RESUMEN
Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Colágeno/farmacología , Dieta/veterinaria , Suplementos Dietéticos , Enfermedades de los Perros/dietoterapia , Osteoartritis/veterinaria , Sustancias Protectoras/farmacología , Animales , Organismos Acuáticos , Colágeno/química , Colágeno/uso terapéutico , Perros , Osteoartritis/dietoterapia , Sustancias Protectoras/química , Sustancias Protectoras/uso terapéuticoRESUMEN
OBJECTIVE: Previous work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA). METHODS: We developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium. RESULTS: Wild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA. CONCLUSIONS: Increased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.
Asunto(s)
Biomarcadores/sangre , Osteoartritis/patología , Sirtuina 1/sangre , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Senescencia Celular/fisiología , Condrocitos/metabolismo , Condrocitos/patología , Humanos , Ratones , Osteoartritis/sangreRESUMEN
In rheumatoid arthritis (RA), cartilage and bone matrix are degraded, and extracellular matrix (ECM) proteins, acting as cellular activators, are liberated. Similar to ECM proteins, matrix-bound chemokines, cytokines, and growth factors (GFs) influence functional properties of key cells in RA, especially synovial fibroblasts. The role of these molecules on attachment, migration, and proinflammatory and prodestructive activation of RASFs was analyzed. Adhesion/migration of RASFs were examined under GF-enriched (GF+) or -reduced (GF-) conditions with or without addition of matrix-associated GFs, TGF-ß, and platelet-derived GF to GF- or culture supernatants. Fibroblast adhesion and alterations in proinflammatory/prodestructive properties (e.g., IL-6/matrix metalloproteinase 3-release) in response to matrix-associated molecules were compared. Effects of GF+, GF-, and other ECM components on human RASF-mediated cartilage invasion were examined in the SCID mouse model. RASF adhesion under GF- conditions was significantly lower compared with GF+ conditions (6.8- versus 8.3-fold). This effect was specific for RA because control cells showed opposite effects (e.g., osteoarthritis synovial fibroblasts [SF]; GF- versus GF+: 10.7- versus 8-fold). Addition of TGF-ß to GF- increased RASF attachment (12.7-fold) compared with other matrices and components. RASF adhesion to GF+ matrix resulted in the strongest IL-6 and matrix metalloproteinase-3 release, and was even more pronounced compared with supplementation of single GFs. In vivo, GF- matrix decreased RASF-mediated cartilage invasion compared with GF+ matrix. ECM components and especially GFs when bound within ECM actively enhance RASF attraction and cartilage adhesion. This observation was specific for RASFs as a reverse behavior was observed for controls.
Asunto(s)
Artritis Reumatoide/inmunología , Adhesión Celular , Movimiento Celular , Fibroblastos/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Membrana Sinovial/citología , Animales , Ensayos de Migración Celular , Movimiento Celular/efectos de los fármacos , Matriz Extracelular , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interleucina-6/metabolismo , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Ratones SCID , Factor de Crecimiento Derivado de Plaquetas/farmacología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Extracellular RNA (exRNA) has been characterized as a molecular alarm signal upon cellular stress or tissue injury and to exert biological functions as a proinflammatory, prothrombotic, and vessel permeability-regulating factor. In this study, we investigated the contribution of exRNA and its antagonist RNase1 in a chronic inflammatory joint disease, rheumatoid arthritis (RA). Upon immunohistochemical inspection of RA, osteoarthritis (OA), and psoriatic arthritis synovium, exRNA was detectable only in the RA synovial lining layer, whereas extracellular DNA was detectable in various areas of synovial tissue. In vitro, exRNA (150-5000 nt) was released by RA synovial fibroblasts (RASF) under hypoxic conditions but not under normoxia or TNF-α treatment. RNase activity was increased in synovial fluid from RA and OA patients compared with psoriatic arthritis patients, whereas RNase activity of RASF and OASF cultures was not altered by hypoxia. Reduction of exRNA by RNase1 treatment decreased adhesion of RASF to cartilage, but it had no influence on their cell proliferation or adhesion to endothelial cells. In vivo, treatment with RNase1 reduced RASF invasion into coimplanted cartilage in the SCID mouse model of RA. We also analyzed the expression of neuropilins in synovial tissue and SF, as they may interact with vascular endothelial growth factor signaling and exRNA. The data support the concepts that the exRNA/RNase1 system participates in RA pathophysiology and that RASF are influenced by exRNA in a prodestructive manner.
Asunto(s)
Artritis Reumatoide/metabolismo , Adhesión Celular , Movimiento Celular , Espacio Extracelular/genética , Fibroblastos/metabolismo , Fibroblastos/patología , ARN/metabolismo , Membrana Sinovial/patología , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
OBJECTIVES: Adiponectin is an effector molecule in the pathophysiology of rheumatoid arthritis, e.g. by inducing cytokines and matrix degrading enzymes in synovial fibroblasts. There is growing evidence that adiponectin affects osteoblasts and osteoclasts although the contribution to the aberrant bone metabolism in rheumatoid arthritis is unclear. Therefore, the adiponectin effects on rheumatoid arthritis-derived osteoblasts and osteoclasts were evaluated. METHODS: Adiponectin and its receptors were examined in bone tissue. Primary human osteoblasts and osteoclasts were stimulated with adiponectin and analysed using realtime polymerase chain-reaction and immunoassays. Effects on matrix-production by osteoblasts and differentiation and resorptive activity of osteoclasts were examined. RESULTS: Immunohistochemistry of rheumatoid arthritis bone tissue showed adiponectin expression in key cells of bone remodelling. Adiponectin altered gene expression and cytokine release in osteoblasts and increased IL-8 secretion by osteoclasts. Adiponectin inhibited osterix and induced osteoprotegerin mRNA in osteoblasts. In osteoclasts, MMP-9 and tartrate resistant acid phosphatase expression was increased. Accordingly, mineralisation capacity of osteoblasts decreased whereas resorptive activity of osteoclasts increased. CONCLUSIONS: The results confirm the proinflammatory potential of adiponectin and support the idea that adiponectin influences rheumatoid arthritis bone remodelling through alterations in osteoblast and osteoclast.
Asunto(s)
Adiponectina/farmacología , Artritis Reumatoide/patología , Remodelación Ósea/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Adulto , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Resorción Ósea/metabolismo , Resorción Ósea/patología , Calcificación Fisiológica/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Receptores de Adiponectina/agonistas , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Factor de Transcripción Sp7 , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Matrix-induced autologous chondrocyte implantation (MACI) has demonstrated effectiveness in treating isolated cartilage defects of the knee but medium- and long-term evidence and information on the management of postoperative complications or partially successful cases are sparse. This study hypothesised that MACI is effective for up to 5 years and that patients with posttreatment problems may go on to obtain clinical benefit from other interventions. METHODS: A follow-on, prospective case series of patients recruited into a previous controlled, randomised, prospective study or newly enroled. Patients were followed up 6, 12, 24 and 60 months after surgery. Outcome measures were Tegner (activity levels) and Lysholm (pain, stability, gait, clinical symptoms) scores. Zone-specific subgroups were analysed 6, 12 and 24 months postoperatively. RESULTS: Sixty-five patients were treated with MACI. Median Tegner score improved from II to IV at 12 months; an improvement maintained to 60 months. Mean Lysholm score improved from 28.5 to 76.6 points (±19.8) at 24 months, settling back to 75.5 points after 5 years (p > 0.0001). No significant differences were identified in the zone-specific analysis. Posttreatment issues (N = 12/18.5 %) were resolved with microfracture, debridement, OATS or bone grafting. CONCLUSIONS: MACI is safe and effective in the majority of patients. Patients in whom treatment is only partially successful can go on to obtain clinical benefit from other cartilage repair options. This study adds to the clinical evidence on the MACI procedure, offers insight into likely treatment outcomes, and highlights MACI's usefulness as part of an armamentarium of surgical approaches to the treatment of isolated knee defects. LEVEL OF EVIDENCE: Prospective case control study with no control group, Level III.
Asunto(s)
Cartílago Articular/cirugía , Condrocitos/trasplante , Traumatismos de la Rodilla/cirugía , Articulación de la Rodilla/cirugía , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Ensayos Clínicos Controlados Aleatorios como Asunto , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.
Asunto(s)
Artritis Reumatoide/enzimología , Movimiento Celular , Citocinas/biosíntesis , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica , Mediadores de Inflamación/metabolismo , Nicotinamida Fosforribosiltransferasa/biosíntesis , Membrana Sinovial/enzimología , Artritis Reumatoide/patología , Quimiocina CCL2/biosíntesis , Femenino , Fibroblastos/patología , Perfilación de la Expresión Génica , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Masculino , Metaloproteinasa 3 de la Matriz/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Membrana Sinovial/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Differential diagnosis of muscle pain and weakness is extensive, including neurological, vertebral, arthrogenic, vascular, traumatic, immunological, endocrine, genetic and infectious aetiologies, as well as medication or toxin-related causes. Muscles are highly sensitive to a large number of drugs, especially with high doses. Although many drug classes can cause toxic myopathy, a significant number of cases are caused by lipid-lowering drugs, long-term use of corticosteroids, and, most often, alcohol misuse. Some drug interactions, e.g. those that are metabolised via the enzyme CYP3A4, can increase the serum levels of the drugs and drug-induced toxicity. A careful history of patient's drug and alcohol consumption is therefore vital. Clinical symptoms depend on the drug, dosage and patient's sensitivity. They can vary from asymptomatic increase in serum levels of creatine kinase, mild myalgia and cramps to muscle weakness, rhabdomyolysis, kidney failure and even death. The pathogenesis is often only partially known and multifactorial. Toxic myopathy is often reversible once the drug is discontinued, alternative drug therapy is started or a different dosage regimen is chosen. Complications such as acute kidney failure must be avoided, and analgesic therapy may be indicated.
Asunto(s)
Enfermedades Musculares , Rabdomiólisis , Humanos , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/terapia , Miotoxicidad , Rabdomiólisis/inducido químicamente , Etanol/efectos adversosRESUMEN
OBJECTIVES: Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage. METHODS: Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay. RESULTS: In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes. CONCLUSION: The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.
Asunto(s)
Adiponectina/metabolismo , Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Adiponectina/inmunología , Artritis Reumatoide/inmunología , Células Cultivadas , Quimiocinas/biosíntesis , Fibroblastos/inmunología , Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/metabolismoRESUMEN
Heterogeneous mixtures of collagen fragments can be used as nutrition supplement or as key ingredients for ointments with therapeutic relevance in wound healing. Some mixtures of collagen fragments are referred to as collagen hydrolysates owing to the production process with hydrolytic enzymes. Since the precise composition of collagen hydrolysates is generally unknown, it is of interest to analyze samples containing various collagen fragments with appropriate biophysical methods. Any product optimization without a profound knowledge concerning the size and the molecular weight distribution of its components is nearly impossible. It turned out that a combination of AFM methods with NMR techniques is exceptionally suited to examine the size range and the aggregation behavior of the collagen fragments in the hydrolysates of fish, jellyfish, chicken, porcine and bovine collagen. Supported by molecular modeling calculations, the AFM and NMR experiments provide a detailed knowledge about the composition of collagen hydrolysates and collagen ointments. Furthermore, the data allow a correlation between the size of the fragments and their potential bioactivity.
Asunto(s)
Colágeno/química , Colágeno/ultraestructura , Animales , Bovinos , Pollos , Hidrólisis , Microscopía de Fuerza Atómica/métodos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Escifozoos , PorcinosRESUMEN
Rheumatoid arthritis (RA) is associated with systemic osteoporosis, which leads to severe disability and low quality of life. Current therapies target osteoclasts to reduce bone degradation, but more treatment options would be required to promote bone protection by acting directly on osteoblasts (OB). Recently, the local production of dopamine in inflamed joints of RA has been observed. Thus, in this project, we aimed to determine the implication of the neurotransmitter dopamine in the bone formation process in RA. Dopamine receptors (DR) in the human bone tissue of RA or osteoarthritis (OA) patients were examined by immunohistochemistry. DR in isolated human osteoblasts (OB) was analyzed by flow cytometry, and dopamine content was evaluated by ELISA. Osteoclasts (OC) were differentiated from the PBMCs of healthy controls (HC) and RA patients. Isolated cells were treated with specific dopamine agonists. The effect of dopamine on mineralization was evaluated by Alizarin red staining. Cytokine release in supernatants was measured by ELISA. Osteoclastogenesis was evaluated with TRAP staining. OC markers were analyzed via real-time PCR and bone resorption via staining of resorption pits with toluidine blue. All DR were observed in bone tissue, especially in the bone remodeling area. Isolated OB maintained DR expression, which allowed their study in vitro. Isolated OB expressed tyrosine hydroxylase, the rate-limiting enzyme for dopamine production, and contained dopamine. The activation of D2-like DR significantly increased bone mineralization in RA osteoblasts and increased osteoclastogenesis but did not alter the expression of OC markers nor bone resorption. DR were found in the bone remodeling area of human bone tissue and dopamine can be produced by osteoblasts themselves, thus suggesting a local autocrine/paracrine pathway of dopamine in the bone. D2-like DRs are responsible for bone mineralization in osteoblasts from RA patients without an increase in bone resorption, thus suggesting the D2-like DR pathway as a possible future therapeutic target to counteract bone resorption in arthritis.
Asunto(s)
Artritis Reumatoide , Resorción Ósea , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Dopamina , Humanos , Osteoblastos/metabolismo , Osteogénesis , Calidad de Vida , Receptores DopaminérgicosRESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. METHODS: Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. RESULTS: Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. CONCLUSION: Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.
Asunto(s)
Adiponectina/fisiología , Artritis Reumatoide/inmunología , Artritis Reumatoide/fisiopatología , Quimiocinas/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/fisiopatología , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/metabolismo , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Humanos , Inflamación/fisiopatología , Articulación de la Rodilla/fisiopatología , Linfocitos/metabolismo , Osteoartritis de la Rodilla , Análisis por Matrices de ProteínasRESUMEN
The current limitations in evaluating synovial fluid (SF) components in health and disease and between species are due in part to the lack of data on normal SF, because of low availability of SF from healthy articular joints. Our study aimed to quantify species-dependent differences in phospholipid (PL) profiles of normal knee SF obtained from equine and human donors. Knee SF was obtained during autopsy by arthrocentesis from 15 and 13 joint-healthy human and equine donors, respectively. PL species extracted from SF were quantitated by mass spectrometry whereas ELISA determined apolipoprotein (Apo) B-100. Wilcoxon's rank sum test with adjustment of scores for tied values was applied followed by Holm´s method to account for multiple testing. Six lipid classes with 89 PL species were quantified, namely phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, phosphatidylethanolamine, plasmalogen, and ceramide. Importantly, equine SF contains about half of the PL content determined in human SF with some characteristic changes in PL composition. Nutritional habits, decreased apolipoprotein levels and altered enzymatic activities may have caused the observed different PL profiles. Our study provides comprehensive quantitative data on PL species levels in normal human and equine knee SF so that research in joint diseases and articular lubrication can be facilitated.
Asunto(s)
Apolipoproteínas B/análisis , Lípidos/análisis , Líquido Sinovial/química , Adulto , Animales , Ceramidas/análisis , Femenino , Caballos , Humanos , Ácido Hialurónico/análisis , Rodilla , Articulación de la Rodilla , Lipidómica/métodos , Masculino , Fosfolípidos/análisis , Especificidad de la Especie , Esfingomielinas/análisis , Líquido Sinovial/citología , Líquido Sinovial/metabolismo , Adulto JovenRESUMEN
Cartilage defects occur in approximately 12% of the population and can result in significant function impairment and reduction in quality of life. Evidence for the variety of surgical treatments available is inconclusive. This study aimed to compare the clinical outcomes of patients with symptomatic cartilage defects treated with matrix-induced autologous chondrocyte implantation (MACI or microfracture (MF). Included patients were >or= 18 and Asunto(s)
Artroplastia Subcondral
, Cartílago Articular/patología
, Cartílago Articular/cirugía
, Condrocitos/trasplante
, Traumatismos de la Rodilla/cirugía
, Adolescente
, Adulto
, Artroscopía
, Cartílago Articular/lesiones
, Femenino
, Estudios de Seguimiento
, Humanos
, Traumatismos de la Rodilla/patología
, Imagen por Resonancia Magnética
, Masculino
, Persona de Mediana Edad
, Estudios Prospectivos
, Trasplante Autólogo
, Resultado del Tratamiento
, Adulto Joven
RESUMEN
Obesity-in which free fatty acid (FFA) levels are chronically elevated-is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and ß-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways.
Asunto(s)
Artritis Reumatoide , Ácido Linoleico/farmacología , Osteoartritis , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ácido Palmítico/farmacología , Anciano , Animales , Células Cultivadas , Femenino , Humanos , Interleucina-8/metabolismo , Leucocitos Mononucleares/citología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Osteoblastos/metabolismo , Osteoclastos/metabolismoRESUMEN
Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.
RESUMEN
OBJECTIVES: Iliac bone grafting and matrix-guided autologous chondrocyte implantation (MACI) can be combined to treat large osteochondral defects of the knee. In this prospective study, we evaluated clinical and magnetic resonance imaging (MRI) findings after one and two years of this treatment method. METHODS: The study included 12 patients who completed a follow-up period of two years. Preoperative arthroscopic and MRI studies revealed grade 3 or 4 osteochondritis dissecans in all the cases. In the first operation, a deep debridement of the sclerotic subchondral bone was performed, followed by press-fit filling of the defect with cancellous bone. In the second operation, a double-layer MACI was fixed within the defect with fibrin glue. The clinical outcomes were evaluated using clinical scores. RESULTS: The clinical outcomes before and 24 months after surgery were as follows: the mean Meyers score increased from 10.2 to 18, Lysholm-Gillquist score increased from 56.6 to 100, and Tegner-Lysholm score increased from 1.8 to 4. These scores did not show notable changes after 12 months. On MRI images, subchondral edema within the bone graft disappeared until the sixth month. Within a year, MRI signal intensity of the cartilage repair tissue well approximated to that of the healthy surrounding cartilage. The thickness of the cartilage repair tissue increased from 1 mm to 1.8 mm within 6 to 12 months. CONCLUSION: Matrix-guided autologous chondrocyte implantation combined with bone grafting may be successfully used in remodeling the joint surface, without causing donor site morbidity within the knee joint. In addition, subchondral pathologic alterations may be effectively treated. Magnetic resonance imaging is a reliable technique to evaluate the repair process.
Asunto(s)
Trasplante Óseo/métodos , Enfermedades de los Cartílagos/cirugía , Cartílago Articular/trasplante , Trasplante de Células/métodos , Condrocitos/trasplante , Adulto , Enfermedades de los Cartílagos/patología , Cartílago Articular/patología , Condrocitos/patología , Estudios de Seguimiento , Humanos , Articulación de la Rodilla/patología , Articulación de la Rodilla/cirugía , Imagen por Resonancia Magnética/métodos , Estudios Prospectivos , Factores de Tiempo , Ingeniería de Tejidos/métodos , Trasplante Autólogo , Resultado del TratamientoRESUMEN
OBJECTIVE: Osteophyte formation in osteoarthritis (OA) is mediated by increased osteoblast activity, which is -in turn- regulated by the Wnt signaling pathway. Obesity is regarded a risk factor in OA, yet little is known about the interaction between adipose tissue-derived factors, the adipokines, and bone formation, although adipokines are associated with the pathogenesis of OA. Therefore, the effect of adipokines on bone and cartilage forming cells and osteophyte development was analyzed. METHODS: Human OA osteophytes were histologically characterized and adipokine expression was evaluated by immunohistochemistry. Osteoblasts and chondrocytes were isolated from OA tissue and stimulated with adiponectin, resistin, or visfatin. Cytokine and osteoblast/chondrocyte markers were quantified and activation of Wnt and p38 MAPK signaling was analyzed. RESULTS: Adiponectin, resistin, and visfatin were expressed in OA osteophytes by various articular cell types. Stimulation of OA osteoblasts with adiponectin and of OA chondrocytes with visfatin led to an increased release of proinflammatory mediators but not to osteoblast differentiation or activation. Additionally, visfatin increased matrix degrading factors in chondrocytes. Wnt signaling was not altered by adipokines, but adiponectin induced p38 MAPK signaling in osteoblasts. CONCLUSION: Adipokines are present in OA osteophytes, and adiponectin and visfatin increase the release of proinflammatory mediators by osteoblasts and chondrocytes. The effects of adiponectin were mediated by p38 MAPK but not Wnt signaling in osteoblasts. Therefore, the results support the idea that adipokines do not directly influence osteophyte development but the proinflammatory conditions in OA.
Asunto(s)
Adiponectina/metabolismo , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Osteoartritis/complicaciones , Osteoblastos/citología , Osteofito/metabolismo , Resistina/metabolismo , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Osteoartritis/metabolismo , Osteoblastos/metabolismo , Vía de Señalización WntRESUMEN
Alleviation of pain and inhibition of inflammation are the primary goals of pharmacotherapy of osteoarthritis (OA). These therapeutic goals can almost always be accomplished by the use of analgesics and nonsteroidal anti-inflammatory drugs (NSAID). One of the main problems of NSAIDs is their gastrointestinal toxicity, for which a prophylactic medication should be considered particularly amongst risk groups. Recent studies have shown that COX-2-selective and maybe also non-selective NSAIDs increase the cardiovascular risk so that their application is getting now drastically restricted. Pharmacological results published until now suggest that a clinically relevant minor analgesic and/or anti-inflammatory effect can be attained with the use of some of the SYmptomatic Slow Acting Drugs in OA (SYSADOAs). However, no clinical studies exist, which can positively confirm prevention, slowing down or reversal of any advanced joint cartilage destruction by any individual medication. Disease modifying therapy is still in its infancy; discovery and development of novel therapeutic targets and agents are an extremely difficult task, currently challenging many pharmaceutical companies and academic institutions.