RESUMEN
The objective of this study is to evaluate inter-reader entheses ultrasound (US) reliability and the influence of the type of image or degree of sonographer experience on US reliability in patients with spondyloarthritis (SpA). Eighteen Latin American ultrasonographers with different experience took part in an US reading exercise evaluating 60 entheseal images (50 % static images and 50 % videos) from healthy controls and SpA patients. The following sonographic lesions were assessed: structure, thickness, bone proliferation/tendon calcification, erosions, bursitis, and Doppler signal. Another group of three experts with significant experience in entheses US read all images too. Inter-reader reliability among participants and experts was calculated by the Cohen's kappa coefficient. Thresholds for kappa values were <0.2 poor, 0.21-0.4 fair, 0.41-0.6 moderate, 0.61-0.8 good, and 0.81-1 excellent. Furthermore, the results for the expert group were stratified based on the type of image. Kappa correlation coefficients among participants, showed variability depending on the type of lesion, being fair for structure and thickness, moderate for calcifications, erosions, and bursitis, and excellent for Doppler signal. Inter-reader reliability among experts was higher, being moderate for structure and thickness, good for calcifications and bursitis, and excellent for erosions and Doppler. Inter-reader reliability for assessing calcification and structure using static images was significantly higher than for videos. Overall inter-reader reliability for assessing entheses by US in SpA is moderate to excellent for most of the lesions. However, special training seems fundamental to achieve better inter-reader reliability. Moreover, the type of image influenced these results, where evaluation of entheses by videos was more difficult than by static images.
Asunto(s)
Entesopatía/diagnóstico por imagen , Espondiloartritis/diagnóstico por imagen , Competencia Clínica , Humanos , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , UltrasonografíaAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Ciclofosfamida/uso terapéutico , Meningitis/tratamiento farmacológico , Artritis Reumatoide/complicaciones , Artritis Reumatoide/patología , Encéfalo/patología , Femenino , Humanos , Infliximab , Imagen por Resonancia Magnética , Meningitis/complicaciones , Meningitis/patología , Persona de Mediana Edad , RecurrenciaRESUMEN
OBJECTIVES: Multiple independent juvenile oligoarthritis susceptibility loci have been identified within the major histocompatibility complex (MHC), including HLA-A, HLA-DRB1 and an as yet unlocalized effect in the centromeric class I region. The discoidin domain receptor 1 (DDR1) gene resides within this region and codes for a receptor tyrosine kinase that plays an important role in regulating cell attachment to collagen, chemotaxis, proliferation and matrix metalloproteinase (MMP) production. DDR1 expression in chondrocytes has not been investigated. The objectives of this study were to investigate expression of DDR1 in healthy chondrocytes and to identify linkage and association of this candidate gene with juvenile oligoarthritis. METHODS: A set of 135 simplex juvenile idiopathic arthritis families consisting of one affected child and healthy parent(s) and 199 healthy unrelated individuals were genotyped for six single nucleotide polymorphisms (SNPs) within the DDR1 gene using the primer extension SNaPshot trade mark method. Single-point and multipoint transmission disequilibrium tests were carried out with the ETDT and TDTPHASE packages. Allele frequency comparisons between cases and controls were carried out with the chi(2) test. DDR1 expression was investigated in normal articular cartilage by RT-PCR and immunofluorescence methods. RESULTS: No linkage and association with any of the six SNPs or their haplotypic combinations were observed in the families studied. No significant differences were observed in allele frequencies between patients and controls. DDR1 expression was found in normal articular cartilage by RT-PCR and by immunofluorescence. CONCLUSIONS: The DDR1 SNPs examined are not involved in susceptibility to juvenile oligoarthritis.
Asunto(s)
Artritis Juvenil/genética , Ligamiento Genético/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Mitogénicos/genética , Cartílago Articular/fisiopatología , Niño , Condrocitos/fisiología , Receptores con Dominio Discoidina , Técnica del Anticuerpo Fluorescente/métodos , Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Genotipo , Haplotipos/genética , Humanos , Desequilibrio de Ligamiento/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.
Asunto(s)
Densidad Ósea/genética , Anomalías del Ojo/genética , Ojo/embriología , Osteoblastos/metabolismo , Osteoporosis/genética , Receptores de LDL/fisiología , Factor de Crecimiento Transformador beta , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Adulto , Animales , Animales no Consanguíneos , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/farmacología , Células COS , Niño , Preescolar , Chlorocebus aethiops , Cromosomas Humanos Par 11/genética , Medios de Cultivo Condicionados/farmacología , ADN Complementario/genética , Proteínas Dishevelled , Femenino , Genes Recesivos , Heterocigoto , Humanos , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Masculino , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Proteínas/genética , Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes , Transducción de Señal , Cráneo/citología , Especificidad de la Especie , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Síndrome , Transfección , Proteínas Wnt , Proteína Wnt-5a , Proteína wnt2 , Proteína Wnt3 , Proteína Wnt4RESUMEN
The spectrum of heterotopic calcification or ossification is expanding because of the reports of several kindreds with calcium pyrophosphate deposition disease, apatite deposition disease, and others with less common syndromes associated with extracellular matrix calcification, such as fibrodysplasia ossificans progressiva and related syndromes. Genomic DNA studies in both humans and mice provide a shortcut to understanding the genetic basis of promotion and prevention of ECM calcification. Mutation in the COL2A1 gene has been identified in one family with spondyloepiphyseal dysplasia and calcium pyrophosphate and apatite crystalline deposits. In another kindred with precocious osteoarthritis without spondyloepiphyseal dysplasia, the phenotype was linked to markers of chromosome 8. In four other kindreds, the phenotypes were linked to an area of chromosome 5p. Two genes located in this region, which are expressed in articular cartilage, are being investigated as possible calcium pyrophosphate deposition disease genes. The results of linkage studies in three kindreds with articular/periarticular ADD with the COL2A1 gene were noninformative. Two different mouse mutations, the ank/ank and the ttw/ttw mice, are associated with intra-articular and ligament apatite deposits caused by a decrease in extracellular pyrophosphate concentrations, mimicking human arthritis caused by apatite deposition disease. Mutations in the matrix GLA protein, both in mice and in humans, are also associated with vascular and articular calcification. These mouse mutations provide cutting-edge information in the investigation of the mechanisms of apatite deposition in humans.
Asunto(s)
Calcinosis/genética , Compuestos de Calcio/metabolismo , Ligamiento Genético/fisiología , Artropatías/genética , Calcinosis/patología , Calcinosis/fisiopatología , Humanos , Artropatías/patología , Artropatías/fisiopatologíaRESUMEN
Early development of the vertebrate skeleton depends on genes that pattern the distribution and proliferation of cells from cranial neural crest, sclerotomes, and lateral plate mesoderm into mesenchymal condensations at sites of future skeletal elements. Within these condensations, cells differentiate to chondrocytes or osteoblasts and form cartilages and bones under the control of various transcription factors. In most of the skeleton, organogenesis results in cartilage models of future bones; in these models cartilage is replaced by bone by the process of endochondral ossification. Lastly, through a controlled process of bone growth and remodeling the final skeleton is shaped and molded. Significant and exciting insights into all aspects of vertebrate skeletal development have been obtained through molecular and genetic studies of animal models and humans with inherited disorders of skeletal morphogenesis, organogenesis, and growth.
Asunto(s)
Desarrollo Óseo/fisiología , Animales , Huesos/embriología , Huesos/metabolismo , Diferenciación Celular , Condrocitos/citología , Condrocitos/fisiología , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Humanos , Osteoblastos/citología , Osteoblastos/fisiologíaRESUMEN
OBJECTIVE: Tibial dyschondroplasia (TD) is an abnormality of growth plate cartilage characterized by the presence of non-vascularized, non-mineralized tissue. The objective of this study was to examine structural and functional alterations of the growth plate-specific type X collagen in TD cartilage. DESIGN: Collagen biosynthesis was examined in organ cultures and in cultured chondrocytes from normal growth plate and TD cartilage. Thermal stability of type X collagen extracted from normal and TD cartilage organ cultures to protease digestions by trypsin plus chymotrypsin or bacterial collagenase was determined. The expression of collagen genes was examined in cultured normal and TD chondrocytes. RESULTS: Synthesis of total collagen and of type X collagen was greater than threefold higher in organ cultures from the TD lesion compared with normal growth plate. The increase in type X collagen synthesis in the lesion was compensated by a reduction in the relative proportions of types II and XI collagens. The thermal denaturation and collagenase cleavage properties of purified types II and X collagens from TD cartilage were normal. The expression of type X collagen gene was threefold higher in cultured TD chondrocytes compared to chondrocytes from normal growth plate. Normal growth plate chondrocytes in primary cultures synthesized predominantly type X collagen (80% of total collagen). In contrast, TD chondrocytes synthesized mainly types I and II collagens and type X collagen represented only 22% of total collagen. TD cells initiated the synthesis of type I collagen within 5 days of primary culture, whereas normal chondrocytes did not synthesize this collagen during the same culture period. Although type X collagen synthesis was reduced in TD chondrocytes, the mRNA levels for type X collagen were substantially higher than in normal chondrocytes. CONCLUSION: Accumulation of type X collagen in TD cartilage results from its increased biosynthesis which is due largely to increased expression of the gene for this collagen, although, the chondrocyte culture studies suggest the possibility of postranscriptional defect in type X collagen synthesis or processing in TD lesion. Moreover, the TD chondrocytes in contrast with normal chondrocytes display evidence of prompt loss of their specific phenotype during short-term primary cultures.
Asunto(s)
Enfermedades de las Aves/metabolismo , Colágeno/biosíntesis , Placa de Crecimiento/metabolismo , Osteocondrodisplasias/veterinaria , Animales , Northern Blotting , Células Cultivadas , Pollos , Quimotripsina/metabolismo , Colágeno/química , Colágeno/genética , Técnicas de Cultivo , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Calor , Masculino , Osteocondrodisplasias/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/análisis , Tibia , Tripsina/metabolismoRESUMEN
In the present studies we found that incubation of human lung fibroblasts with transforming growth factor-beta 1 (TGF-beta 1) potentiated the interleukin-1 beta (IL-1 beta) and/or tumor necrosis factor-alpha (TNF-alpha)-stimulated production of prostaglandin E2 (PGE2). Analysis of fibroblast proteins showed the induction of cyclooxygenase-1 (Cox-1) by TGF-beta 1 and the induction of Cox-2 by IL-1 beta and TNF-alpha. The levels of transcripts for Cox-1 were minimally modified by IL-1 beta or TNF-alpha, however, they were increased by 12-fold by TGF-beta 1. Transcripts for Cox-2 were induced by IL-1 beta or TNF-alpha and their induction was potentiated by TGF-beta 1. TGF-beta 1 alone did not induce Cox-2 transcripts. In vitro transcription assays showed that IL-1 beta and TNF-alpha increased the transcription of the Cox-2 gene, whereas TGF-beta 1 had no effect. Addition of TGF-beta did not increase further the transcription of Cox-2 in IL-1 beta-treated cells, but increased the stability of the corresponding transcripts. The transcription rate of the Cox-1 gene was not increased by any of the cytokines studied. In summary, we demonstrate that the potentiation of PGE2 production by TGF-beta 1 in IL-1 beta and TNF-alpha-treated fibroblasts is the result of transcriptional stimulation of the Cox-2 gene by IL-1 beta and TNF-alpha and the stabilization of the resulting transcripts by TGF-beta 1.
Asunto(s)
Interleucina-1/farmacología , Isoenzimas/efectos de los fármacos , Pulmón/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/genética , Pulmón/citología , Pulmón/enzimología , Proteínas de la Membrana , Persona de Mediana Edad , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
OBJECTIVE: To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. METHODS: Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. RESULTS: The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. CONCLUSION: These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.
Asunto(s)
Condrocitos/fisiología , Proteínas de la Matriz Extracelular , Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Proteoglicanos/genética , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Agrecanos , Biglicano , Núcleo Celular/fisiología , Células Cultivadas , Cromatografía en Gel , Decorina , Combinación de Medicamentos , Feto , Humanos , Lectinas Tipo C , Persona de Mediana Edad , ARN Mensajero/metabolismo , Sulfatos/metabolismo , Transcripción Genética/fisiologíaRESUMEN
We examined in vitro collagen biosynthesis by organ cultures from human fetal epiphyseal growth plate cartilage. The biosynthetic products were characterized by NaCl fractional precipitation, limited proteolytic digestion, and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Organ cultures of human fetal epiphyseal growth plate cartilage synthesized large amounts of type X collagen in addition to type II, type IX, and type XI collagens. The individual polypeptide chains of human type X collagen migrated with an apparent M(r) of 45 kDa after proteolytic digestion with pepsin. The migration pattern of these molecules did not change when examined under reducing and nonreducing conditions, indicating that they did not contain intrahelical disfulfide bonds. Comparison of the rates at type X collagen biosynthesis at weeks 20 and 24 of human fetal development showed a marked increase of 24 weeks. Northern hybridization analysis of total RNA from freshly isolated epiphyseal growth plate chondrocytes with a cDNA corresponding to the carboxyl terminus of human type X collagen indicated that the developmental increase of type X collagen production is determined by pre-translational mechanisms.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno/metabolismo , Epífisis/metabolismo , Feto/metabolismo , Placa de Crecimiento/metabolismo , Secuencia de Bases , Northern Blotting , Cartílago Articular/embriología , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Epífisis/embriología , Feto/citología , Placa de Crecimiento/citología , Homeostasis , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , SolubilidadRESUMEN
OBJECTIVE: To establish long-term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage-specific phenotype. METHODS: Chondrocytes isolated from 20-24-week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly-(2-hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, II, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human-specific complementary DNAs. RESULTS: Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosynthetic studies demonstrated the maintenance of a cartilage-specific phenotype throughout the 180 days of culture, with the production of aggrecan and types II, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type II procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture. CONCLUSION: The human chondrocyte culture system described here allows the maintenance of a chondrocyte-specific phenotype for prolonged periods (up to 180 days). The long-term chondrocyte cultures formed nodular structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage-specific gene expression.
Asunto(s)
Cartílago Articular/fisiología , Feto/citología , Placa de Crecimiento/embriología , Northern Blotting , Cartílago Articular/citología , Recuento de Células , Células Cultivadas , Colágeno/biosíntesis , ADN/metabolismo , Feto/fisiología , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Histocitoquímica , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Microscopía de Contraste de Fase , Polietilenglicoles , Biosíntesis de Proteínas , Factores de TiempoRESUMEN
Maintenance of chick embryos in long-term culture without their calcareous egg-shell is a useful method for studying the relationship between calcium homeostasis and cell differentiation during skeletogenesis. Previously, we have shown that in shell-less (SL) embryos, calcium deficiency induces a cartilage-like phenotype in osteogenic tissues, such as calvaria (Jacenko and Tuan [1986] Dev. Biol. 115:215). In this investigation, we have studied the relationship between cartilage calcification and hypertrophy, and the expression of type X collagen, a specific product of hypertrophic chondrocytes. For this study, the cephalic (calcifying) and caudal (permanently cartilaginous) regions of sterna from day 18 and day 20 normal (NL) and SL embryos were metabolically labeled with [14C]-proline. Analysis of the biosynthetic products revealed significant differences in type X collagen expression in the cephalic region of sternal cartilage. In NL tissues, type X collagen production increased from 13.1% of total collagen at day 18 to 43.7% at day 20. In contrast, in SL embryos, type X collagen was not detectable until day 20, when it represented only 1% of total collagen. Comparison of the NL and SL embryos with respect to their serum calcium level and sternal calcium content and histology revealed a direct relationship between low systemic calcium and limited cartilage hypertrophy, undermineralization, and decreased type X collagen production in the sternal cephalic cartilage. Supplementation of CaCO3 to SL embryos increased their serum and sternal calcium, and restored cartilage hypertrophy, mineralization, and type X collagen synthesis in the cephalic portion of the sterna. These findings confirm that a critical relationship exists between calcium homeostasis, chondrocyte hypertrophy, mineralization, and type X collagen synthesis in the cephalic region of sternal cartilage. These results further demonstrate the importance of calcium in the morphogenetic events of endochondral ossification, in particular the transition from hyaline cartilage to hypertrophic cartilage, and eventually to bone.
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Calcio/deficiencia , Cartílago/embriología , Embrión de Pollo/anatomía & histología , Embrión de Pollo/metabolismo , Colágeno/metabolismo , Esternón/embriología , Animales , Matriz Ósea/metabolismo , Calcificación Fisiológica , Calcio/metabolismo , Cartílago/metabolismo , Cartílago/patología , Electroforesis , Hipertrofia , Técnicas de Cultivo de Órganos , Esternón/metabolismoRESUMEN
To examine the possibility that cytokines produced in inflamed joint tissues may contribute to the loss of articular cartilage by causing inhibition of synthesis of cartilage-specific matrix macromolecules, we studied the effects of interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha), alone and in combination, on the expression of the genes for types-II, -IX and -XI collagens in cultured human chondrocytes. Chondrocytes isolated from human fetal epiphyseal cartilage by sequential enzymic digestions were cultured in the presence of IFN gamma (30 pM), TNF alpha (15 pM) or a combination of suboptimal concentrations of both cytokines (1.5 pM IFN gamma plus 0.3 pM TNF alpha). IFN gamma caused a maximal decrease of 23.3-32.6% in the biosynthesis of collagen by chondrocytes. TNF alpha was a more potent inhibitor causing a 42.8-45.3% decrease at one-half the concentration of IFN gamma. A synergistic inhibitory effect of 58.2% was observed with the combination of 1.5 pM IFN gamma plus 0.3 pM TNF alpha. Electrophoretic analysis of the biosynthesized proteins showed a co-ordinate decrease in the production of the three cartilage-specific collagen types II, IX and XI. These effects were accompanied by parallel changes in the steady-state levels of their corresponding mRNAs. In vitro transcription assays showed that the collagen inhibitory effects of the cytokines occurred largely at the transcriptional level. Similar effects of the cytokines were observed on biosynthesis of types-II, -IX and -XI collagens and steady-state mRNA levels for type-II collagen by chondrocytes obtained from adult articular cartilage. These observations indicate that IFN gamma and TNF alpha can induce a synergistic inhibition of the synthesis of cartilage-specific collagens by fetal and adult human chondrocytes and suggest that these effects may contribute to the articular cartilage loss that occurs in inflammatory joint diseases.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno/genética , Interferón gamma/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Núcleo Celular/metabolismo , Células Cultivadas , Colágeno/biosíntesis , Expresión Génica , Humanos , Técnicas In Vitro , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The identification of mutations in cartilage-specific collagen genes in inherited forms of osteoarthritis (OA) and other heritable cartilage diseases has been hampered by the difficulty in obtaining sufficient tissue to isolate RNA and by the loss of the cartilage phenotype during in vitro chondrocyte culture. To overcome these limitations we employed RNA Polymerase Chain Reaction (PCR) to amplify cDNAs for the cartilage-specific collagens types II, IX and XI from mRNA obtained from small numbers of chondrocytes. We also amplified cDNAs for these collagens from "illegitimate transcripts" from Epstein-Barr virus (EBV)-transformed lymphocytes. Total RNA was obtained from freshly isolated human fetal and adult chondrocytes and from long-term (90 days) chondrocyte cultures. The RNA was reverse transcribed to cDNA and the cDNA amplified in the same tube with oligonucleotide primers specific for types II, IX and XI collagens. The amplified double-stranded products were cloned and sequenced. Successful amplification of the entire 4.4 kb of the type II procollagen cDNA was obtained from as little as 6 ng of RNA from freshly isolated fetal human chondrocytes. Seven hundred and eighty base pairs of the alpha 1 (IX) collagen cDNA and the entire published sequence of alpha 2 (XI) collagen cDNA, were also amplified from these cells. We were also able to amplify cDNAs for the three cartilage-specific collagens from "illegitimate transcripts" from EBV-transformed lymphocytes. Thus, these methods will allow the identification of mutations in cartilage-specific collagen genes in patients with inherited OA and other heritable cartilage diseases from small amounts of cartilage or chondrocyte RNA or from non-cartilaginous sources.
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Cartílago/metabolismo , Colágeno/metabolismo , ADN/genética , Linfocitos/metabolismo , Reacción en Cadena de la Polimerasa , Secuencia de Bases , Cartílago/citología , Línea Celular Transformada , Colágeno/clasificación , Colágeno/genética , Herpesvirus Humano 4 , Humanos , Datos de Secuencia MolecularRESUMEN
Prostaglandin G/H synthase (PGG/HS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins and thromboxanes. We screened a human lung fibroblast cDNA library with an ovine PGG/HS cDNA and isolated a 2.3-kilobase clone (HCO-T9). Sequence analysis of this clone showed that (a) it contained the entire translated region of PGG/HS and (b) it displayed an in-frame splicing of the last 111 base pairs encoded by exon 9, which resulted in the elimination of the N-glycosylation site at residue 409. Polymerase chain reaction amplification with specific oligonucleotides of reverse-transcribed mRNA from diverse human tissues and cultured cells yielded 400- and 300-base pair fragments that corresponded, respectively, to the intact and spliced transcripts. The expression of these two transcripts in cultured human lung fibroblasts was differentially regulated by serum, transforming growth factor beta 1, interleukin 1 beta, tumor necrosis factor alpha, and phorbol 12-myristate 13-acetate, as each of these conditions stimulated preferentially the expression of the unspliced transcripts. The elimination of one of the four N-glycosylation sites by the alternative splicing of exon 9 and the differential regulation of this process by relevant cytokines and growth factors may represent a mechanism for the regulation of PGG/HS enzymatic activity under physiological or pathological conditions.
Asunto(s)
Interleucina-1/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Empalme del ARN , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/genética , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Pulmón/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas/metabolismo , Mapeo Restrictivo , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
We have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of chondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, chondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 micrograms/ml) produced a dose-dependent decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.
Asunto(s)
Hormona del Crecimiento/farmacología , Placa de Crecimiento/citología , Factor I del Crecimiento Similar a la Insulina/farmacología , Animales , Ácido Ascórbico/farmacología , Recuento de Células , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Pollos , Matriz Extracelular/fisiología , Placa de Crecimiento/fisiología , Masculino , Microscopía Electrónica de RastreoRESUMEN
We explored the use of Testsimplet (TS) in synovial fluid (SF) analysis. TS is a glass slide coated with a dry mixture of methylene blue and cresyl violet, which in contact with one drop of SF provides a stained fresh preparation. We applied the TS to the study of 159 SFs of patients with different rheumatic diseases. In those SFs of patients with crystal-associated diseases, the crystal search was performed both on unstained preparations and with TS. TS was as good as the Wright's and Papanicolaou stain in characterizing SF cells, lupus erythematosus cells, and detection of occasional bacteria. TS allowed a better visualization of Reiter's cells, cartilage fragments, synovial villi, fat droplets, and fibrin. Crystals were identified in every TS of those patients with crystal-associated diseases. TS is a rapid and reproducible method of SF supravital staining. Crystals are well preserved for simultaneous examination with compensated polarized light.
Asunto(s)
Coloración y Etiquetado , Líquido Sinovial/citología , Benzoxazinas , Humanos , Azul de Metileno , Oxazinas , Juego de Reactivos para DiagnósticoRESUMEN
Primary cultures of chondrocytes isolated from the proliferative and hypertrophic zones of bovine growth plate cartilage were established, and their morphological and biosynthetic characteristics were compared. High-cell-density monolayer cultures maintained a predominantly chondrocytic morphology, preserved their zone-specific collagen phenotype, and produced an abundant proteoglycan matrix. In contrast, monolayer cultures plated at low cell densities contained many cells with fibroblastic morphology and initiated the synthesis of type I collagen within 72 h following seeding. On a per cell basis, the proliferative zone chondrocytes displayed greater biosynthetic activity than chondrocytes from the hypertrophic region. Characterization of newly synthesized collagens showed that type II collagen was the major biosynthetic product of both types of cells. In contrast, type X collagen was synthesized exclusively by hypertrophic chondrocytes and represented about 20% of the total collagen produced by these cells. The type X collagen molecules synthesized by the hypertrophic cells contained disulfide bonds within their triple-helical domains and assembled into a high-Mr aggregate. When polymerase chain reaction (PCR) was employed to amplify type X collagen sequences from cDNA obtained by reverse transcription of mRNA from cultures of both types of chondrocytes, only the hypertrophic chondrocyte mRNA yielded PCR amplification products indicative of the presence of type X collagen transcripts.