RESUMO
OBJECTIVE AND DESIGN: Interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF-alpha on gene expression in the human chondrosarcoma cell line, SW1353. MATERIALS AND METHODS: SW1353 cells were stimulated singularly with IL-1alpha, TNF-alpha, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. RESULTS: Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-alpha. The expression pattern of proIL-1beta, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-alpha, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1beta is produced in the proform in IL-1alpha-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. CONCLUSIONS: IL-1 and TNF-alpha each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1alpha appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.
Assuntos
Condrossarcoma/genética , Condrossarcoma/metabolismo , Citocinas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Metaloproteinases da Matriz/genética , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Condrossarcoma/enzimologia , Análise por Conglomerados , Citocinas/biossíntese , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interleucina-1/biossíntese , Metaloproteinases da Matriz/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.
Assuntos
Apoptose , Caspases/imunologia , Cisteína Endopeptidases/imunologia , Fragmentos de Imunoglobulinas/imunologia , Animais , Caspase 7 , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular , Cricetinae , Expressão Gênica , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Líquido Intracelular/metabolismo , Células Jurkat , TransfecçãoRESUMO
From June 1984 to December 1990, 96 patients underwent "open" coronary endarterectomy and reconstruction. In 50 patients (group 1), a saphenous vein (SV) graft was used to reconstruct and bypass 54 coronary vessels. In 46 patients (group 2), 46 coronary vessels were reconstructed with an SV patch and then bypassed with the internal mammary artery (IMA): Seventy-four LAD coronary arteries (36 in group 1 and 38 in group 2) were treated with these procedures. Operative mortality was 8% in group 1 and 2.1% in group 2. Five patients (10%) in group 1 and 1 patient (2.1%) in group 2 developed perioperative myocardial infarction. The early postoperative patency of the reconstructed vessels was 84.6% in group 1 and 92.5% in group 2. Angiographic controls were performed between 30 and 36 months after operation in 18 patients (72%) of group 1 and in 16 patients (69%) of group 2 with patency rates of 66.7% and 81.5%, respectively. A further angiographic study performed between 54 and 60 months after operation of 9/22 patients of group 1 and 5/9 patients of group 2 did not show any additional closure of the endarterectomized vessels. Three- and 5-year survival analyzed by the Kaplan-Meier method was 79.6% and 69.7%, respectively, in group 1 and 86.8% for both the 3- and 5-year survival in group 2. After a mean follow-up of 51.0 and 35.5 months, 62.8% of the surviving patients of group 1 and 75.6% of group 2 were asymptomatic.(ABSTRACT TRUNCATED AT 250 WORDS)