RESUMO
Zika fever is a reemerging arthropod-borne viral disease; however, Zika virus (ZIKV) can be transmitted by other, non-vector means. Severe Zika fever is characterized by neurological disorders, autoimmunity, or congenital Zika syndrome. Monocytes are primary ZIKV targets in humans and, in response to infection, release extracellular vesicles like exosomes. Exosomes mediate intercellular communication and are involved in the virus's ability to circumvent the immune response, promoting pathological processes. This study aimed to evaluate the role of monocyte exosomes in cell-to-cell viral transmission. We isolated exosomes from ZIKV-infected monocytes (Mø exo ZIKV) by differential ultracentrifugation and identified them by nanoparticle tracking analysis; transmission electron microscopy; and CD63, CD81, TSG101, and Alix detection by cytofluorometry. Purified exosome isolates were obtained by uncoupling from paramagnetic beads or by treatment with UV radiation and RNase A. We found that Mø exo ZIKV carry viral RNA and E/NS1 proteins and that their interaction with naïve cells favors viral transmission, infection, and cell differentiation/activation. These data suggest that Mø exo ZIKV are an efficient alternative pathway for ZIKV infection. Knowledge of these mechanisms contributes to understanding the pathogenesis of severe disease and to the development of new vaccines and therapies.
Assuntos
Exossomos , Vesículas Extracelulares , Infecção por Zika virus , Zika virus , Humanos , MonócitosRESUMO
To date, no safe vaccine or antivirals for Zika virus (ZIKV) infection have been found. The pathogenesis of severe Zika, where host and viral factors participate, remains unclear. For the control of Zika, it is important to understand how ZIKV interacts with different host cells. Knowledge of the targeted cellular pathways which allow ZIKV to productively replicate and/or establish prolonged viral persistence contributes to novel vaccines and therapies. Monocytes and endothelial vascular cells are the main ZIKV targets. During the infection process, cells are capable of releasing extracellular vesicles (EVs). EVs are mediators of intercellular communication. We found that mosquito EVs released from ZIKV-infected (C6/36) cells carry viral RNA and ZIKV-E protein and are able to infect and activate naïve mosquito and mammalian cells. ZIKV C6/36 EVs promote the differentiation of naïve monocytes and induce a pro-inflammatory state with tumor necrosis factor-alpha (TNF-α) mRNA expression. ZIKV C6/36 EVs participate in endothelial vascular cell damage by inducing coagulation (TF) and inflammation (PAR-1) receptors at the endothelial surface of the cell membranes and promote a pro-inflammatory state with increased endothelial permeability. These data suggest that ZIKV C6/36 EVs may contribute to the pathogenesis of ZIKV infection in human hosts.
Assuntos
Aedes/virologia , Vesículas Extracelulares/metabolismo , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Células Endoteliais/patologia , Células Endoteliais/virologia , Humanos , Monócitos/virologia , Fenótipo , Fosfatidilserinas/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Inativação de VírusRESUMO
Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biossíntese de Proteínas/genética , Multimerização Proteica/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Chlorocebus aethiops , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Mutação Puntual/genética , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Viral/genética , Rotavirus/genética , Infecções por Rotavirus/virologia , Alinhamento de Sequência , Replicação Viral/genéticaRESUMO
Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.
Assuntos
Aedes/metabolismo , Vírus da Dengue/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Ácido N-Acetilneuramínico/biossíntese , Aedes/genética , Aedes/virologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ácido N-Acetilneuramínico/genética , Saliva/enzimologia , Saliva/virologia , Sialiltransferases/genética , Sialiltransferases/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
BACKGROUND: Dengue fever (DF) is the most prevalent arthropod-borne viral disease affecting humans. The World Health Organization (WHO) proposed a revised classification in 2009 to enable the more effective identification of cases of severe dengue (SD). This was designed primarily as a clinical tool, but it also enables cases of SD to be differentiated into three specific subcategories (severe vascular leakage, severe bleeding, and severe organ dysfunction). However, no study has addressed whether this classification has advantage in estimating factors associated with the progression of disease severity or dengue pathogenesis. We evaluate in a dengue outbreak associated risk factors that could contribute to the development of SD according to the 2009 WHO classification. METHODS: A prospective cross-sectional study was performed during an epidemic of dengue in 2009 in Chiapas, Mexico. Data were analyzed for host and viral factors associated with dengue cases, using the 1997 and 2009 WHO classifications. The cost-benefit ratio (CBR) was also estimated. RESULTS: The sensitivity in the 1997 WHO classification for determining SD was 75%, and the specificity was 97.7%. For the 2009 scheme, these were 100% and 81.1%, respectively. The 2009 classification showed a higher benefit (537%) with a lower cost (10.2%) than the 1997 WHO scheme. A secondary antibody response was strongly associated with SD. Early viral load was higher in cases of SD than in those with DF. Logistic regression analysis identified predictive SD factors (secondary infection, disease phase, viral load) within the 2009 classification. However, within the 1997 scheme it was not possible to differentiate risk factors between DF and dengue hemorrhagic fever or dengue shock syndrome. The critical clinical stage for determining SD progression was the transition from fever to defervescence in which plasma leakage can occur. CONCLUSIONS: The clinical phenotype of SD is influenced by the host (secondary response) and viral factors (viral load). The 2009 WHO classification showed greater sensitivity to identify SD in real time. Timely identification of SD enables accurate early decisions, allowing proper management of health resources for the benefit of patients at risk for SD. This is possible based on the 2009 WHO classification.
Assuntos
Vírus da Dengue/fisiologia , Dengue Grave/virologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Estudos Transversais , Vírus da Dengue/genética , Progressão da Doença , Feminino , Humanos , Lactente , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Estudos Prospectivos , Dengue Grave/epidemiologia , Dengue Grave/imunologia , Índice de Gravidade de Doença , Organização Mundial da Saúde , Adulto JovemRESUMO
OBJECTIVES: Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. AIM: To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). METHODS: Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. RESULTS: Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1ß, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. CONCLUSION: Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA.
Assuntos
Artroscopia , Colágeno Tipo I/uso terapêutico , Inflamação/tratamento farmacológico , Osteoartrite do Joelho/tratamento farmacológico , Irrigação Terapêutica , Adulto , Terapia Combinada , Método Duplo-Cego , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/cirurgia , Placebos , Resultado do TratamentoRESUMO
Dengue fever (DF) is the most prevalent arthropod-borne viral disease of humans. No safe vaccine is available, there is no experimental animal model and no specific treatment (antiviral) for Dengue virus (DV) infection exists. The pathogenic mechanisms of the severe forms of the disease, such as Dengue shock syndrome (DSS) and Dengue haemorrhagic fever (DHF), in which endothelial damage is the pathognomonic sign, are not fully understood. Clinical observations have revealed significant abnormalities in the coagulation and inflammation systems, with increased levels of soluble thrombomodulin (sTM) in the plasma of patients with DHF/DSS (grade III or IV). Blood sTM was proposed as an early predictor of DSS during the febrile stage. However, the role of the DV in endothelial injury during DSS is unclear. Here, we present novel insights into the participation of DV in the downregulation of the thrombomodulin-thrombin-protein C complex formation at the endothelial surface, with a reduction in activated protein C (APC). APC is the most important vasoprotective protein because it downregulates thrombin generation (by the inactivation of procoagulant factors Va and VIIIa) and has anti-inflammatory, antiapoptotic, and barrier protection properties. These biological functions of APC are associated with the endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1) signalling pathways, which link the coagulation-inflammation responses. We found alterations in the antithrombotic and cytoprotective protein C pathways during DV infection of human endothelial vascular cells, which may explain the vasculopathy observed during DHF/DSS. Clarification of the basic principles that underlie these processes has important implications for the design of new therapeutic strategies for DHF/DSS.
Assuntos
Vírus da Dengue/patogenicidade , Células Endoteliais/virologia , Proteína C/metabolismo , Dengue Grave/virologia , Transdução de Sinais , Trombina/metabolismo , Trombomodulina/metabolismo , Animais , Antígenos CD/metabolismo , Apoptose , Permeabilidade Capilar , Linhagem Celular , Chlorocebus aethiops , Citoproteção , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Receptor de Proteína C Endotelial , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Receptor PAR-1/metabolismo , Receptores de Superfície Celular/metabolismo , Dengue Grave/sangue , Dengue Grave/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dengue fever is the most prevalent viral disease transmitted by vectors (Aedes aegypti, Aedes albopictus) in worldwide. More than 100 million cases occur annually with a mortality rate of 5% and no safe vaccine is available. The pathogenesis of Dengue, where host and viral factors participate in the establishment of Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS) remains unresolved. Clinical observations have revealed significant abnormalities in coagulation and inflammation systems, with increased levels of tissue factor (TF) and the chemokine IL-8, correlating with the severity of the disease and implicating damage to endothelial vascular cells (EVC). Here we present novel insights concerning the crosstalk between the regulatory signaling pathways of the coagulation-inflammation processes, during Dengue virus (DV) infection of EVC. We found that DV up-regulates Protease Activated receptor type-1 (inflammation) and TF (coagulation) receptors, via the phosphorylation of p38 and ERK1/2 MAPKs, which favor the activation of NF-kappaB transcription factor. This induces pro-inflammatory (IL-8) or pro-adhesive (VCAM-1) gene expression which may lead to EVC activation. The elucidation of the basic principles that signal these processes has important implications for the design of new therapeutic strategies for DHF/DSS.
Assuntos
Coagulação Sanguínea , Vírus da Dengue/patogenicidade , Células Endoteliais/virologia , Inflamação/virologia , Dengue Grave , Transdução de Sinais , Células Cultivadas , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Humanos , Inflamação/sangue , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Protrombina/metabolismo , Receptor PAR-1/metabolismo , Dengue Grave/sangue , Dengue Grave/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismo , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Virulência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Beta2-glycoprotein I (beta2GPI) is a glycoprotein of unknown physiological function. It is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome (APS). beta2GPI binds with high affinity to the atherogenic lipoprotein Lp(a) which shares structural homology with plasminogen, a key molecule in the fibrinolytic system. Impaired fibrinolysis has been described in APS. The present work reports the interaction between beta2GPI and Glu-Plasminogen which may explain the recently described proteolytic effect of plasmin on beta2GPI. In the process of Glu-Plasminogen activation, we found an increase in plasmin generation both at fibrin and cellular surface level as a function of the concentration of beta2GPI added, suggesting an important role as a cofactor in the trimolecular complex beta2GPI-Plasminogen-tPA. This phenomenon represents a novel regulatory step both in the positive feedback mechanism for extrinsic fibrinolysis and in antithrombotic regulation. IgG anti-beta2GPI antibodies recognized the beta2GPI at the endothelial surface inducing its activation with an increase of ICAM-I and a decrease in the expression of thrombomodulin favoring a pro-thrombotic state in the vascular endothelium. The interference in the plasmin conversion by anti-beta2GPI antibodies could generate thrombosis as observed in APS.
Assuntos
Glicoproteínas/fisiologia , Plasminogênio/metabolismo , Anticorpos , Células Cultivadas , Endotélio Vascular/metabolismo , Ativação Enzimática , Fibrinólise , Glicoproteínas/imunologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Kringles/fisiologia , Trombomodulina/biossíntese , beta 2-Glicoproteína IRESUMO
Autosomal recessive hypercholesterolemia (ARH) is characterized by elevated LDL serum levels, xanthomatosis, and premature coronary artery disease. Three loci have been described for this condition (1p35, 15q25-q26 and 13q). Recently, the responsible gene at the 1p35 locus, encoding an LDL receptor adaptor protein (ARH) has been identified. We studied a Mexican ARH family with two affected siblings. Sequence analysis of the ARH gene (1p35 locus) revealed that the affected siblings are homozygous for a novel mutation (IVS4+2T>G) affecting the donor splice site in intron 4, whereas both the parents and an unaffected sister are heterozygous for this mutation. The IVS4+2T>G mutation results in a major alternative transcript derived from a cryptic splice site, which carries an in-frame deletion of 78 nucleotides in the mature mRNA. The translation of this mRNA yields a mutant protein product (ARH-26) lacking 26 amino acids, resulting in the loss of beta-strands beta6 and beta7 from the PTB domain. This is the first case where a naturally occurring mutant with an altered PTB domain has been identified.