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1.
PLoS One ; 6(9): e24715, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931826

RESUMO

BACKGROUND: Genetic and environmental factors influence susceptibility to Crohn's disease (CD): NOD2 is the strongest individual genetic determinant and smoking the best-characterised environmental factor. Carriage of NOD2 mutations predispose to small-intestinal, stricturing CD, a phenotype also associated with smoking. We hypothesised that cigarette smoke extract (CSE) altered NOD2 expression and function in intestinal epithelial cells. METHODS AND FINDINGS: Intestinal epithelial cell-lines (SW480, HT29, HCT116) were stimulated with CSE and nicotine (to mimic smoking) ±TNFα (to mimic inflammation). NOD2 expression was measured by qRT-PCR and western blotting; NOD2-RIPK2 interactions by co-immunoprecipitation (CoIP); nuclear NFκB-p65 by ELISA; NFκB activity by luciferase reporter assays and chemokines (CCL20, IL8) in culture supernatants by ELISA. In SW480 and HT29 cells the TNFα-induced NOD2 expression at 4 hours was reduced by CSE (p = 0.0226), a response that was dose-dependent (p = 0.003) and time-dependent (p = 0.0004). Similar effects of CSE on NOD2 expression were seen in cultured ileal biopsies from healthy individuals. In SW480 cells CSE reduced TNFα-induced NFκB-p65 translocation at 15 minutes post-stimulation, upstream of NOD2. Levels of the NOD2-RIPK2 complex were no different at 8 hours post-stimulation with combinations of CSE, nicotine and TNFα, but at 18 hours it was increased in cells stimulated with TNFα+CSE but decreased with TNFα alone (p = 0.0330); CSE reduced TNFα-induced NFκB activity (p = 0.0014) at the same time-point. At 24 hours, basal CCL20 and IL8 (p<0.001 for both) and TNFα-induced CCL20 (p = 0.0330) production were decreased by CSE. CSE also reduced NOD2 expression, CCL20 and IL8 production seen with MDP-stimulation of SW480 cells pre-treated with combinations of TNFα and CSE. CONCLUSIONS: CSE delayed TNFα-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFκB activity and decreased chemokine production. These effects may be involved in the pathogenesis of small-intestinal CD and may have wider implications for the effects of smoking in NOD2-mediated responses.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Intestinos/citologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Fumar/efeitos adversos , Western Blotting , Células HCT116 , Células HT29 , Humanos , Proteína Adaptadora de Sinalização NOD2/genética , Ligação Proteica/efeitos dos fármacos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia
2.
Hum Mol Genet ; 19(24): 4930-8, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20858604

RESUMO

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.


Assuntos
Doença de Crohn/genética , Variações do Número de Cópias de DNA/genética , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , beta-Defensinas/genética , Estudos de Casos e Controles , Genoma Humano/genética , Humanos , Londres , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escócia , Homologia de Sequência do Ácido Nucleico
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