RESUMO
OBJECTIVES: In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays. METHODS: The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3). RESULTS: For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2â¯%. Reported levels were between 4.9 ± 1.2 and 33.9 ± 1.6â¯pmol/L (group mean ± standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4â¯%. Reported concentrations were between 25.3 ± 18.1 and 45.7 ± 19.4â¯pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4â¯%, and reported concentrations were from 0.06 ± 0.00 to 1.00 ± 0.11â¯nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8â¯%. Concentrations were between 0.12 ± 0.11 and 0.85 ± 0.23â¯nmol/L. CONCLUSIONS: For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended.
Assuntos
Hormônios Esteroides Gonadais , Espectrometria de Massas em Tandem , Humanos , Criança , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Imunoensaio/métodos , Testosterona , EstradiolRESUMO
The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1ß in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1ß-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, complement components and IL-6. Extracellular matrix (ECM) molecules were released at different time points, and fragmentation of aggrecan and cartilage oligomeric matrix protein was observed at days 3 and 6, similar to early-stage OA in vivo. Degradation products of the collagenous network were observed at days 18 and 22, similar to late-stage OA. This model displays a longitudinal quantification of released molecules from the ECM of articular cartilage. Identification of dynamic changes of extracellular matrix molecules in the secretome of equine explants stimulated with IL-1ß over time may be useful for identifying components released at different time points during the spontaneous OA process.
Assuntos
Cartilagem Articular/metabolismo , Matriz Extracelular/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Osteoartrite/metabolismo , Animais , Cartilagem Articular/patologia , Matriz Extracelular/patologia , Cavalos , Inflamação/metabolismo , Inflamação/patologia , Osteoartrite/patologiaRESUMO
Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell-like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor-like domain 7 (EGFL7), the stem cell-indicating factor Stro-1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin-1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro-1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro-1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell-like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage.
Assuntos
Biomarcadores/metabolismo , Cartilagem Articular/crescimento & desenvolvimento , Condrócitos/fisiologia , Lâmina de Crescimento/citologia , Cavalos/crescimento & desenvolvimento , Animais , Cartilagem Articular/citologia , Diferenciação Celular , Condrócitos/citologia , HomeostaseRESUMO
BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.
HISTORIAL: Suero autólogo acondicionado (ACS) es usado para tartar osteoartritis en caballos, aunque sus efectos no han sido completamente investigados. OBJETIVOS: Investigar los efectos de suero equino y suero acondicionado en condrocitos estimulados con interleukina (IL)-1ß y explantes de cartílago con osteoartritis leve. DISEÑO DEL ESTUDIO: Estudio experimental in vitro. MÉTODOS: El efecto de tres preparaciones séricas diferentes (control no incubado (PS), suero incubado 24 h (PS24h), y suero incubado 24 h en frascos ACS (PCS)) combinados y obtenidos de caballos cojos fueron probados en dos modelos in vitro. Las concentraciones de IL-1ß y de receptor antagonista de IL-1 (IL-1Ra) fueron medidas en todos los sueros. En el modelo 1, los cultivos de pellets de condrocitos fueron estimulados con IL-1ß antes de ser tratados con las preparaciones séricas durante 2 y 48 h. Se realizaron análisis de micromatrices, reacciones de polimerasa en cadena y de matriz de metalopeptidasa-13. En el modelo 2, explantaciones de cartílago proveniente de caballos con osteoartritis estructural fueron tratados con PS o PCS en los días 0, 6 y 12, o dejados sin tartar, y evaluados al día 24 usando la escala de graduación OARSI para evaluación histológica de cartílago articular. RESULTADOS: La concentración de IL-1Ra en PS24h y PCS fue significativamente mayor que en PS. En el modelo 1, los genes relacionados a la inflamación y a la degradación de la matriz cartilaginosa estaban aumentados después de 48 h en todos los grupos tratados en comparación a los controles no tratados. Las moléculas de matriz cartilaginosa, agrecanos y colágenos estaban disminuidos en los pellets PS24h y PCS versus los controles no tratados. Los genes de señales de factores de crecimiento FGF7 estaban aumentados en todos los grupos tratados, BMP2 en PS24h y INHBA in PCS en comparación con los controles no tratados. En el modelo 2, la escala OARSI al día 24 no fue significativamente distinta entre los grupos de tratamientos. LIMITACIONES PRINCIPALES: Los resultados de modelos in vitro no pueden ser directamente aplicados a situaciones in vivo. CONCLUSIONES: El tratamiento in vitro con suero acondicionado no alivió las respuestas inducidas por IL-1ß en pellets de condrocitos o llevo a mejoramiento morfológico en explantes de cartílago con osteoartritis.
Assuntos
Cartilagem Articular , Doenças dos Cavalos , Osteoartrite , Cavalos , Animais , Condrócitos/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Osteoartrite/terapia , Osteoartrite/veterinária , Inflamação/metabolismo , Inflamação/veterinária , Células Cultivadas , Doenças dos Cavalos/metabolismoRESUMO
BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h], and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6, and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS- treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated- compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.
RESUMO
The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1ß, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1ß treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1ß-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1ß, whereas slight anabolic effects were induced by IL-6 and HMGB-1.
Assuntos
Cartilagem Articular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteína HMGB1/farmacologia , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Agrecanas/metabolismo , Animais , Cartilagem Articular/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Expressão Gênica/efeitos dos fármacos , Cavalos , Proteínas Matrilinas , Metaloproteinase 13 da Matriz/metabolismoRESUMO
BACKGROUND: The quality of control materials is crucial for evaluating external quality assessment (EQA) results. To detect method differences, the EQA material should behave the same as a patient sample, meaning the material must be commutable. Noncommutable materials may cause misinterpretations of EQA results. Here, we examined the commutability of EQA materials used in 3 Nordic EQA schemes for lipids. METHODS: The study was designed according to the procedures recommended for assessing commutability by the International Federation of Clinical Chemistry and Laboratory Medicine. Commutability was assessed based on the difference in bias between a control material (CM) and clinical samples (CS) consisting of human plasma using 2 different measurement procedures (MPs). Measurands: LDL-cholesterol (LDL-C), total cholesterol (TC), HDL-cholesterol (HDL-C), and triglycerides (TG). Four CMs (CM1-4) were assessed for commutability by using 40 CS and 3 MPs (Abbott Architect, Roche Cobas, and Siemens Atellica). RESULTS: Unmodified native CMs (CM1 and CM3), stored at -80 °C, were commutable for all included measurands, except for LDL-C that was indeterminate, when comparing MPs pairwise. Modified CM2 was noncommutable for HDL-C, LDL-C, non-HDL-C, and LDL-C calculations. Unmodified native CM4, stored at -20°C, was noncommutable for LDL-C. CONCLUSIONS: Unmodified serum samples stored at -80 °C were commutable for lipids on the evaluated MPs, and therefore suitable as CMs in EQA schemes. Moreover, the study demonstrated that minor modifications of samples may lead to noncommutability.
Assuntos
Lipoproteínas , Projetos de Pesquisa , Humanos , Controle de QualidadeRESUMO
OBJECTIVE: Chondrocytes are responsible for remodeling and maintaining the structural and functional integrity of the cartilage extracellular matrix. Because of the absence of a vascular supply, chondrocytes survive in a relatively hypoxic environment and thus have limited regenerative capacity during conditions of cellular stress associated with inflammation and matrix degradation, such as osteoarthritis (OA). Glucose is essential to sustain chondrocyte metabolism and is a precursor for key matrix components. In this study, we investigated the importance of glucose as a fuel source for matrix repair during inflammation as well as the effect of glucose on inflammatory mediators associated with osteoarthritis. DESIGN: To create an OA model, we used equine chondrocytes from 4 individual horses that were differentiated into cartilage pellets in vitro followed by interleukin-1ß (IL-1ß) stimulation for 72 hours. The cells were kept at either normoglycemic conditions (5 mM glucose) or supraphysiological glucose concentrations (25 mM glucose) during the stimulation with IL-1ß. RESULTS: We found that elevated glucose levels preserve glucose uptake, hyaluronan synthesis, and matrix integrity, as well as induce anti-inflammatory actions by maintaining low expression of Toll-like receptor-4 and low secretion of glutamate. CONCLUSIONS: Adequate supply of glucose to chondrocytes during conditions of inflammation and matrix degradation interrupts the detrimental inflammatory cycle and induces synthesis of hyaluronan, thereby promoting cartilage repair.
Assuntos
Condrócitos/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Ácido Hialurônico/biossíntese , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicólise/fisiologia , Cavalos , Hialuronan Sintases/biossíntese , Hialuronan Sintases/genética , Interleucina-1beta/imunologiaRESUMO
Osteoarthritis is an inflammatory and degenerative joint disease commonly affecting horses. To identify genes of relevance for cartilage pathology in osteoarthritis we studied the time-course effects of interleukin (IL)-1ß on equine articular cartilage. Articular cartilage explants from the distal third metacarpal bone were collected postmortem from three horses without evidence of joint disease. The explants were stimulated with IL-1ß for 27â¯days and global gene expression was measured by microarray. Gene expression was compared to that of unstimulated explants at days 3, 9, 15, 21 and 27. Release of inflammatory proteins was measured using Proximity Extension Assay. Stimulation with IL-1ß led to time-dependent changes in gene expression related to inflammation, the extracellular matrix (ECM), and phenotypic alterations. Gene expression and protein release of cytokines, chemokines, and matrix-degrading enzymes increased in the stimulated explants. Collagen type II was downregulated from day 15, whereas other ECM molecules were downregulated earlier. In contrast molecules involved in ECM signaling (perlecan, chondroitin sulfate proteoglycan 4, and syndecan 4) were upregulated. At the late time points, genes related to a chondrogenic phenotype were downregulated, and genes related to a hypertrophic phenotype were upregulated, suggesting a transition towards hypertrophy later in the culturing period. The data suggest that this in vitro model mimics time course events of in vivo inflammation in OA and it may be valuable as an in vitro tool to test treatments and to study disease mechanisms.
Assuntos
Cartilagem Articular/metabolismo , Regulação da Expressão Gênica , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Animais , Células Cultivadas , Condrócitos , Matriz Extracelular , Cavalos , OsteoartriteRESUMO
OBJECTIVE: To determine the effects of interleukin (IL)-6 and IL-1ß stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. SAMPLE: Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. PROCEDURES: Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1ß for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3ß (GSK-3ß), and ß-catenin proteins in macroscopically normal cartilage samples and OCFs. RESULTS: Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3ß and coiled-coil domain containing 88C increased after 1 hour and expression of ß-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. CONCLUSION AND CLINICAL RELEVANCE: Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator ß-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.
Assuntos
Condrócitos/efeitos dos fármacos , Fator 5 de Diferenciação de Crescimento/metabolismo , Cavalos/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
STUDY DESIGN: Descriptive experimental study performed in rabbits of 2 age groups. OBJECTIVE: To study and investigate presence of prechondrocytic cells and cell migration routes (MR) in the intervertebral disc (IVD) region to gain knowledge about the normal IVD regeneration pattern. SUMMARY OF BACKGROUND DATA: Disc degeneration is thought to play a major role in patients with chronic lumbar pain. Regeneration processes and cell migration within the IVD have been sparsely described. Therefore, it is of interest to increase knowledge of these processes in order to understand pathological conditions of the IVD. METHODS: At the beginning of the experiment, 5-bromo-2-deoxyuridine (BrdU) in vivo labeling was performed in 2 groups of rabbits, 3 and 9 months old (total 27 rabbits). BrdU is incorporated into DNA during mitosis, and then it is gradually diluted with each cell division until it finally disappears. Incorporation of BrdU was then visualized by immunohistochemistry (IHC) at different time points providing cell division pattern and presence of slow-cycling cells in the IVD region. IVD tissue was investigated by IHC for growth and differentiation factor-5 (GDF5), SOX9 (chondrogenic lineage markers), SNAIL homolog 1 (SNAI1), SNAIL homolog 2 (SLUG) (migration markers), and ß1-INTEGRIN (cellular adhesion marker). In addition, GDF5, SOX9, and BMPRIB expression were investigated on genetic level. RESULTS: BrdU cells were observed in early time points in the IVD niche, adjacent to the epiphyseal plate, at later time points mainly in outer region of the annulus fibrosus for both age groups of rabbits, indicating a gradual migration of cells. The presence of SLUG, SNAI1, GDF5, SOX9, and ß1-INTEGRIN was found in same regions. CONCLUSION: The results suggest a cellular MR from the IVD stem cell niche toward the annulus fibrosus and the inner parts of the IVD. These findings may be of importance for understanding IVD regenerative mechanisms and for future development of biological treatment strategies.