RESUMEN
OBJECTIVES: Intervertebral disc (IVD) related diseases and age-related IVD degeneration are responsible for significant morbidity. Inflammatory mediators and pro-inflammatory cytokines, including interleukin (IL)-17, show elevated expression in degenerated disc tissue. IL-17 is reported to transduce signals across the cell membrane predominantly via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway, leading to transcriptional activation of target genes. METHODS: In this study, we investigated whether the JAK/STAT pathway plays a role in IL-17-mediated signaling in the nucleus pulposus (NP) cells of IVDs. Vascular endothelial growth factor (VEGF) and IL-17 were found to be highly expressed in human degenerated NP tissue. In isolated rat NP cells, IL-17-induced VEGF expression in a time- and dose-dependent manner. Rat NP cells were co-transfected with VEGF promoter plasmid along with constitutively active STAT1, STAT3 or JAK2 plasmid. VEGF promoter activity was found to be increased by STAT1, STAT3 and JAK2 in IL-17-treated cells. Transfection of cultured rat NP cells with STAT1 or STAT3 lentiviral short hairpin RNAs or treatment with the JAK2 inhibitor AG490 significantly reduced IL-17-stimulated VEGF expression. CONCLUSIONS: IL-17 upregulated VEGF expression in rat NP cells mediated by the JAK/STAT pathway, and elevated levels of IL-17 and VEGF are present in human degenerated NP tissue. These findings provide new insight into the pathology of IVD degeneration.
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Interleucina-17/biosíntesis , Degeneración del Disco Intervertebral/metabolismo , Quinasas Janus/biosíntesis , Núcleo Pulposo/metabolismo , Factores de Transcripción STAT/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Animales , Femenino , Humanos , Interleucina-17/análisis , Degeneración del Disco Intervertebral/patología , Quinasas Janus/análisis , Masculino , Núcleo Pulposo/química , Núcleo Pulposo/patología , Ratas , Factores de Transcripción STAT/análisis , Transducción de Señal , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
BACKGROUND: Research is still being conducted in order to determine the mechanisms responsible for the initiation of rheumatoid arthritis (RA) as well as for its persistence and progression. OBJECTIVES: The aim of this work was to establish the expression of the signal transducer and activator of transcription (STAT) transcription factors and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) transcription factor in peripheral blood leukocytes and synovial fluid cells. The correlations between the activation level of the transcription factors and the activity of the disease were also analyzed. MATERIAL AND METHODS: In total, the study included 34 RA patients and 19 healthy individuals as controls. The expression of NFκB, STAT1, STAT3, STAT4, STAT5 and STAT6 in peripheral blood leukocytes and synovial fluid cells was established. The immunocytochemistry method was used to determine the degree of activation of STAT and NF-κB transcription factors. For the location of the factors, primary polyclonal anti-STATs and monoclonal anti-NF-κB antibodies were used. RESULTS: The expression of STAT1, STAT3, STAT4, STAT5, STAT6 and NFκB was significantly higher in the group of RA patients than in the controls. No statistically significant differences were found between the expression of STATs in peripheral blood leukocytes and synovial fluid cells. CONCLUSIONS: In comparison with the control group, the expression of the STAT and NFκB transcription factors in RA patients was higher, which may be helpful in better understanding the etiopathogenesis of the disease in the future, and may potentially have important therapeutic implications.
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Artritis Reumatoide/metabolismo , Quinasas Janus/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción STAT/metabolismo , Adulto , Anciano , Femenino , Humanos , Quinasas Janus/análisis , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , Factores de Transcripción STAT/análisis , Adulto JovenRESUMEN
Interleukin-6 (IL-6) is one of the most important cytokines which has been shown to play a critical role in the pathogenesis of cholesteatoma. In this study, we aimed to investigate the expression of interleukin-6 (IL-6) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in middle ear cholesteatoma epithelium in an effort to determine the role of IL-6/JAK/STAT3 signaling pathway in the pathogenesis of cholesteatoma. Immunohistochemistry was used to examine the expression of IL-6 and p-STAT3 in 25 human middle ear cholesteatoma samples and 15 normal external auditory canal (EAC) epithelium specimens. We also analyzed the relation of IL-6 and p-STAT3 expression levels to the degree of bone destruction in cholesteatoma. We found that the expression of IL-6 and p-STAT3 were significantly higher in cholesteatoma epithelium than in normal EAC epithelium (p<0.05). In cholesteatoma epithelium, a significant positive association was observed between IL-6 and p-STAT3 expression (p<0.05). However, no significant relationships were observed between the degree of bone destruction and the levels of IL-6 and p-STAT3 expression (p>0.05). To conclude, our results support the concept that IL-6/JAK/STAT3 signaling pathway is active and may play an important role in the mechanisms of epithelial hyper-proliferation responsible for cholesteatoma.
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Colesteatoma del Oído Medio/metabolismo , Oído Medio/química , Células Epiteliales/química , Interleucina-6/análisis , Quinasas Janus/análisis , Factor de Transcripción STAT3/análisis , Transducción de Señal , Adulto , Estudios de Casos y Controles , Proliferación Celular , Colesteatoma del Oído Medio/patología , Osículos del Oído/patología , Oído Medio/patología , Activación Enzimática , Células Epiteliales/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , FosforilaciónRESUMEN
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Quinasas Janus/análisis , Receptores de Interferón/análisis , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Interferón gamma/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos , Receptores de Interferón/metabolismo , Coloración y Etiquetado , Receptor de Interferón gamma , Proteína Fluorescente RojaRESUMEN
Fundamento y objetivo: La identificación de la mutación V617F del gen JAK2 en pacientes diagnosticados de neoplasias mieloproliferativas crónicas (NMPC) es fundamental en el cribado diagnóstico. Nuestro objetivo fue investigar la asociación entre la cuantificación de la mutación V617F (carga alélica JAK2V617F) y el fenotipo clínico al diagnóstico, y definir el papel de la carga alélica en la predicción de complicaciones. Pacientes y métodos: En el Servicio de Hematología del Hospital Universitario La Paz realizamos un estudio observacional retrospectivo (1987-2011) en 114 pacientes diagnosticados de NMPC y análisis de detección de la mutación V617F: 39 policitemias vera (PV), 71 trombocitemias esenciales y 6 mielofibrosis primarias. El porcentaje de alelos mutados fue evaluado en polimorfonucleares de sangre periférica mediante la técnica quantitative real time polymerase chain reaction (qRT-PCR, «reacción en cadena de la polimerasa cuantitativa en tiempo real»). En función de si la carga tumoral se encuentra entre 1-50% o es>50% los pacientes fueron diagnosticados de JAK2mut heterocigoto u homocigoto, respectivamente. Resultados: Se realizó el análisis cuantitativo por qRT-PCR en los 114 pacientes incluidos en el estudio, detectando la mutación V617F en 69 casos y siendo negativa para los 45 pacientes restantes. Encontramos que la presencia de mutación se asocia con la trombosis, y especialmente con la trombosis arterial. Además, y en la serie completa, la carga alélica homocigótica se asocia con diagnóstico de PV, edad avanzada, leucocitosis, mayor hematopoyesis y esplenomegalia. Conclusiones: La detección de la mutación V617F está asociada a una mayor incidencia de trombosis, leucocitosis y esplenomegalia. Su identificación nos permitiría una mejor estratificación pronóstica de los pacientes diagnosticados de NMPC (AU)
Background and objectives: Our study has investigated the presence of the mutation V617F in the JAK2 gene in patients diagnosed with chronic myeloproliferative neoplasms (MPNs). Furthermore, we determined if JAK2 (V617F) allelic burden associates with a specific clinical phenotype and if its quantification can be used as a marker to predict outcome and complications in patients with MPNs.Patients and methods: A retrospective observational study was conducted from 1987 to 2011 in the Haematology Department of La Paz University Hospital. The allelic burden was measured in 114 patients diagnosed with MPNs: 39 polycythemia vera (PV) patients, 71 essential thrombocythaemia patients and 6 primary myelofibrosis patients. The quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to determinate the percentage of mutated alleles in peripheral blood neutrophils. Patients were divided in 2 groups: heterozygous if the result was≤50% of the tested cells, and homozygous if it was positive in>50% of the cells. Results: Sixty-nine patients were positive for the JAK2 mutation and 45 were negative. Among those positive, the mutation was associated with arterial thrombosis. In addition, we demonstrate in the homozygous group that the V617F mutation is associated to PV, advanced age, leukocytosis, marked haematopoiesis and splenomegaly. Conclusions: The presence of V617F mutation is associated with a higher incidence of thrombosis, leukocytosis and splenomegaly. The identification of mutation on the JAK2 gene could help in a better definition of evolution and prognostic stratification of the myeloproliferative disorders (AU)
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Humanos , Trastornos Mieloproliferativos/genética , Neoplasias Hematológicas/genética , Cromosoma Filadelfia , Quinasas Janus/análisis , Mutación , Estudios Retrospectivos , Leucocitosis/genética , Esplenomegalia/etiologíaRESUMEN
No disponible
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Humanos , Cromosoma Filadelfia , Quinasas Janus/análisis , Trastornos Mieloproliferativos/genética , Neoplasias Hematológicas/genética , MutaciónRESUMEN
Aberrant activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway may predispose to leukemia due to deregulation of proliferation, differentiation or apoptosis. This study was conducted to investigate whether any association exists between genetic polymorphisms in the JAK2, STAT3 and STAT5 genes and individual susceptibility to leukemia. A case-control study was carried out using a Chinese sample set with 344 cases of leukemia and 346 controls matched by age and ethnicity. Genomic DNA was assayed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) on 13 single nucleotide polymorphisms (SNPs). Genotype analyses showed that two SNPs, namely rs17886724 and rs2293157 located in STAT3 and STAT5, respectively, were significantly associated with leukemia (p < 0.05 for all). Interaction analyses of SNPs (rs17886724|rs2293157; rs11079041| rs2293157) showed that there were inferior associations in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) compared to the control group (0.1 > p > 0.05). Linkage disequilibrium existed between rs11079041 and rs2293157 in both leukemia and control groups (r(2) = 0.7). The haplotypes displayed significant association between rs11079041 and rs2293157 in both leukemia and control groups (p < 0.05). The accuracy rate of the support vector machine (SVM) classification model in making a prediction of leukemia was 97%. The results indicated that STAT3 and STAT5 gene SNPs may be prognostic of leukemia.
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Quinasas Janus/genética , Leucemia/diagnóstico , Leucemia/genética , Factores de Transcripción STAT/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico/genética , Estudios de Casos y Controles , Análisis Mutacional de ADN/métodos , Femenino , Predisposición Genética a la Enfermedad , Humanos , Janus Quinasa 2/análisis , Janus Quinasa 2/genética , Quinasas Janus/análisis , Leucemia/etnología , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Factores de Transcripción STAT/análisis , Factor de Transcripción STAT3/análisis , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT5/análisis , Factor de Transcripción STAT5/genética , Adulto JovenRESUMEN
OBJECTIVE: Evaluate expression of inducible negative regulators of JAK/STAT pathway and their target proteins during the course of ligature-induced experimental periodontal disease in rats. DESIGN: Rats were sacrificed 07, 15 and 30 days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western blot. RESULTS: Ligature-induced model presented significant progressive bone loss from 7 to 30 days. Inflammation was evident and similar for 07 and 15 days; however, a decrease on severity at the end of the experimental period was observed. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control group at 15 and 30days. The SOCS1 and SOCS3 protein expression and activation of STAT1 and STAT3 were increased in earlier periods in the ligature model. CONCLUSION: This study suggests that SOCS1 and SOCS3 were directly correlated with regulatory mechanism of the inflammatory process responsible for the periodontal disease destruction.
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Periodontitis/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/análisis , Pérdida de Hueso Alveolar/metabolismo , Pérdida de Hueso Alveolar/patología , Animales , Colágeno/análisis , Tejido Conectivo/patología , Modelos Animales de Enfermedad , Fibroblastos/patología , Encía/metabolismo , Encía/patología , Procesamiento de Imagen Asistido por Computador/métodos , Interleucina-10/análisis , Interleucina-6/análisis , Quinasas Janus/análisis , Masculino , Osteoprotegerina/análisis , Periodontitis/patología , Ligando RANK/análisis , Ratas , Ratas Wistar , Factores de Transcripción STAT/análisis , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisisRESUMEN
INTRODUCTION: Periapical inflammation is initiated by insult to the dental pulp and mediated by inflammatory cytokines in the periodontal tissue. On the other hand, the destruction of tissue can be prevented by the suppression of pro-inflammatory cytokine activity. The balance between these cytokines and their counterregulatory molecules has been suggested to regulate tissue destruction. Suppressors of cytokine signaling (SOCS) proteins are known to suppress inflammatory cytokine signaling via the classic negative feedback loop. However, the mechanism by which they are induced by inflammatory cytokines and regulated during the development of periodontal disease remains to be clarified. We investigated the effects of inflammatory cytokines on SOCS protein expression and their signaling pathways in human periodontal ligament (PDL) cells. METHODS: We examined the effect of inflammatory cytokines on SOCSs expression and its signaling pathway in human PDL cells using reverse transcription- and real-time polymerase chain reaction, Western blot methods. Furthermore, we also examined whether these cytokines-induced SOCS-3 suppress chemokines secretion using ELISA methods. RESULTS: We found that inflammatory cytokines interleukin (IL)-1beta and IL-6 induced expression of SOCS-3 but not that of SOCS-2 in human PDL cells. IL-1beta and IL-6 simultaneously induced IL-8 and monocyte chemoattractant protein-1 secretion in PDL cells, whereas SOCS-3 overexpression suppressed secretion of these chemokines through inhibition of phosphorylation in downstream signaling. CONCLUSION: The results suggest that pro-inflammatory cytokines induced SOCS-3 expression. The SOCS-3 induction suggests playing an important role in negative feedback, suppressing serious destruction of periodontal tissue in apical periodontitis through a chemokine-dependent mechanism.
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Citocinas/inmunología , Ligamento Periodontal/inmunología , Transducción de Señal/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Western Blotting , Células Cultivadas , Quimiocina CCL2/análisis , Quimiocina CCL2/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Interleucina-8/análisis , Interleucina-8/inmunología , Quinasas Janus/análisis , Quinasas Janus/inmunología , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteínas Quinasas Activadas por Mitógenos/inmunología , Ligamento Periodontal/citología , Fosforilación , ARN Mensajero/análisis , Receptores de Interleucina-6/análisis , Receptores de Interleucina-6/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción STAT/análisis , Factores de Transcripción STAT/inmunología , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/análisis , Factores de Tiempo , Regulación hacia Arriba/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
c-Met is responsible for cell motility and tumour spreading. c-Met expression and signal transducers reflecting c-Met functionality were investigated in breast carcinomas, in correlation with patient outcome and tumour vasculature. Tissue microarrays of 930 breast carcinomas were constructed, categorised according to patients' follow-up (4- to 10-year follow-up; median, 6.5 years). Standardised immunocytochemical procedures were performed using anti-c-Met, -PI3K, -FAK, -JAK, and -CD146, -FYN and an automated autostainer (Ventana). High-throughput densitometry measuring the extent of immunoprecipitates was assessed by image analysis (SAMBA). c-Met overexpression correlated with poor survival along with PI3K and FAK reflecting c-Met functionality and CD146 and FYN expression in endothelial cells. Automated quantification of immunocytochemical precipitates using image analysis was shown to provide an objective means of measuring cellular proteins that are potentially relevant for current practice in pathological diagnosis and for specific therapy combining inhibitors of both c-Met and downstream transducer pathways, and of tumour angiogenesis.