RESUMO
OBJECTIVE: To describe histological and metabolic characteristics of glenohumeral joint (GHJ) articular cartilage and compare to knee and ankle joints. DESIGN: Macroscopically healthy human humeral head, glenoid, knee, and ankle articular cartilage were obtained from tissue donors (N = 16, 9 males, 7 females; age 45-78 years), within 24 hours of death. Gross morphology of each joint was assessed using Collins grading. Cartilage explants were removed from the entire surface of each joint, cultured for 48 hours with or without interleukin-1ß and processed for histology with Safranin O, proteoglycan (PG) synthesis/content, and polymerase chain reaction for collagen II, aggrecan, and SOX9. Results were compared between uncultured and cultured controls and across all 3 joints. RESULTS: Structural differences were seen on histology between GHJ cartilage and knee and ankle cartilage of the same Collins grade, specifically, depletion of Safranin O staining in the extracellular matrix. Treatment of glenoid and humerus specimens with IL-1ß demonstrated a trend toward decreased PG synthesis in each explant but this decrease did not reach significance. There was no significant difference in PG synthesis between humerus, glenoid, knee, and ankle samples at baseline, day-0 control, 48-hour control, and 48 hours after treatment with 0.1 ng or 10 ng of IL-1ß. There were no significant increases in collagen II, SOX9, and aggrecan expression in glenoid and humeral head cartilage samples treated with IL-1ß compared to baseline controls. CONCLUSIONS: GHJ articular cartilage did not significantly differ from ankle or knee cartilage with regard to PG synthesis and gene expression. However, it did differ in its histological appearance in normal state.
Assuntos
Produtos Biológicos , Cartilagem Articular , Idoso , Agrecanas , Tornozelo , Articulação do Tornozelo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoglicanas , Ombro , Doadores de TecidosRESUMO
Mechanisms that govern the shift from joint homeostasis to osteoarthritis (OA) remain unknown. Here, we identify a pathway used for joint development and homeostasis, and its role in OA. Using a combination of transgenic, pharmacological, and surgical conditions in mouse and human tissues, we found that TGF-ß signaling promotes joint homeostasis through regulation of the IL-36 family. We identified IL-36 receptor antagonist (IL-36 in mice and IL-36RN in humans) as a potential disease-modifying OA drug. Specifically, OA development was associated with IL-36α up-regulation and IL-36Ra down-regulation in mice with tissue-specific postnatally induced ablation of Tgfbr2, mice treated with a TGF-ß signaling inhibitor, mice with posttraumatic OA, and aging mice with naturally occurring OA. In human cartilage, OA severity was associated with decreased TGFBR2 and IL-36RN, whereas IL-36α increased. Functionally, intra-articular treatment with IL-36Ra attenuated OA development in mice, and IL-36RN reduced MMP13 in human OA chondrocytes. These findings highlight the relevance of TGFBR2-IL-36 interplay in joint homeostasis and IL-36RN as a potential therapeutic agent for OA.
Assuntos
Interleucina-1/metabolismo , Terapia de Alvo Molecular , Osteoartrite/metabolismo , Osteoartrite/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Envelhecimento/patologia , Animais , Condrócitos/metabolismo , Condrócitos/patologia , Progressão da Doença , Regulação para Baixo/genética , Humanos , Injeções Intra-Articulares , Articulações/patologia , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Regulação para Cima/genéticaRESUMO
Objective To investigate the responses of refrigerated osteochondral allograft cartilage (OCA) and fresh cartilage (FC), including cell survival and metabolism, to surgical impaction and proinflammatory cytokines. Design Osteochondral plugs (8 mm diameter) were harvested from prolonged-refrigerated (14-28 days) and fresh (≤24 hours postmortem) human femoral hemicondyles and subjected to a 0.2 N s pneumatic impaction impulse. Cartilage explants were removed from subchondral bone and randomized to 1 of 6 treatment groups: (1) Unimpacted control (UIC), (2) Impacted control (IC), (3) Impacted + interleukin (IL)-1ß (0.1 ng/mL), (4) Impacted + IL-1ß (0.1 ng/mL) + IL-6, (5) Impacted + IL-1ß (10 ng/mL), and (6) Impacted + IL-1ß (10 ng/mL) + IL-6. Samples were measured for cell viability, histology, and proteoglycan (PG) content at days 0, 2, 7, and 14 of culture. Results In UIC, cell viability was indistinguishable between OCA and FC and remained constant. Impaction alone decreased cell viability by 30% ( P < 0.01) in the OCA superficial layer and by 26% ( P < 0.01) in the entire tissue, but did not affect viability in FC. Cytokine addition did not further influence cell viability. Impaction alone did not affect PG synthesis. Addition of cytokines to impacted tissue decreased PG synthesis by ~3-fold in both tissue types in comparison with corresponding impacted controls ( P < 0.01). Throughout 2-week culture, PG release remained stable in all FC groups, but peaked at day 14 in OCA cartilage subjected to cytokines. Conclusions Mechanical impaction, mimicking surgical insertion, has a more profound effect on cell viability in OCA than in FC. Addition of proinflammatory cytokines further decreases OCA tissue metabolism and integrity.
Assuntos
Aloenxertos/transplante , Cartilagem Articular/metabolismo , Sobrevivência Celular/fisiologia , Citocinas/metabolismo , Transplante Homólogo/métodos , Aloenxertos/metabolismo , Cartilagem Articular/patologia , Fêmur/citologia , Fêmur/transplante , Humanos , Interleucinas/metabolismo , Avaliação de Resultados em Cuidados de Saúde , Proteoglicanas/metabolismo , Líquido Sinovial/metabolismo , Transplante Homólogo/efeitos adversosRESUMO
INTRODUCTION: The purpose of this study was to correlate the level of anabolic and catabolic biomarkers in synovial fluid (SF) from patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA) and asymptomatic organ donors. METHODS: SF was collected from the knees of 45 OA, 22 RA patients and 20 asymptomatic organ donors. Eight biomarkers were selected and analyzed by using an enzyme-linked immunosorbent assay: interleukin (IL)-1, IL-6, IL-8 and IL-11; leukemia-inhibitory factor (LIF); cartilage oligomeric protein (COMP); osteocalcin; and osteogenic protein 1 (OP-1). Data are expressed as medians (interquartile ranges). The effects of sex and disease activity were assessed on the basis of the Western Ontario and McMaster Universities index score for patients with OA and on the basis of white blood cell count, erythrocyte sedimentation rate and C-reactive protein level for patients with RA. RESULTS: The mean ages (± SD) of the patients were as follows: 53 ± 9 years for patients with OA, 54 ± 11 years for patients with RA and 52 ± 7 years for asymptomatic organ donors. No effect of participants' sex was identified. In the SF of patients with RA, four of five cytokines were higher than those in the SF of patients with OA and those of asymptomatic organ donors. The most significant differences were found for IL-6 and IL-8, where IL-6 concentration in SF of patients with RA was almost threefold higher than that in patients with OA and fourfold higher than that in asymptomatic donor controls: 354.7 pg/ml (1,851.6) vs. 119.4 pg/ml (193.2) vs. 86.97 pg/ml (82.0) (P < 0.05 and P < 0.05, respectively). IL-8 concentrations were higher in SF of patients with RA than that in patients with OA as well as that in asymptomatic donor controls: 583.6 pg/ml (1,086.4) vs. 429 pg/ml (87.3) vs. 451 pg/ml (170.1) (P < 0.05 and P < 0.05, respectively). No differences were found for IL-11 in the SF of patients with RA and that of patients with OA, while a 1.4-fold difference was detected in the SF of patients with OA and that of asymptomatic donor controls: 296.2 pg/ml (257.2) vs. 211.6 pg/ml (40.8) (P < 0.05). IL-1 concentrations were the highest in the SF of RA patients (9.26 pg/ml (11.1)); in the SF of asymptomatic donors, it was significantly higher than that in patients with OA (9.083 pg/ml (1.6) vs. 7.76 pg/ml (2.6); P < 0.05). Conversely, asymptomatic donor control samples had the highest LIF concentrations: 228.5 pg/ml (131.6) vs. 128.4 pg/ml (222.7) in the SF of patients with RA vs. 107.5 pg/ml (136.9) in the SF of patients with OA (P < 0.05). OP-1 concentrations were twofold higher in the SF of patients with RA than those in patients with OA and threefold higher than those in asymptomatic donor control samples (167.1 ng/ml (194.8) vs. 81.79 ng/ml (116.0) vs. 54.49 ng/ml (29.3), respectively; P < 0.05). The differences in COMP and osteocalcin were indistinguishable between the groups, as were the differences between active and inactive OA and RA. CONCLUSIONS: Activation of selected biomarkers corresponds to the mechanisms that drive each disease. IL-11, LIF and OP-1 may be viewed as a cluster of biomarkers significant for OA; while profiling of IL-1, IL-6, IL-8, LIF and OP-1 may be more significant in RA. Larger, better-defined patient cohorts are necessary to develop a biomarker algorithm for prognostic use.
Assuntos
Artrite Reumatoide/diagnóstico , Biomarcadores/análise , Citocinas/análise , Osteoartrite/diagnóstico , Líquido Sinovial/química , Artrite Reumatoide/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Articulação do Joelho/metabolismo , Masculino , Metabolismo , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Líquido Sinovial/metabolismo , Doadores de TecidosRESUMO
OBJECTIVE: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. DESIGN: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. RESULTS: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. CONCLUSION: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Fraturas de Cartilagem/tratamento farmacológico , Poloxâmero/farmacologia , Tensoativos/farmacologia , Traumatismos do Tornozelo/tratamento farmacológico , Traumatismos do Tornozelo/metabolismo , Traumatismos do Tornozelo/patologia , Articulação do Tornozelo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Cartilagem Articular/lesões , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Inibidores Enzimáticos/farmacologia , Fraturas de Cartilagem/metabolismo , Fraturas de Cartilagem/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Imidazóis/farmacologia , Interleucina-6/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tálus/efeitos dos fármacos , Tálus/lesões , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1beta (IL-1beta)-induced cartilage degeneration to investigate the effect of IL-1beta on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes. METHODS: Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1beta on BMP receptors was studied by reverse transcription-polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1beta (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1beta and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1beta activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38). RESULTS: IL-1beta reduced the number of activin receptor-like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1-mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1beta offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38. CONCLUSION: These findings describe new mechanisms of the crosstalk between OP-1 and IL-1beta in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Adulto , Proteína Morfogenética Óssea 7/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/citologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Interleucina-1beta/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Smad1/metabolismo , Proteína Smad4/metabolismo , Proteína Smad6/metabolismoRESUMO
This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Condrócitos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Adulto , Proteína Morfogenética Óssea 7 , Cartilagem Articular , Células Cultivadas , Condrócitos/fisiologia , Condrócitos/transplante , Humanos , Transplante AutólogoRESUMO
We compared anabolic and anti-catabolic activities of selected bone morphogenetic proteins (BMP-2, -4, -6, and -7) and cartilage-derived morphogenetic proteins (CDMP-1 and -2) in human normal adult articular chondrocytes. Ankle chondrocytes were cultured in alginate beads in the presence of 10% serum and treated with either growth factors only (each at 100 ng/ml) or the combination of interleukin-1 (IL-1 beta) (0.1 ng/ml) and BMPs. Chondrocyte metabolism was assessed by proteoglycan (PG) synthesis, content, DNA content, and cell survival. The results showed that BMP-2, -4, and -7 were more potent in stimulating PGs than other growth factors tested. The highest levels of PG synthesis were detected at day 9 in the presence of BMP-7. With regard to anti-catabolic properties, the effect depended upon treatment scheme (simultaneous or sequential). Under simultaneous cultures, BMP-2, -4, and -6 failed to counteract IL-1 beta induced inhibition of PG synthesis, while the CDMPs restored this parameter to serum control levels. Only BMP-7 showed consistent and pronounced anti-catabolic activity in either culture treatment scheme. None of the factors induced cell death or chondrocyte proliferation. In conclusion, the growth factors tested showed different levels of effects on human chondrocytes in culture, but only BMP-7 displayed both strong anabolic and anti-catabolic properties.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alginatos , Proteínas Morfogenéticas Ósseas/genética , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Sinergismo Farmacológico , Ácido Glucurônico , Fator 5 de Diferenciação de Crescimento/farmacologia , Ácidos Hexurônicos , Humanos , Interleucina-1beta/farmacologia , Pessoa de Meia-Idade , Proteoglicanas/biossíntese , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Carbamazepine intoxication is associated with seizures, coma, arrhythmias and death. In acute intoxication, charcoal hemoperfusion is employed for removal of the drug. This can be associated with thrombocytopenia, coagulopathy, hypothermia and hypocalcemia. Alternatively, we used high-efficiency hemodialysis with a batch dialysis system (Genius), lowering not only serum levels of carbamazepine but removing a considerable amount of the drug as measured in the dialysate. This treatment regimen was compared to treatment by hemoperfusion. A 3.5-hour high-flux hemodialysis was as effective as a 2-hour hemoperfusion. We conclude that high-efficiency hemodialysis is a safe and effective alternative for treating life-threatening carbamazepine intoxication.
Assuntos
Antimaníacos/intoxicação , Carbamazepina/intoxicação , Hemoperfusão , Intoxicação/terapia , Diálise Renal , Adulto , Feminino , Humanos , Fatores de TempoRESUMO
BACKGROUND: Growth factors like IGF-I have been implicated in the pathogenesis of diabetic retinopathy. To investigate the role and bioavailability of IGF-I we measured not only total but also free serum levels of IGF-I in diabetic patients. METHODS: A total of 159 patients with and without diabetic retinopathy were investigated (60 diabetic patients without and 99 with clinically and angiographically diagnosed diabetic retinopathy). One hundred ten healthy volunteers served as controls. The data were analyzed by Student's t-test, analysis of variance and correlation; P < 0.05 was considered statistically significant. RESULTS: Diabetic patients with retinopathy showed significantly lower serum levels of free IGF-I than diabetics without retinopathy. Free IGF-I was negatively correlated with glycosylated hemoglobin 1c. IGFBP-3 levels were diminished in type 2 patients with diabetic retinopathy, while type 1 patients with diabetic retinopathy showed higher IGFBP-1 levels. CONCLUSION: Diabetic patients with retinopathy showed significantly reduced serum levels of free IGF-I. Low IGF-I was associated with poor metabolic control.
Assuntos
Retinopatia Diabética/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Disponibilidade Biológica , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Valores de ReferênciaRESUMO
Transcranial Doppler ultrasound was used to measure mean flow velocities in both middle cerebral arteries while 16 young subjects performed a visual task involving the processing of hierarchically structured stimuli. Specifically, large (global) letters composed of smaller (local) letters were presented, with the subjects' task being to attend either to the local or to the global level and press a button whenever a target on the designated level occurred. Each run was comprised of a 35-sec period of passive stimulation, followed by 65 sec of active task. A highly significant increase of blood flow was detected upon initiation of the active task, which was clearly present after ca. 4 sec. The flow velocity reached a maximum after 20 sec and remained stable for the remainder of the active condition. No hemispheric differences with respect to global or local conditions were observed.
Assuntos
Artérias Cerebrais/fisiologia , Cognição/fisiologia , Adulto , Velocidade do Fluxo Sanguíneo , Feminino , Humanos , Masculino , Percepção Visual/fisiologiaRESUMO
BACKGROUND: Wegener's granulomatosis is a focal necrotizing granulomatous vasculitis of unknown etiology. It manifests initially as localized inflammation, usually of the lungs, and preceeds to the state of generalisation which can be life-threatening due to renal failure. Ocular and orbital involvement occurs in 28-58% of patients with Wegener's granulomatosis. More often, the anterior segments of the eye are involved whereas inflammation of the posterior segments is quite uncommon. PATIENTS AND METHODS: We report a case of posterior scleritis as the initial manifestation of Wegener's granulomatosis in a 65-year-old woman. RESULTS: Posterior scleritis responded favourably to therapy with systemic steroids and cyclophosphamide. CONCLUSIONS: Wegener's granulomatosis can have a poor prognosis if not diagnosed and treated in time. Knowledge of posterior scleritis as a possible presenting symptom may therefore facilitate early diagnosis and treatment and prevent progression of the disease.
Assuntos
Granulomatose com Poliangiite/diagnóstico , Esclerite/diagnóstico , Idoso , Feminino , Angiofluoresceinografia , Humanos , Descolamento Retiniano/diagnóstico , Esclera/patologiaRESUMO
Blood flow velocities in both middle cerebral arteries (MCA) were measured using transcranial Doppler ultrasound (TCD) in healthy subjects engaged in a continuous static visual vigilance task. Stimuli comprised white vertical gratings on a black background with a size of 5 x 5" (non-targets) or 5 x 3.5" (targets). Button presses were required to the rare (8.5%) targets. Over the 30 min session a decrease in hit rate and an increase in reaction time were seen, indicating a decrease in vigilance. These performance changes were paralleled by a decrease in flow velocity in both MCAs. No hemispheric difference was seen. These data suggest a close coupling of performance and blood flow in vigilance tasks. Modulation of cholinergic activity during the vigilance task might be the common underlying mechanism.