RESUMO
RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.
Assuntos
Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Família , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Heterozigoto , Homozigoto , Adolescente , Adulto , Plaquetas/metabolismo , Domínio Catalítico/genética , Criança , Feminino , Triagem de Portadores Genéticos/métodos , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Agregação Plaquetária , Portugal , Adulto JovemRESUMO
DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293 T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250 kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Éxons , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/imunologia , Células HEK293 , Humanos , Soros Imunes/química , Soros Imunes/metabolismo , Interleucina-4/imunologia , Células Jurkat , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas , RNA Mensageiro/genética , TranscriptomaRESUMO
BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (PREX1) was reported to be overexpressed in some cancers and involved in cancer development, but its expression and significance in gastric cancer remain unclear. AIM: To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer, especially to evaluate the potential mechanism of PREX1 in gastric cancer. METHODS: Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer. The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal. The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor (TGF) ß1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics. Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFß1 on the expression of PREX1. Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFß1 pathway. Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells. RESULTS: PREX1 was overexpressed in the gastric tumors, and the expression levels were positively associated with the development of gastric cancer. Also, the high expression of PREX1 revealed poor prognosis, especially for those advanced and specific intestinal gastric cancer patients. PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFß1-related mediators. Furthermore, TGFß1 could induce the expression of PREX1 at both the protein and mRNA level. Also, PREX1 could activate the TGFß1 pathway. The induced PREX1 could increase the migration and invasion activity of gastric cancer cells. CONCLUSION: PREX1 is overexpressed in gastric cancer, and the high level of PREX1 predicts poor prognosis. PREX1 is closely associated with TGFß signaling and promotes the metastasis of gastric cancer cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/sangue , Fosfatos de Fosfatidilinositol/sangue , Neoplasias Gástricas/genética , Fator de Crescimento Transformador beta1/sangue , Adulto , Biologia Computacional , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , RNA Mensageiro/sangue , Transdução de Sinais/genética , Neoplasias Gástricas/sangue , Neoplasias Gástricas/mortalidadeRESUMO
Friedreich's ataxia (FA) is a neurodegenerative disease with no approved therapy that is the result of frataxin deficiency. The identification of human FA blood biomarkers related to disease severity and neuro-pathomechanism could support clinical trials of drug efficacy. To try to identify human biomarkers of neuro-pathomechanistic relevance, we compared the overlapping gene expression changes of primary blood and skin cells of FA patients with changes in the Dorsal Root Ganglion (DRG) of the KIKO FA mouse model. As DRG is the primary site of neurodegeneration in FA, our goal was to identify which changes in blood and skin of FA patients provide a 'window' into the FA neuropathomechanism inside the nervous system. In addition, gene expression in frataxin-deficient neuroglial cells and FA mouse hearts were compared for a total of 5 data sets. The overlap of these changes strongly supports mitochondrial changes, apoptosis and alterations of selenium metabolism. Consistent biomarkers were observed, including three genes of mitochondrial stress (MTIF2, ENO2), apoptosis (DDIT3/CHOP), oxidative stress (PREX1), and selenometabolism (SEPW1). These results prompted our investigation of the GPX1 activity as a marker of selenium and oxidative stress, in which we observed a significant change in FA patients. We believe these lead biomarkers that could be assayed in FA patient blood as indicators of disease severity and progression, and also support the involvement of mitochondria, apoptosis and selenium in the neurodegenerative process.
Assuntos
Biomarcadores/sangue , Ataxia de Friedreich/sangue , Gânglios Espinais/metabolismo , Estresse Oxidativo/genética , Animais , Antioxidantes/metabolismo , Apoptose/genética , Modelos Animais de Doenças , Fatores de Iniciação em Eucariotos/sangue , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Gânglios Espinais/patologia , Regulação da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Proteínas de Ligação ao Ferro/genética , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/sangue , Miocárdio/metabolismo , Selênio/metabolismo , Fator de Transcrição CHOP/sangue , FrataxinaRESUMO
Objective: The aim of the study was to determine the expression profile of long noncoding RNAs (lncRNAs) in CD4+ T cells from COPD patients and explore the clinical value of the lncRNAs. Methods: First, microarray analysis was performed. Differentially expressed lncRNAs were validated by quantitative real-time reverse transcription-PCR (qRT-PCR) in samples from 56 patients with acute exacerbations of COPD (AECOPD), 56 patients with stable COPD, and 35 healthy controls. Meanwhile, the clinical value was tested by receiver operating characteristic curve analysis. The functions of lncRNAs were analyzed by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database. The potential target genes that might be regulated by NR-026690 and ENST00000447867 were identified by the lncRNA-mRNA network and competing endogenous RNA network. The transcriptional expression level of rap guanine nucleotide exchange factor 3 (RAPGEF3) was tested by qRT-PCR. The correlation of the expression between NR-026690, ENST00000447867, and RAPGEF3 was analyzed by Spearman's correlation test. Results: We found that the relative expression levels of ENST00000447867 and NR-026690 in the CD4+ T cells of AECOPD patients were significantly higher than in the stable COPD patients and control subjects by microarray and qRT-PCR validation. The transcriptional expression level of RAPGEF3 in the CD4+ T cells was significantly higher in the AECOPD group compared to the control group (P<0.01) and the stable COPD group (P<0.05). RAPGEF3 expression was positively associated with NR-026690 (r=0.4925, P<0.01) and ENST00000447867 (r=0.4065, P<0.01). Conclusion: NR-026690 and ENST00000447867 might be potential biomarkers for COPD. They might affect RAPGEF3 as miRNA sponges to regulate COPD development.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , RNA Longo não Codificante/sangue , Idoso , Estudos de Casos e Controles , Citocinas/sangue , Citocinas/genética , Bases de Dados Genéticas , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Marcadores Genéticos , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Regulação para CimaRESUMO
CONTEXT: Osteoporosis (OP) is a progressive systemic bone disease. Dual-energy X-ray absorptiometry (DXA) is routinely employed and is considered the gold standard method for the diagnosis of OP. OBJECTIVE: We aimed to investigate the potential use of combined information from multiple bone turnover markers (BTMs) as a clinical diagnostic tool for OP. MATERIALS AND METHODS: A total of 9053 Chinese postmenopausal women (2464 primary OP patients and 6589 healthy controls) were recruited. Serum levels of six common BTMs, including BAP, BSP, CTX, OPG, OST and sRANKL were assayed. Models based on support vector machine (SVM) were constructed to explore the efficiency of different combinations of multiple BTMs for OP diagnosis. RESULTS: Increasing the number of BTMs used in generating the models increased the predictive power of the SVM models for determining the disease status of study subjects. The highest kappa coefficient for the model with one BTM (BAP) compared to DXA was 0.7783. The full model incorporating all six BTMs resulted in a high kappa coefficient of 0.9786. CONCLUSION: Our findings showed that although single BTMs were not sufficient for OP diagnosis, appropriate combinations of multiple BTMs incorporated into the SVM models showed almost perfect agreement with the DXA.
Assuntos
Biomarcadores/sangue , Remodelação Óssea/genética , Osteoporose/sangue , Absorciometria de Fóton , Idoso , Densidade Óssea/genética , China/epidemiologia , Enzimas de Restrição do DNA/sangue , Feminino , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Pessoa de Meia-Idade , Osteoporose/diagnóstico por imagem , Osteoporose/patologia , Ligante RANK/sangue , Fatores de Troca de Nucleotídeo Guanina Rho/sangue , Máquina de Vetores de SuporteRESUMO
Multiplexed biosensors hold great promise for early diagnosis of diseases where the detection of multiple biomarkers is required. Hyper Immunoglobulin E syndromes (HIES) are rare primary immunodeficiency disorders associated with mutations either in the signal transducer and activator of transcription 3 (STAT3), dedicator of cytokinesis 8 DOCK8) or phosphoglucomutase 3 (PGM3) genes. Yet, the diagnosis of HIES is challenged by the complexity of the existing laboratory assays. Here, we report for the first time the development of a multiplexed electrochemical immunosensor for the simultaneous detection of DOCK8, STAT3 and PGM3 proteins. The immunosensor was constructed on carbon array electrodes that were first modified by electrodeposition of gold nanoparticles (AuNPs). The array electrodes were then used to immobilize specific antibodies for the three proteins after the functionalization of the electrodes with cysteamine/glutaraldehyde linkers. The simultaneous detection of the DOCK8, PGM3 and STAT3 proteins was successfully realized by the immunosensor with respective limits of detections of 3.1, 2.2 and 3.5â¯pg/ml. The immunosensor has shown good sensitivity as well as selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and Duchenne Muscular Dystrophy (DMD). Moreover, the immunosensor was successfully applied in human serum samples showing capability to distinguish the HIES from the control samples.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Análise Química do Sangue/instrumentação , Análise Química do Sangue/métodos , Síndrome de Job/diagnóstico , Ouro/química , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Síndrome de Job/sangue , Nanopartículas Metálicas/química , Fosfoglucomutase/sangue , Fator de Transcrição STAT3/sangueRESUMO
The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbß3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.
Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/sangue , Transtornos Hemorrágicos/sangue , Transtornos Hemorrágicos/genética , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Plaquetas/patologia , Criança , Pré-Escolar , Feminino , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , LinhagemRESUMO
Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.
Assuntos
Barreira Hematoencefálica/imunologia , Interleucina-9/imunologia , Acidente Vascular Cerebral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Complexo CD3/sangue , Estudos de Casos e Controles , Hipóxia Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Expressão Gênica , Glucose/metabolismo , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Interleucina-9/sangue , Interleucina-9/genética , Interleucina-9/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/biossíntese , Proteínas Nucleares/sangue , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Índice de Gravidade de Doença , Acidente Vascular Cerebral/patologia , Subpopulações de Linfócitos T/imunologia , Proteínas de Junções Íntimas/metabolismo , Transativadores/sangue , Transativadores/genética , Adulto JovemRESUMO
Cardiovascular complications determine morbidity/mortality in chronic kidney disease (CKD). We hypothesized that progressive CKD drives the release of cathepsin-S (Cat-S), a cysteine protease that promotes endothelial dysfunction and cardiovascular complications. Therefore, Cat-S, soluble tumor-necrosis-factor receptor (sTNFR) 1/2 and glomerular filtration rate (GFR) were measured in a CKD mouse model, a German CKD-cohort (MCKD, n = 421) and two Swedish community-based cohorts (ULSAM, n = 764 and PIVUS, n = 804). Association between Cat-S and sTNFR1/2/GFR was assessed using multivariable linear regression. In the mouse model, Cat-S and sTNFR1/2 concentrations were increased following the progressive decline of GFR, showing a strong correlation between Cat-S and GFR (r = -0.746, p < 0.001) and Cat-S and sTNFR1/sTNFR2 (r = 0.837/0.916, p < 0.001, respectively). In the human cohorts, an increase of one standard deviation of estimated GFR was associated with a decrease of 1.008 ng/ml (95%-confidence interval (95%-CI) -1.576-(-0.439), p < 0.001) in Cat-S levels in MCKD; in ULSAM and PIVUS, results were similar. In all three cohorts, Cat-S and sTNFR1/sTNFR2 levels were associated in multivariable linear regression (p < 0.001). In conclusion, as GFR declines Cat-S and markers of inflammation-related endothelial dysfunction increase. The present data indicating that Cat-S activity increases with CKD progression suggest that Cat-S might be a therapeutic target to prevent cardiovascular complications in CKD.
Assuntos
Catepsinas/sangue , Fatores de Troca do Nucleotídeo Guanina/sangue , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Adulto , Idoso , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Alemanha , Taxa de Filtração Glomerular , Humanos , Testes de Função Renal , Masculino , Camundongos , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , Fatores de Risco , SuéciaRESUMO
Panels composed of Single Nucleotide Polymorphisms (SNPs) in genes related to pigmentation, when associated with different phenotypes, may assist in predicting the physical appearance of an individual, being very useful in forensic caseworks. We evaluated the association of seven OCA2-HERC2 SNPs and haplotypes with pigmentation characteristics (eye, skin, hair and freckles) in the highly admixed and phenotypically heterogeneous Brazilian population. All the seven SNPs evaluated presented one allele associated with phenotypes from at least two pigmentation features and the alternative allele associated with the opposite phenotypes from the same trait. The genotypic associations followed the same pattern for all seven SNPs. Nine haplotypes were observed in our sample and eight were associated with at least two pigmentation traits. Such SNPs and haplotypes could be deemed as good predictors for the presence of freckles and for skin, eye and hair pigmentation in the Brazilian population.
Assuntos
Albinismo Oculocutâneo/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Haplótipos/genética , Polimorfismo de Nucleotídeo Único , Pigmentação da Pele/genética , Albinismo Oculocutâneo/sangue , Brasil , Feminino , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Masculino , Ubiquitina-Proteína LigasesRESUMO
Dedicator of cytokinesis 2 (DOCK2) is a CDM family protein containing DOCK homology region (DHR)-1 and DHR-2, Src-homology 3 (SH3) domain, and C-terminal polybasic amino acid cluster. The CDM family consists of 11 mammalian members and is classified into four subfamilies, the DOCK-A, -B, -C, and -D. DOCK2 is a member of DOCK-A subfamily and an atypical guanine exchange factor regulating the loading of GTP to activate Rac. It is primarily found in peripheral blood, spleen, and thymus and mainly expressed in lymphocytes and macrophages of various organs. DOCK2 is also expressed in microglial in brain and is induced in neointima smooth muscle following vascular injury. Functionally, DOCK2 is involved in cell motility, polarity, adhesion, proliferation, and apoptosis. It is essential for lymphocyte migration and activation as well as neutrophil chemotaxis. DOCK2 also regulates the differentiation of natural killer T cells, type 2 T helper cells, and plasmacytoid dendritic cells. In addition, it is important for the growth of B cell lymphoma and prostate cancer cells. Deletion of DOCK2 enables long-term cardiac allograft survival. Moreover, DOCK2 is associated with the Alzheimer Disease, HIV development, and the early-onset of invasive infections. Recently, we found that DOCK2 plays a critical role in SMC phenotypic modulation and vascular remodeling. In this review, we will briefly summarize recent advancement of DOCK2 function. J. Cell. Physiol. 232: 1931-1940, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema Imunitário/metabolismo , Transdução de Sinais , Animais , Doença , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Sistema Imunitário/imunologia , Ligação ProteicaRESUMO
OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.
Assuntos
Plaquetas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Hemostasia , Ativação Plaquetária , Trombose/prevenção & controle , Animais , Modelos Animais de Doenças , Genótipo , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos Transgênicos , Mutação , Fenótipo , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Trombose/sangue , Trombose/genética , Fatores de Tempo , Proteínas rap1 de Ligação ao GTP/sangueRESUMO
BACKGROUND: B and T cells play a key role in rheumatoid arthritis (RA) pathophysiology. RasGRP1 and RasGRP3 are involved in T and B cell receptors signaling, and belong to gene combination able to predict infliximab responsiveness, leading to the question of RasGRP1 and RasGRP3 involvement in RA. METHODS: RasGRP1 and RasGRP3 expression levels were measured by qRT-PCR and/or western-blot in peripheral blood mononuclear cells (PBMCs), in T and B cells from untreated RA patients and in RA patients treated by TNFα inhibitors. T and B cells from healthy controls (HC) were cultured with TNFα, and TNFα receptors neutralizing antibodies to highlight the TNFα effects on RasGRP1 and RasGRP3 pathways. MAPK pathways and apoptosis were respectively analyzed using the Proteome Profiler arrays and flow cytometry. RESULTS: In PBMCs from RA patients, gene expression levels of RasGRP1 were invariant while RasGRP3 was downregulated under TNFα inhibitors and upregulated under TNFα. In T cells from RA patients, RasGRP1 was decreased and its gene expression level was correlated with disease activity. In T cells from HC, TNFα stimulation increased RasGRP1 gene expression level while it reduced RasGRP1 protein expression level. Bryostatin-1 experiments have confirmed that the TNFα effect observed on T cells proliferation was due to the decrease of RasGRP1 expression. Besides, RasGRP3 expression level increased in PBMCs from RA patients under TNFα and in B cells from HC leading us to conclude that RasGRP3 in B cells was modulated by TNFα. CONCLUSION: This study demonstrates RasGRP1 dysregulation in RA patients while RasGRP3 is characterized as a biomarker linked to TNFα inhibitors. After binding to TNFR1, TNFα reduced RasGRP1 protein expression resulting in inhibition of T cell activation. TRIAL REGISTRATION: Clinicaltrials.gov NCT00234234 , registered 04 November 2008; NCT00767325 , registered 05 October 2005.
Assuntos
Artrite Reumatoide/sangue , Proteínas de Ligação a DNA/sangue , Fatores de Troca do Nucleotídeo Guanina/sangue , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/sangue , Adalimumab/farmacologia , Adalimumab/uso terapêutico , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Biomarcadores/sangue , Células Cultivadas , Etanercepte/farmacologia , Etanercepte/uso terapêutico , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Adulto Jovem , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.
Assuntos
Ativação Plaquetária/fisiologia , Proteínas R-SNARE/sangue , Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/sangue , Animais , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Grânulos Citoplasmáticos/fisiologia , Exocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/sangue , Agregação Plaquetária/fisiologia , Proteínas R-SNARE/deficiência , Proteínas R-SNARE/genéticaRESUMO
BACKGROUND: To evaluate effects of neoadjuvant chemotherapy on the bone turnover markers of preoperational breast cancer patients. METHODS: Forty-one breast cancer patients (29 premenopausal and 12 postmenopausal) and 60 healthy women (30 premenopausal and 30 postmenopausal) aged 30-64 years, were evaluated for their bone status. Serum levels of the bone formation markers PINP and BAP, as well as the resorption markers ICTP and ß-Crosslaps in addition to E2, FSH, 25(OH)D and PTH were measured at the initial diagnosis and at 24 hours after each four chemotherapy cycles. BMD T-scores were determined in 12 patients 6 months after the neoadjuvant chemotherapies. RESULTS: The baseline levels of both bone formation and resorption markers in premenopausal patients were higher than in premenopausal healthy women (p<0.05), while no statistic difference was observed between postmenopausal patients and postmenopausal healthy women. Regardless of the menopausal status, chemotherapy increased the ICTP and ß-Crosslaps levels (p<0.05), but decreased the BAP and PINP levels (p<0.05), the later one significantly more with Taxane medication (p<0.01, p<0.05). Chemotherapy caused significant decreases of 25(OH)D levels in premenopausal (p<0.01) and postmenopausal (p<0.05) patients, however, did not affect the PTH concentrations. In premenopausal patients the E2 level decreased, while the FSH level increased after chemotherapy (p<0.05). Patients with pronounced ICTP and ß-Crosslaps combined with reduced BAP and PINP serum concentrations after neoadjuvant chemotherapies were prone to develop osteoporosis 6 month later. CONCLUSIONS: Neoadjuvant chemotherapy appeared to promote bone resorption and inhibit bone formation in both postmenopausal and premenopausal early-stage breast patients.
Assuntos
Remodelação Óssea , Neoplasias da Mama , Colágeno Tipo I/sangue , Colágeno/sangue , Fatores de Troca do Nucleotídeo Guanina/sangue , Terapia Neoadjuvante , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Pró-Colágeno/sangue , Adulto , Neoplasias da Mama/sangue , Neoplasias da Mama/terapia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The incidence of osteoporotic fractures increases as our population ages. Until now, the exact biochemical processes that occur during the healing of metaphyseal fractures remain unclear. Diagnostic instruments that allow a dynamic insight into the fracture healing process are as yet unavailable. In the present matched pair analysis, we study the time course of the osteoanabolic markers bone specific alkaline phosphatase (BAP) and transforming growth factor ß1 (TGFß1), as well as the osteocatabolic markers crosslinked C-telopeptide of type-I-collagen (ß-CTX) and serum band 5 tartrate-resistant acid phosphatase (TRAP5b), during the healing of fractures that have a low level of bone mineral density (BMD) compared with fractures that have a normal BMD. Between March 2007 and February 2009, 30 patients aged older than 50 years who suffered a metaphyseal fracture were included in our study. BMDs were verified by dual energy Xray absorptiometry (DXEA) scans. The levels of BTMs were examined over an 8-week period. Osteoanabolic BAP levels in those with low levels of BMD were significantly different from the BAP levels in those with normal BMD. BAP levels in the former group increased constantly, whereas the latter group showed an initial strong decrease in BAP followed by slowly rising values. Osteocatabolic ß-CTX increased in the bone of the normal BMD group constantly, whereas these levels decreased significantly in the bone of the group with low BMD from the first week. TRAP5b was significantly reduced in the low level BMD group. With this work, we conduct first insights into the molecular biology of the fracture healing process in patients with low levels of BMD that explains the mechanism of its fracture healing. The results may be one reason for the reduced healing qualities in bones with low BMD.
Assuntos
Densidade Óssea , Osso e Ossos/metabolismo , Fraturas Ósseas/etiologia , Fraturas Ósseas/metabolismo , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Fraturas Ósseas/epidemiologia , Fatores de Troca do Nucleotídeo Guanina/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Fator de Crescimento Transformador beta/sangue , CicatrizaçãoRESUMO
This postmarketing surveillance study assessed the safety and effectiveness of daily teriparatide treatment in patients with osteoporosis in a Japanese clinical setting. In this prospective, multicenter, observational study, patients with osteoporosis at high risk for fracture received subcutaneous injections of teriparatide (20 µg/day) for a maximum of 24 months. For this interim report, data from 1,671 patients were eligible for analysis at the cutoff date. The mean age was 75.3 years; 93% of patients (1,552/1,671 patients) were women. There were 117 adverse drug reactions (ADRs) reported in 101 of 1,671 patients (6.04%); the most common reported ADRs were nausea, dizziness, headache, and palpitations. No clinically significant safety issues were identified, although 5 serious ADRs were reported in 4/1,671 (0.24 %) patients. At 12 months, 71.9% of patients remained on teriparatide treatment. From 1 month, there were rapid increases in the biomarkers of bone formation P1NP and, to a lesser extent, BAP. In contrast, increases in the biomarkers of bone resorption, serum NTX, urinary NTX, and TRACP5b, were smaller. After 12 months of treatment, there was an increase in bone mineral density at the lumbar spine, femoral neck, and total hip, and a decrease in the Visual Analog Scale score for back pain. The incidence of new vertebral and nonvertebral fractures was 1.21% and 3.18%, respectively. In conclusion, the favorable safety profile and effectiveness of teriparatide observed in this population of Japanese patients with osteoporosis were accompanied by relatively high persistence with treatment, which is a key factor in the success of osteoporosis treatment.
Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Osteoporose/tratamento farmacológico , Teriparatida/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/urina , Conservadores da Densidade Óssea/efeitos adversos , Reabsorção Óssea/sangue , Reabsorção Óssea/urina , Feminino , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/urina , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/sangue , Osteoporose/urina , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/urina , Pró-Colágeno/sangue , Pró-Colágeno/urina , Estudos Prospectivos , Teriparatida/efeitos adversosRESUMO
BACKGROUND: Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene is thought to be a key regulator of E6AP. METHODS AND RESULTS: Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. CONCLUSIONS: Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.
Assuntos
Amish/genética , Síndrome de Angelman/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Adolescente , Adulto , Proteínas de Ciclo Celular/química , Linhagem Celular , Criança , Pré-Escolar , DNA/análise , DNA/genética , Análise Mutacional de DNA , Feminino , Fibroblastos/química , Fibroblastos/metabolismo , Estudo de Associação Genômica Ampla , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Lactente , Masculino , Modelos Moleculares , Proteínas Nucleares/química , Linhagem , Ubiquitina-Proteína LigasesRESUMO
The Rho family of GTP binding proteins, also commonly referred to as the Rho GTPases, are master regulators of the platelet cytoskeleton and platelet function. These low-molecular-weight or 'small' GTPases act as signaling switches in the spatial and temporal transduction, and amplification of signals from platelet cell surface receptors to the intracellular signaling pathways that drive platelet function. The Rho GTPase family members RhoA, Cdc42 and Rac1 have emerged as key regulators in the dynamics of the actin cytoskeleton in platelets and play key roles in platelet aggregation, secretion, spreading and thrombus formation. Rho GTPase regulators, including GEFs and GAPs and downstream effectors, such as the WASPs, formins and PAKs, may also regulate platelet activation and function. In this review, we provide an overview of Rho GTPase signaling in platelet physiology. Previous studies of Rho GTPases and platelets have had a shared history, as platelets have served as an ideal, non-transformed cellular model to characterize Rho function. Likewise, recent studies of the cell biology of Rho GTPase family members have helped to build an understanding of the molecular regulation of platelet function and will continue to do so through the further characterization of Rho GTPases as well as Rho GAPs, GEFs, RhoGDIs and Rho effectors in actin reorganization and other Rho-driven cellular processes.