RESUMO
Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a challenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001-2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-antithrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lymphocytes (%) and thrombin time were increased. In dengue patients, the absolute lymphocytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamination events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies.
Assuntos
Biomarcadores/sangue , Dengue/diagnóstico , Dengue/patologia , Testes Diagnósticos de Rotina/métodos , Adolescente , Adulto , Idoso , Bioestatística , Análise Química do Sangue , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Curva ROC , Venezuela , Adulto JovemRESUMO
A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.
Assuntos
Arrestina/metabolismo , Membrana Celular/metabolismo , Luz , Segmento Externo da Célula Bastonete/metabolismo , Animais , Arrestina/química , Arrestina/isolamento & purificação , Bovinos , Membrana Celular/efeitos da radiação , Peso Molecular , Fosforilação/efeitos da radiação , Ligação Proteica/efeitos da radiação , Segmento Externo da Célula Bastonete/efeitos da radiação , Solubilidade , Especificidade por SubstratoRESUMO
We used gene expression profiling of human primary cells infected in vitro with dengue virus (DENV) as a tool to identify secreted mediators induced in response to the infection. Affymetrix GeneChip analysis of human primary monocytes, B cells and dendritic cells infected with DENV in vitro showed strong induction of monocyte chemotactic protein 2 (MCP-2/CCL8), interferon gamma-induced protein 10 (IP-10/CXCL10) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10). The expression of these genes was confirmed in dendritic cells infected with DENV in vitro at mRNA and protein levels. A prospectively enrolled cohort of DENV-infected Venezuelan patients was used to measure the levels of these proteins in serum during three different periods of the disease. Results showed significant increase of MCP-2, IP-10, and TRAIL levels in patients infected with DENV during the febrile period, when compared to healthy donors and patients with other febrile illnesses. MCP-2 and IP-10 levels were still elevated during the post-febrile period while TRAIL levels dropped close to normal after defervescense. Patients with primary infections had higher TRAIL levels than patients with secondary infections during the febrile period of the disease. Increased levels of IP-10, TRAIL and MCP-2 in acute DENV infections suggest a role for these mediators in the immune response to the infection. MCP-2 was identified in this work as a new unreported and important dengue-related protein and IP-10 was confirmed as a novel and strong pro-inflammatory marker in acute disease.
Assuntos
Vírus da Dengue/imunologia , Vírus da Dengue/fisiologia , Dengue/imunologia , Perfilação da Expressão Gênica , Adolescente , Adulto , Linfócitos B/virologia , Células Cultivadas , Quimiocina CCL8/biossíntese , Quimiocina CCL8/sangue , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/sangue , Criança , Estudos de Coortes , Células Dendríticas/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/virologia , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Venezuela , Adulto JovemRESUMO
Endothelial-mesenchymal transdifferentiation (EMT) is believed to play a crucial role in embryonic vascular development and intimal thickening, which contributes to the pathogenesis of atherosclerotic lesions. However, the mechanisms by which it occurs, as well as the signals that control it, have not yet been elucidated. Given the important role played by the CD40-CD40 ligand (CD40L) system during the initiation and progress of atherosclerosis, we investigated whether both CD40 and CD40L were present in the aortic wall during EMT and the advanced stages of chicken embryo development. CD40-CD40L expression was found on endothelial cells (ECs), mesenchymal cells, and smooth muscle cells (SMCs) at all stages examined, and appeared to be distributed across the aortic wall. However, some notable differences between the expression patterns were observed. CD40 had a more restricted distribution compared to CD40L, and did not stain every cell type of the aortic wall. According to immunoblotting and enzyme-linked immunosorbent assay (ELISA) analyses, the CD40L content was highest at day 7 of development. An important and novel finding was the expression of CD40L in areas where ECs transdifferentiate into mesenchymal cells. Specifically, CD40L was associated to the surface of cells that were detaching and migrating from the monolayer of ECs, whereas for CD40 a very diffuse subcellular localization was seen at the monolayer and the detaching and migrating cells. These data suggest a possible role for CD40-CD40L interactions during EMT and the remodeling of the aorta.