Mechanisms of programmed cell death associated to severe dengue in human renal lesions.

Dengue virus (DENV) is a global health problem. Severe dengue can manifest with hemorrhage and signs of organ dysfunction, including the kidneys. The innate immune system is an important barrier against arbovirus infection and, specifically in dengue, the cytokines IL1ß and IL18 and caspase-1 activation make up a set of host immune strategies. Cell death mechanisms include pyroptosis, necroptosis and autophagy, each with peculiar markers: gasdermin, RIPK3/MLKL, LC3, respectively. In DENV infection, necrosis and apoptosis are involved and, when infecting monocytes and macrophages in vitro, DENV is capable of inducing pyroptosis. Our objective was to explore the presence of markers of necroptosis, pyroptosis and autophagy in renal lesions caused by DENV. MATERIAL AND METHODS: twenty specimens of lesions from patients who died due to DENV infection, from the pathology department of Hospital Guilherme Álvaro, Santos, SP, were subjected to histological and immunohistochemical studies. Histological sections were stained with hematoxylin-eosin to evaluate tissue changes or collected for research with antibodies: anti-DENV (Instituto Evandro Chagas-PA), RIPK3 (NBP2-45592), MLKL (ab184718), gasdermin D (#36425), LC3 (14600-AP), caspase 1 (#98033), IL1ß (AF201-NA) and IL18 (SC6178). Semi-quantitative analysis was performed on 20 glomeruli and evaluation on tubules and mononuclear cells. This study was approved by the ethics committee of the USP Faculty of Medicine. RESULTS: histological analysis demonstrated glomerular congestion, glomerulitis (medium to severe), acute kidney injury and hyalinization of the glomeruli. Viral antigens were visualized on mononuclear cells. LC3 (autophagy) expression ranged from moderate to intense (++/+++) in glomeruli, tubules and mononuclear cells. The expression of gasdermin (pyroptosis) was mild (+) in most cases in the glomeruli and moderate (++) in the tubules. RIPK3 and MLKL (necroptosis) mild in tubules and mononuclear cells (+). The expression of the cytokines IL1ß and IL18 and caspase 1 was moderate (++). Statistical analysis showed greater expression of LC3 over the others. CONCLUSIONS: Our results contribute to the understanding of the pathogenesis of renal involvement in severe dengue, considering the likely anti-viral mechanism of autophagy. To a lesser extent, pyroptosis is also present, corroborating previous data.

Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice.

Typically occurring during secondary dengue virus (DENV) infections, dengue hemorrhagic fever (DHF) causes abnormal immune responses, as well as endothelial vascular dysfunction, for which the responsible viral factor remains unclear. During peak viremia, the plasma levels of virion-associated envelope protein domain III (EIII) increases to a point at which cell death is sufficiently induced in megakaryocytes in vitro. Thus, EIII may constitute a virulence factor for endothelial damage. In this study, we examined endothelial cell death induced by treatment with DENV and EIII in vitro. Notably, pyroptosis, the major type of endothelial cell death observed, was attenuated through treatment with Nlrp3 inflammasome inhibitors. EIII injection effectively induced endothelial abnormalities, and sequential injection of EIII and DENV-NS1 autoantibodies induced further vascular damage, liver dysfunction, thrombocytopenia, and hemorrhage, which are typical manifestations in DHF. Under the same treatments, pathophysiological changes in the Nlrp3 inflammasome-deficient mice were notably reduced compared with those in the wild-type mice. These results suggest that the Nlrp3 inflammasome constitutes a potential therapeutic target for treating DENV-induced hemorrhage in DHF.

Effects of chloroquine or hydroxychloroquine treatment on non-SARS-CoV2 viral infections: A systematic review of clinical studies.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used as antiviral agents for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We performed a systematic review to examine whether prior clinical studies that compared the effects of CQ and HCQ to a control for the treatment of non-SARS-CoV2 infection supported the use of these agents in the present SARS-CoV2 outbreak. PubMed, EMBASE, Scopus and Web of Science (PROSPERO CRD42020183429) were searched from inception through 2 April 2020 without language restrictions. Of 1766 retrieved reports, 18 studies met our inclusion criteria, including 17 prospective controlled studies and one retrospective study. CQ or HCQ were compared to control for the treatment of infectious mononucleosis (EBV, n = 4), warts (human papillomavirus, n = 2), chronic HIV infection (n = 6), acute chikungunya infection (n = 1), acute dengue virus infection (n = 2), chronic HCV (n = 2), and as preventive measures for influenza infection (n = 1). Survival was not evaluated in any study. For HIV, the virus that was most investigated, while two early studies suggested HCQ reduced viral levels, four subsequent ones did not, and in two of these CQ or HCQ increased viral levels and reduced CD4 counts. Overall, three studies concluded CQ or HCQ were effective; four concluded further research was needed to assess the treatments' effectiveness; and 11 concluded that treatment was ineffective or potentially harmful. Prior controlled clinical trials with CQ and HCQ for non-SARS-CoV2 viral infections do not support these agents' use for the SARS-CoV2 outbreak.

Cytokine Expression in Dengue Fever and Dengue Hemorrhagic Fever Patients with Bleeding and Severe Hepatitis.

Dengue is the most common mosquito-borne flaviviral infection in the world today. Several factors contribute and act synergistically to cause severe infection. One of these is dysregulated host immunological mediators that cause transient pathophysiology during infection. These mediators act on the endothelium to increase vascular permeability, which leads to plasma leakage compromising hemodynamics and coagulopathy. We conducted a prospective study to explore the expression of pro- and anti-inflammatory cytokines and how they relate to clinical dengue manifestations, by assessing their dynamics through acute dengue infection in adults admitted to the Hospital for Tropical Diseases, Bangkok, Thailand. We performed cytokine analysis at three phases of infection for 96 hospitalized adults together with serotyping of confirmed dengue infection during the outbreaks of 2015 and 2016. The serum concentrations of seven cytokines (interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha, and interferon gamma) were measured in duplicate using a commercial kit (Bio-Plex Human Cytokine Assay). In this study, the cytokine profile was suggestive of a T-helper 2 response. Most patients had secondary infection, and the levels of viremia were higher in patients with plasma leakage than those without plasma leakage. In addition, we observed that bleeding and hepatitis were associated with significantly higher levels of IL-8 during the early phases of infection. Furthermore, IL-6 levels in the early phase of infection were also elevated in bleeding patients with plasma leakage. These results suggest that IL-6 and IL-8 may act in synergy to cause bleeding in patients with plasma leakage.

Molecular Signatures of Dengue Virus-Specific IL-10/IFN-γ Co-producing CD4 T Cells and Their Association with Dengue Disease.

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.

Association of tumour necrosis factor-α (TNF-α) gene polymorphisms (-308 G>A and -238 G>A) and the risk of severe dengue: A meta-analysis and trial sequential analysis.

Individual studies have assessed the association between TNF-α-308G>A and TNF-α-238 G>A polymorphisms and severity of dengue infection. However, the results are inconclusive and most studies had small sample sizes. The objective of this study was to summarize the evidence of association between TNF-α-308 G>A and TNF-α-238 G>A and severity of dengue infection. This study follows the preferred reporting items for systematic reviews and meta- analyses of genetic association studies, recommended by PLOS One. We calculated pooled odds ratio and its 95% confidence interval (CI) to estimate the association between TNF-α-308 G>A or TNF-α-238 G>A and the risk of severe dengue infections. To determine the information size required for this meta-analysis study, a trial sequential analysis (TSA) was done. Eight studies (640 cases and 1275 controls), which assessed the association of TNF-α-308 G>A or TNF-α-238 G>A and the risk of DHF were included. Overall, we found no significant association between TNF-α-308 G>A and the DHF risk in the allelic model (OR, 0.91; 95% CI, 0.51-1.63), the recessive model (OR,1.32;95%CI,0.73-2.37), the dominant model (OR,0.93;95%CI:0.59-1.47) or the additive model (OR,1.43,95;95%CI:0.79-2.59). There was also no significant association between TNF-α-238 G>A and DHF risk under the allele contrast model (OR:1.51;95%CI:0.88-2.58), the recessive model (OR,1.48,95% CI:0.33-6.58), the dominant model (OR,1.48;95%CI:0.56-3.92), or the additive model (OR:1.5;95%CI:0.34-6.69). On subgroup analysis, neither the Asian population nor the non-Asian population showed significant association between TNF-α-308 G>A/TNF-α-238 G>A and the DHF risk under any genetic models. Leave-one-out meta-analysis showed stability of the results. TSA plots suggested that the sample size in this meta-analysis study was below the required information size. The findings suggest an inclusive evidence of the association between TNF-α-308/ TNF-α-238 G>A and the risk of developing severe dengue infection. Large studies with evidence of Hardy-Weinberg equilibrium, assessing gene-gene interactions are recommended.

The predictive and diagnostic accuracy of vascular endothelial growth factor and pentraxin-3 in severe dengue.

This study aimed to evaluate vascular endothelial growth factor (VEGF) and pentraxin 3 (PTX-3) as predictive and diagnostic markers in differentiating severe dengue from non-severe dengue. The study was conducted in Ampang Health Clinic, Ampang Hospital and Serdang Hospital. The plasma levels of VEGF and PTX-3 were compared between severe dengue and non-severe dengue by ELISA from the day of presentation until discharged. Multiple logistic regression was used to develop predictive and diagnostic models by incorporating other clinical parameters. The receiver operating characteristics (ROC) analysis was used to assess the accuracy of the biomarkers and the developed models. Eighty-two patients were recruited, 29 with severe dengue and four died. The Area Under the Curve (AUC) was statistically significant in VEGF as diagnostic marker at Day 2 and 3 of illness with sensitivity of 80.00%-100.00% and specificity of 76.47%-80.00%. The predictive model with AUC of 0.84 (p < 0.01) has a sensitivity of 100.00% and specificity of 79.25% for predicting severe dengue. The diagnostic model with AUC of 0.71 (p < 0.01) has a sensitivity of 76.19% and specificity of 73.58% for diagnosing severe dengue. The AUC for PTX-3 was not statistically significant. VEGF may be used in combination with other clinical parameters to predict the severity of the disease. As a single biomarker, it may be used as an adjunct investigation to support the diagnosis of severe dengue. PTX-3 was not able to differentiate severe dengue from non-severe dengue.

Host Genetic Polymorphisms Influencing Susceptibility to Dengue.

Dengue is a pandemic-prone viral disease which is endemic in more than 100 countries and which puts half of the world's population at risk. While the disease presents as subclinical infection or mild fever in the majority of cases, approximately a quarter of the infected individuals experience severe forms of disease like dengue hemorrhagic fever/dengue shock syndrome that cause significant rates of mortality and morbidity. The pathogenesis of this differential outcome of infection is determined by a complex interplay of factors associated with the virus, vector, and host; much of which is not completely understood. In this review, we present an update on the various host genetic polymorphisms that have been reported to influence the susceptibility to dengue. For the convenience of discussion, we have categorized the genetic factors according to the different arms of the immune system with which the corresponding immune determinants are associated.

A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

BACKGROUND: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. METHODOLOGY/PRINCIPAL FINDINGS: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. CONCLUSIONS/SIGNIFICANCE: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate.

JNK1/2 inhibitor reduces dengue virus-induced liver injury.

High viral load with liver injury is exhibited in severe dengue virus (DENV) infection. Mitogen activated protein kinases (MAPKs) including ERK1/2 and p38 MAPK were previously found to be involved in the animal models of DENV-induced liver injury. However, the role of JNK1/2 signaling in DENV-induced liver injury has never been investigated. JNK1/2 inhibitor, SP600125, was used to investigate the role of JNK1/2 signaling in the BALB/c mouse model of DENV-induced liver injury. SP600125-treated DENV-infected mice ameliorated leucopenia, thrombocytopenia, hemoconcentration, liver transaminases and liver histopathology. DENV-induced liver injury exhibited induced phosphorylation of JNK1/2, whereas SP600125 reduced this phosphorylation. An apoptotic real-time PCR array profiler was used to screen how SP600125 affects the expression of 84 cell death-associated genes to minimize DENV-induced liver injury. Modulation of caspase-3, caspase-8 and caspase-9 expressions by SP600125 in DENV-infected mice suggests its efficiency in restricting apoptosis via both extrinsic and intrinsic pathways. Reduced expressions of TNF-α and TRAIL are suggestive to modulate the extrinsic apoptotic signals, where reduced p53 phosphorylation and induced anti-apoptotic Bcl-2 expression indicate the involvement of the intrinsic apoptotic pathway. This study thus demonstrates the pivotal role of JNK1/2 signaling in DENV-induced liver injury and how SP600125 modulates this pathogenesis.

Dengue-associated hemophagocytic lymphohistiocytosis in an adult: A case report and literature review.

BACKGROUND: Infection-associated hemophagocytic syndrome (IAHS) is potentially a fatal disease caused by systemic infection complicated by hemophagocyticlymphohistiocytosis (HLH). Here, we report a case of HLH associated with dengue hemorrhagic fever (DHF) after a trip to Thailand. CASE SUMMARY: A 33-year-old healthy female patient presented with 3 days of fever, myalgia, and skin rash. Serotype 3 dengue virus was isolated. Clinical and laboratory findings fulfilled the criteria of HLH. After the initiation of corticosteroid therapy, the patient recovered and laboratory findings were normalized. CONCLUSION: It would be important to differentially diagnose dengue-associated HLH from severe DHF. Early recognition and initiation of steroid treatment would be crucial for the successful treatment of dengue fever complicated by HLH.

The Pattern of Adipose Tissue Accumulation during Early Infancy Provides an Environment for the Development of Dengue Hemorrhagic Fever.

BACKGROUND: Dengue is the most prevalent arthropod-borne viral illness in humans with half of the world's population at risk. During early infancy, severe dengue can develop after a primary dengue virus infection. There has been a clinical observation that severe dengue during the first year of life is seen only in chubby infants. METHODOLOGY/PRINCIPAL FINDINGS: We examined the associations between the development of severe dengue and adipose tissue accumulation patterns during the first year of life in a prospective observational clinical study of infants and dengue virus infections. We found that adipose tissue contains two potential targets for dengue virus infection and production- adipocytes and adipose tissue macrophages. During the first year of life, total body adiposity and visceral adipose tissue stores were at their highest levels in early infancy. Early infancy was also characterized by a relative decrease in alternatively activated (anti-inflammatory) macrophages, and a relative increase in circulating pro-inflammatory cytokines. CONCLUSIONS/SIGNIFICANCE: The data has been used to propose a model where the adipose tissue accumulation pattern and pro-inflammatory environment during early infancy provide the conditions for the potential development of severe dengue in immune-susceptible infants.

Endothelial Cell Sensitization by Death Receptor Fractions of an Anti-Dengue Nonstructural Protein 1 Antibody Induced Plasma Leakage, Coagulopathy, and Mortality in Mice.

The mechanisms leading to the life-threatening dengue hemorrhagic fever (DHF) remain elusive. DHF preferentially occurs during secondary dengue infections, suggesting that aberrant immune responses are involved in its development. We previously demonstrated that the autoantibodies elicited by dengue virus (DENV) nonstructural protein 1 (NS1; anti-NS1 Igs) induce plasma leakage and mortality in mice with warfarinized anticoagulant suppression. However, the involved pathogenic Ig fractions of anti-NS1 Igs remain unclear. In this study, the autoreactive Igs in patients with DHF and in NS1-immunized rabbits crossreacted with TNF-related apoptosis-inducing ligand receptor 1 (death receptor [DR]4). Challenges with the DENV in a subcytotoxic dose sensitized endothelial cells to apoptosis. Treatments with the autoantibodies induced proapoptotic activities and suppressed the surface expression of endothelial anticoagulant thrombomodulin. Combined treatments comprising the DENV and DR4 affinity-purified fractions of anti-NS1 IgGs (anti-NS1-DR4 Ig), but not preimmune control IgGs, in subcytotoxic doses led to apoptosis in endothelial cells. Treatments with the anti-NS1-DR4 Ig led to plasma leakage, coagulopathy, and morality in mice with warfarinized anticoagulant suppression. These results suggest that DR4-induced endothelial cell sensitization through NS1-elicited autoantibodies exacerbates anticoagulant suppression, vascular injury, and plasma leakage. Detecting and blocking anti-DR Igs in patients may be novel strategies for managing severe DENV infection.

Intervenciones de enfermería para la atención del paciente con dengue grave en el segundo y tercer nivel de atención / Nursing interventions for the care of the patient with dengre grave in the second and third level

Introducción. El dengue es una enfermedad infecciosa sistémica, viral transmitida por el mosquito “Aedes”. La OMS calcula que cada año se producen entre 50 y 100 millones de infecciones por este virus en el mundo. En México en el transcurso de la última década ha habido un incremento de casos, especialmente de tipo hemorrágico.Métodos. Se realizó una búsqueda sistemática de artículos en cuatro etapas, retrospectiva a 10 años relacionada a dengue grave en PubMed, BVS, Tripdatabase y sitios Web; obteniendo 149 resultados, siendo útiles 40 para el desarrollo de esta guía: revisiones sistemáticas, meta análisis, ensayos clínicos aleatorizados, estudios observacionales y Guías de Práctica Clínica.Resultados. Revisiones sistemáticas mencionan que la desnutrición grado 2 o 3 proteico calórica severa protege contra la vasculopatía del dengue y que la fase crítica comienza alrededor del período de desaparición de fiebre, la leucopenia avanza y hay una rápida disminución de plaquetas, esto precede a la manifestación más específica y potencialmente mortal de esta fase: incrementa la permeabilidad capilar conduciendo a la pérdida de plasma y aumento en él hematocrito; un estudio transversal prospectivo encontró que infecciones sucesivas del virus del dengue aumentan el riesgo de dengue grave y un estudio multicéntrico refiere que las fallas orgánicas más desarrolladas fueron respiratorias, cardiovasculares, neurológicas y hepáticas.Conclusiones. La implementación de las recomendaciones están dirigidas principalmente a las intervenciones de enfermería que contribuyen a la prevención, recuperación, disminución de complicaciones y limitación de daños para el paciente con dengue grave.

Introduction. Dengue fever is a systemic, viral infectious disease transmitted by the ""Aedes"" mosquito. WHO estimates that each year occur between 50 and 100 million infections by this virus in the world. In Mexico over the course of the last decade, there has been an increase in cases, especially of hemorrhagic type. Methods. A systematic search of articles was carried out in four stages, back at 10 years related to severe dengue in PubMed, VHL, Tripdatabase and Web sites; 149 results, being useful 40 for the development of this guide: systematic reviews, meta analysis, clinical trials randomized, observational studies and clinical practice guidelines. Results. Systematic reviews mentioned malnutrition grade 2 or severe caloric protein 3 protects against vascular disease of dengue and the critical phase begins around the time of the disappearance of fever, leukopenia advances and there is a rapid decrease in platelets, this precedes the manifestation more specific and potentially deadly this phase: increases capillary permeability, leading to the loss of plasma and increase the hematocrit; a prospective cross-sectional study found that successive infections of the dengue virus increases the risk of serious dengue and a multicenter study concerns that more developed organic failures were respiratory, cardiovascular, neurological and hepatic.Conclusions. The implementation of the recommendations are directed mainly to the nursing interventions that contribute to the prevention, recovery, reduction of complications and limitation of patient with severe dengue.

Introdução. Dengue é uma doença infecciosa sistêmica, viral transmitida pelo mosquito ""Aedes"". O que estima-se que cada ano produzirá entre 50 e 100 milhões de infecções por este vírus no mundo. No México, durante a última década tem sido um aumento de casos, especialmente do tipo hemorrágico. Métodos. Uma busca sistemática dos artigos foi realizada em quatro etapas, em 10 anos relacionados com graves da dengue no PubMed, BVS, Tripdatabase e Web sites; 149 resultados, sendo 40 útil para o desenvolvimento deste guia: revisões sistemáticas, meta análise, ensaios clínicos randomizados, estudos observacionais e diretrizes de prática clínica.Resultados. Revisões sistemáticas mencionado o grau de desnutrição 2 ou proteína calórica grave 3 protege contra doença vascular de dengue e a fase crítica começa na época do desaparecimento da febre, leucopenia avança e há uma rápida diminuição de plaquetas, isto precede a manifestação mais específica e potencialmente mortal nesta fase: aumenta a permeabilidade capilar, levando à perda de plasma e aumento do hematócrito; um estudo transversal prospectivo descobriu que infecções sucessivas do vírus da dengue a aumentam o risco de dengue grave e um estudo multicêntrico refere-se que os defeitos orgânicos mais específica e potencialmente mortal nesta fase: aumenta a permeabilidade capilar, levando à perda de plasma e aumento do hematócrito; um estudo transversal prospectivo descobriu que infecções sucessivas do vírus da dengue a aumentam o risco de dengue grave e um estudo multicêntrico refere-se que os defeitos orgânicos mais desenvolvidos eram respiratórias, cardiovasculares, neurológicas e hepáticas. Conclusões. A implementação das recomendações são direcionados principalmente para eles, as intervenções de enfermagem que contribuem para a prevenção, recuperação, redução de complicações e a limitação de danos para o paciente com dengue grave.

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.

Association between proinflammatory cytokines and lipid peroxidation in patients with severe dengue disease around defervescence.

OBJECTIVES: Proinflammatory cytokines and the oxidative stress response are reported to be involved in dengue viral disease. The present study investigated the correlation of proinflammatory cytokines and lipid peroxidation with dengue severity. METHODS: Clinical samples from 27 dengue fever (DF) cases, 30 dengue haemorrhagic fever (DHF) cases, and 24 dengue shock syndrome (DSS) cases were studied around defervescence, along with samples from 30 healthy controls. Plasma samples were analysed for tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) by ELISA and for malondialdehyde (MDA) by thiobarbituric acid assay. RESULTS: Dengue-infected individuals had significantly higher levels of TNF-α, IFN-γ, and MDA in comparison to controls. The ratio of TNF-α to IFN-γ was significantly higher in DHF and DSS than in DF. A TNF-α/IFN-γ ratio value of 5.69 around defervescence predicted DHF and DSS with moderate accuracy and thus may serve as an indicator to study dengue severity. The study observed a significant positive correlation of lipid peroxides with TNF-α levels and the TNF-α/IFN-γ ratio in severe dengue cases. CONCLUSIONS: We propose that the oxidative stress response induced by the dengue virus may trigger the inflammatory cytokine responses in dengue severity and thereby contributes to the pathogenesis of the disease; however the interplay between the oxidative response and inflammatory activity in disease virulence needs further study.

Los cambios histopatológicos del hígado durante dengue severo: [Editorial] / Histopathological changes in the liver during severe dengue: [Editorial]

El dengue es una enfermedad producida por el virus dengue perteneciente a la familia Flaviviridae , género Flavivirus y que es transmitido a los humanos por el vector Aedes , principalmente Aedes aegypti y Aedes albopictus . A la fecha se reconocen cuatro serotipos del virus, aunque recientemente el Doctor Nikos Vasilakis, virólogo de la Universidad de Texas durante la Tercera Conferencia internacional de Dengue y Dengue Hemorrágico, reportó el que podría ser el quinto serotipo (http://www.dengue2013bangkok.com/home/index/en). Esta sería la primera descripción de un nuevo serotipo para este virus en los últimos 50 años, lo que viene a opacar el panorama del desarrollo de una posible vacuna para esta infección, cada vez más prevalente en el mundo, particularmente en las regiones tropicales y subtropicales del planeta.

Dengue is a disease caused by the dengue virus belonging to the Flaviviridae family, genus Flavivirus, which is transmitted to humans by the Aedes vector, mainly Aedes aegypti and Aedes albopictus. To date, four serotypes of the virus are recognised, although recently Dr. Nikos Vasilakis, a virologist at the University of Texas during the Third International Conference on Dengue and Dengue Haemorrhagic Fever, reported what could be the fifth serotype (http://www.dengue2013bangkok.com/home/index/en). This would be the first description of a new serotype for this virus in the last 50 years, which would overshadow the outlook for the development of a possible vaccine for this increasingly prevalent infection worldwide, particularly in tropical and subtropical regions of the world.

Association of HLA-DRB1 and TNF genotypes with dengue hemorrhagic fever.

Genes coding for human leukocyte antigen (HLA) class II molecules and tumor necrosis factor (TNF)-α are polymorphic and have been shown to influence susceptibility to infectious diseases. In the present study, HLA-DRB1, -DQB1 and TNF gene polymorphisms were investigated in 114 dengue patients (DEN) [85 dengue fever (DF) cases and 29 dengue hemorrhagic fever (DHF) cases] and 110 healthy controls (HCs) using PCR based methods. The frequencies of HLA-DRB1*07 allele [DF vs. DHF, P=0.0034, Pc=0.044, OR 3.79] and HLA-DRB1*07/*15 genotype [DF vs. DHF, P=0.00071, Pc=0.038, OR 10.41] were significantly higher in DHF cases as compared to HCs and DF cases. Higher frequency of rs1800629 'G/A' genotype was observed in DHF cases as compared to DF cases. The frequency of rs1799964 'C/C' genotype of the TNF gene was found to be significantly higher in all patient groups compared to HCs [HCs vs. DEN, P=0.0054, Pc=0.0162, OR 3.57; HCs vs. DF, P=0.036, OR 2.89; HCs vs. DHF, P=0.0088, Pc=0.0240, OR 5.11]. Presence of combination of HLA-DRB1*07/*15 with either rs1799964 'C/C' genotype or rs1800629 'G/A' genotype or both was present in 17.2% of DHF cases and 1.2% in HCs while this combination was not observed in DF cases. The results suggest that HLA-DRB1*07/*15 genotype in combination with TNF polymorphisms influence progression to DHF.

Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage.

BACKGROUND: Although most symptomatic dengue infections follow an uncomplicated course, complications and unusual manifestations are increasingly being reported due to rising disease burden. Expanded dengue syndrome is a new entity added into World Health Organization (WHO) classification system to incorporate this wide spectrum of unusual manifestations. We report a case of expanded dengue syndrome with subacute thyroiditis and intracerebral hemorrhage. This is the first case report of thyroiditis in dengue infection. CASE PRESENTATION: A 20 years old man presented with fever, myalgias, arthralgias, retro-orbital pain, vomiting and gum bleeding during a large dengue outbreak in Lahore, Pakistan. On 7th day of illness patient became afebrile, but he developed severe headaches, unconsciousness followed by altered behavior. On 9th day of illness patient developed painful neck swelling accompanied by fever, tremors, palpitations, hoarseness of voice and odynophagia. Examination revealed acutely swollen, tender thyroid gland along with features of hyperthyroidism. Laboratory evaluation revealed stable hematocrit, thrombocytopenia and leukopenia. Patient had seroconverted for anti-dengue IgM antibodies on the 10th day of illness. A non-contrast Computed Tomogram (CT) of the brain showed right frontal lobe hematoma. Thyroid profile showed increased free T3 and T4 and low TSH. Technetium thyroid scan showed reduced tracer uptake. He was diagnosed as having subacute thyroiditis and treated with oral prednisolone and propranolol. Follow up CT brain showed resolving hematoma. Patient's recovery was uneventful. CONCLUSION: Subacute thyroiditis may develop during the course of dengue fever and should be included as a manifestation of expanded dengue syndrome. It should be suspected in patients with dengue fever who develop painful thyroid swelling and clinical features of hyperthyroidism.