina CCL5/fisiología , Dengue/complicaciones , Interferón gamma/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Adulto , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Citocinas/fisiología , Dengue/inmunología , Dengue/mortalidad , Femenino , Humanos , Inmunidad Celular , Interferón gamma/metabolismo , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.
Asunto(s)
Virus del Dengue/inmunología , Inmunidad Celular , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Inyecciones Intraventriculares , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/administración & dosificaciónRESUMEN
The flavivirus non-structural protein 1 (NS1) is a conserved glycoprotein with as yet undefined biological function. This protein dimerizes when inside infected cells or associated to cell membranes but also forms lipid-associated hexamers when secreted to the extracellular space. A single amino acid substitution (P250L) is capable of preventing the dimerization of NS1 resulting in lower virulence and slower virus replication. In this work, based on molecular dynamics simulations of the dengue-2 virus NS1 [Formula: see text]-ladder monomer as a core model, we found that this mutation can induce several conformational changes that importantly affect critical monomer-monomer interactions. Based on additional simulations, we suggest a mechanism by which a highly orchestrated sequence of events propagate the local perturbations around the mutation site towards the dimer interface. The elucidation of such a mechanism could potentially support new strategies for rational production of live-attenuated vaccines and highlights a step forward in the development of novel anti-flavivirus measures.
Asunto(s)
Flavivirus , Simulación de Dinámica Molecular , Mutación , Multimerización de Proteína , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Conformación Proteica en Lámina beta , Estructura Cuaternaria de ProteínaRESUMEN
Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.
RESUMEN
The interferon (IFN) family of cytokines is recognized as a key component of the innate immune response and the first line of defense against viral infection. The usage of the IFN-alpha as a biopharmaceutical has been mainly applied in the treatment of chronic hepatitis C. In the literature it is possible to find a great variety of methods to determine the potency of these cytokines, and many efforts have been made in order to develop practical bioassays to study the biological activity of IFNs. In this technical note, we present a different approach to determine the potency of a recombinant IFN-alpha preparation based on the activation of the signal transducers and activators of transcription 1 (STAT1) using flow cytometry technique. Under the conditions of this study, this new approach proved to be useful and promising to assess the potency of these biopharmaceuticals and may also be used as an important tool in the quality control of such biological products.