RESUMO
OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).
Assuntos
Cartilagem Articular/fisiologia , Etiquetas de Sequências Expressas , Biblioteca Gênica , Dados de Sequência Molecular , Osteoartrite do Joelho/genética , Adulto , Northern Blotting/métodos , Estudos de Casos e Controles , Expressão Gênica , Humanos , Hibridização In Situ/métodosRESUMO
Traumatic injury to a joint is known to increase the risk for the development of secondary osteoarthritis, but it is unclear how this process occurs. The existence of such a discrete event that can lead to an increased risk of osteoarthritis has spurred interest in developing in vitro models of traumatic joint injury. The current authors review some of the recent insights gained from these model systems into the pathogenesis of osteoarthritis, including the evidence for an initial, irreversible insult to chondrocytes during mechanical injury, the occurrence of apoptotic chondrocyte death, and attempts to identify the effects of trauma on chondrocyte metabolic response. Results also are presented from the authors' ongoing studies of the degradative pathways initiated by traumatic mechanical loads, the mechanism by which chondrocytes are affected during compression, and possible contributions of the joint capsule to posttraumatic cartilage degradation.
Assuntos
Cartilagem Articular/lesões , Doença Aguda , Fenômenos Biomecânicos , Cartilagem Articular/fisiopatologia , Técnicas de Cultura , Expressão Gênica , Humanos , Metaloproteinase 3 da Matriz/biossíntese , Metaloproteinase 3 da Matriz/genéticaRESUMO
The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Arginina/farmacologia , Compostos Benzidrílicos/farmacologia , Complemento C3a/metabolismo , Proteínas Inativadoras do Complemento/farmacologia , Proteínas de Membrana , Receptores de Complemento/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacocinética , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacocinética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Compostos Benzidrílicos/metabolismo , Compostos Benzidrílicos/farmacocinética , Ligação Competitiva , Linhagem Celular , Proteínas Inativadoras do Complemento/metabolismo , Proteínas Inativadoras do Complemento/farmacocinética , Modelos Animais de Doenças , Edema/patologia , Edema/prevenção & controle , Cobaias , Membro Posterior , Humanos , Injeções Intraperitoneais , Leucocitose/imunologia , Leucocitose/patologia , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores de Complemento/metabolismo , Células Tumorais CultivadasRESUMO
We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).
Assuntos
Reabsorção Óssea/enzimologia , Interleucina-1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Bioensaio , Radioisótopos de Cálcio/análise , Radioisótopos de Cálcio/metabolismo , Bovinos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Técnicas de Cultura , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Interleucina-1/farmacologia , Interleucina-6/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Rádio (Anatomia)/citologia , Rádio (Anatomia)/embriologia , Rádio (Anatomia)/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Ulna/citologia , Ulna/embriologia , Ulna/enzimologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
OBJECTIVE: To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin avbeta3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally dosed either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. RESULTS: SB 273005 is a potent antagonist of the closely related integrins, avbeta3 (Ki = 1.2 nM) and alphavbeta5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. CONCLUSION: Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the alphavbeta3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.
Assuntos
Artrite Experimental/prevenção & controle , Piridinas/uso terapêutico , Receptores de Vitronectina/antagonistas & inibidores , Administração Oral , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Edema/prevenção & controle , Imageamento por Ressonância Magnética , Masculino , Ratos , Ratos Endogâmicos Lew , Receptores de Vitronectina/administração & dosagemRESUMO
Mitogen-activated protein kinases (MAPKs) are involved in many cellular processes. The stress-activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke-induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre- and post-stroke and intravenously post-stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28-41%, and neurological deficits by 25-35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke-induced expression of IL-1beta and TNFalpha (i.e., cytokines known to contribute to stroke-induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Imidazóis/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Isquemia Encefálica/metabolismo , Células Cultivadas , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacocinética , Interleucina-1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The stress-activated mitogen-activated protein kinase (MAPK) p38 has been linked to the production of inflammatory cytokines/mediators/inflammation and death/apoptosis following cell stress. In these studies, a second-generation p38 MAPK inhibitor, SB 239063 (IC(50) = 44 nM), was found to exhibit improved kinase selectivity and increased cellular (3-fold) and in vivo (3- to 10-fold) activity over first-generation inhibitors. Oral SB 239063 inhibited lipopolysaccharide-induced plasma tumor necrosis factor production (IC(50) = 2.6 mg/kg) and reduced adjuvant-induced arthritis (51% at 10 mg/kg) in rats. SB 239063 reduced infarct volume (48%) and neurological deficits (42%) when administered orally (15 mg/kg, b.i.d.) before moderate stroke. Intravenous SB 239063 exhibited a clearance of 34 ml/min/kg, a volume of distribution of 3 l/kg, and a plasma half-life of 75 min. An i.v. dosing regimen that provided effective plasma concentrations of 0.38, 0.75, or 1.5 microg/ml (i.e., begun 15 min poststroke and continuing over the initial 6-h p38 activation period) was used. Significant and dose-proportional brain penetration of SB 239063 was demonstrated during these infusion periods. In both moderate and severe stroke, intravenous SB 239063 produced a maximum reduction of infarct size by 41 and 27% and neurological deficits by 35 and 33%, respectively. No effects of the drug were observed on cerebral perfusion, hemodynamics, or body temperature. Direct neuroprotective effects from oxygen and glucose deprivation were also demonstrated in organotypic cultures of rat brain tissue. This robust in vitro and in vivo SB 239063-induced neuroprotection emphasizes the potential role of MAPK pathways in ischemic stroke and also suggests that p38 inhibition warrants further study, including protection in other models of nervous system injury and neurodegeneration.
Assuntos
Encéfalo/patologia , Inibidores Enzimáticos/uso terapêutico , Imidazóis/uso terapêutico , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/patologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Temperatura Corporal/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipocampo/patologia , Inflamação/patologia , Inflamação/prevenção & controle , Técnicas de Cultura de Órgãos , Piridinas/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos SHR , Proteínas Quinases p38 Ativadas por MitógenoAssuntos
Anti-Inflamatórios/farmacologia , Citocinas/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/uso terapêutico , Apoptose/fisiologia , Artrite/tratamento farmacológico , Artrite/enzimologia , Osso e Ossos/enzimologia , Cartilagem/enzimologia , Ensaios Clínicos Fase I como Assunto , Inibidores das Enzimas do Citocromo P-450 , Ativação Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Imidazóis/uso terapêutico , Pulmão/enzimologia , Pneumopatias/tratamento farmacológico , Pneumopatias/enzimologia , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Animais , Dados de Sequência Molecular , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/enzimologia , Neovascularização Patológica , Psoríase/enzimologia , Piridinas/uso terapêutico , Alinhamento de Sequência , Choque Séptico/tratamento farmacológico , Choque Séptico/enzimologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/enzimologia , Viroses/tratamento farmacológico , Viroses/enzimologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The p38 MAP kinase inhibitor, SB 242235, was evaluated for its effects on the metabolism of bovine and human cartilage and primary chondrocyte cultures. SB 242235 had no effect on proteoglycan synthesis (PG) in bovine articular cartilage explants (BAC), as measured by [(35)S]-sulfate incorporation into glycosaminoglycans (GAGs). In addition, the compound had no effect on IL-1 alpha-induced GAG release from these cultures. However, there was a potent, dose-dependent inhibition of nitric oxide (NO) release from IL-1 alpha-stimulated BAC with an IC(50)of approximately 0.6 microM, with similar effects observed in primary chondrocytes. The effect on BAC was time dependent, and mechanistically did not appear to be the result of inhibition of protein kinase C (PKC), protein kinase A (PKA) or MEK-1. The effect on NO release in bovine chondrocytes was at the level of inducible nitric oxide synthase (iNOS) gene expression, which was inhibited at similar concentrations as nitrite production. In primary human chondrocytes, IL-1 beta induction of p38 MAP kinase was inhibited by SB 242235 with an IC(50)of approximately 1 microM. Surprisingly, however, treatment of IL-beta-stimulated human cartilage or chondrocytes with SB 242235 did not inhibit either NO production or the induction of iNOS. On the other hand, the natural product hymenialdisine (HYM), a protein tyrosine kinase (PTK) inhibitor, inhibited NO production and iNOS in both species. In contrast to the differential control of iNOS, PGE(2)was inhibited by SB 242235 in both IL-1-stimulated bovine and human chondrocyte cultures. These studies indicate that there are species differences in the control of iNOS by p38 inhibitors and also that different pathways may control IL-1-induced proteoglycan breakdown and NO production.
Assuntos
Condrócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Interleucina-1/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Bovinos , Técnicas de Cultura de Células , Condrócitos/metabolismo , Técnicas de Cultura , Dinoprostona/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Proteoglicanas/biossíntese , RNA Mensageiro/genética , Especificidade da Espécie , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.
Assuntos
Apoptose , Cartilagem Articular/lesões , Cartilagem Articular/patologia , Condrócitos/patologia , Animais , Animais Recém-Nascidos , Cartilagem Articular/metabolismo , Bovinos , Colágeno/metabolismo , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Cinética , Estresse MecânicoRESUMO
OBJECTIVE: The purpose of this study was to evaluate the ossification state of the meniscus in the guinea-pig stifle joint using micro-computerized tomography. DESIGN: Hind limbs from six (N=12) and 24 (N=11) month-old male Hartley guinea-pigs were removed and the joints were imaged using high resolution micro-computerized tomography. The ossified volume of the medial and lateral menisci from both groups of animals was quantified. RESULTS: Ossification of both the medial and lateral menisci of the both the 6- and 24-month-old animals was observed. In both age groups, the ossified region of the medial meniscus was significantly larger than the lateral meniscus. In addition, there is a significant increase in ossified volume of the medial meniscus between 6 and 24 months of age. CONCLUSIONS: There is a significant amount of ossification of the menisci in the male Hartley guinea-pig, with the medial compartment showing more bone than the lateral. In addition, as the animals age, there is an increase in ossification within the medial compartment. Bone remodeling and cartilage degeneration is evident in the medial compartment within these animals as they age. It is possible that the increased ossification of the medial meniscus could alter the joint biomechanics and, in part, stimulate this medial compartment joint destruction.
Assuntos
Doenças das Cartilagens/complicações , Meniscos Tibiais , Ossificação Heterotópica/complicações , Osteoartrite/etiologia , Envelhecimento/patologia , Animais , Doenças das Cartilagens/diagnóstico por imagem , Doenças das Cartilagens/patologia , Cobaias , Membro Posterior , Masculino , Meniscos Tibiais/diagnóstico por imagem , Ossificação Heterotópica/diagnóstico por imagem , Ossificação Heterotópica/patologia , Tomografia Computadorizada por Raios XRESUMO
Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.
Assuntos
Apoptose , Caspases/fisiologia , Condrócitos/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 3 , Inibidores de Caspase , Linhagem Celular , Colágeno/genética , Humanos , Oligopeptídeos/farmacologia , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.
Assuntos
Anticorpos Monoclonais/toxicidade , Antígenos CD4/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Células CHO , Candidíase/imunologia , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Feminino , Citometria de Fluxo , Humanos , Hipersensibilidade Tardia/imunologia , Sistema Imunitário/crescimento & desenvolvimento , Hibridização in Situ Fluorescente , Teste de Cultura Mista de Linfócitos , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Testes para Micronúcleos , Infecções por Pneumocystis/imunologia , Reprodução/efeitos dos fármacosRESUMO
Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.
Assuntos
Apoptose , Inibidores de Caspase , Inibidores Enzimáticos/química , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Sítios de Ligação , Camptotecina/farmacologia , Caspase 3 , Caspase 7 , Condrócitos/efeitos dos fármacos , Colágeno/genética , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Isatina/análogos & derivados , Camundongos , Modelos Moleculares , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Osteoartrite/tratamento farmacológico , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
OBJECTIVE: To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS: SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION: Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.
Assuntos
Artrite Experimental/tratamento farmacológico , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Absorciometria de Fóton , Animais , Anti-Inflamatórios/farmacologia , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/enzimologia , Artrografia , Densidade Óssea , Extremidades , Humanos , Processamento de Imagem Assistida por Computador , Interleucina-6/sangue , Lipopolissacarídeos/farmacologia , Imageamento por Ressonância Magnética , Masculino , Ratos , Ratos Endogâmicos Lew , Tarso Animal , Tíbia , Tomografia Computadorizada por Raios X , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Idoxifene, a selective estrogen receptor modulator, was evaluated in male and female rats with adjuvant-induced arthritis (AA). AA was induced in Lewis rats with Mycobacterium butyricum in paraffin oil injected into the base of the tail, and the animals were treated with idoxifene prophylactically (days 0-21) or therapeutically (days 10-21). Efficacy was determined by measurements of paw inflammation, bone mineral content, and bone mineral density (BMD) with dual X-ray absorptiometry and by histological evaluation. Serum interleukin-6 levels were measured as a marker of the anti-inflammatory effects of the compound. Estrogen was included for comparison and was administered at 5 mg/kg, three times a week s.c. Prophylactic treatment of male AA rats with idoxifene at 10, 3, and 1 mg/kg and estrogen at 5 mg/kg significantly inhibited paw inflammation. There was improved joint integrity measured by BMD and reduced serum interleukin-6 levels in animals treated with 10 mg/kg/day idoxifene. Idoxifene and estrogen were as effective for AA in female Lewis rats as in male rats, significantly inhibiting paw inflammation and improving BMD. Histological evaluation of the tibiotarsal joints of female rats treated with 10 mg/kg showed protection of bone, cartilage, and soft tissue. Therapeutic treatment with either idoxifene or estrogen (starting on day 10 of disease) of male and female Lewis rats also was effective in reducing paw inflammation in these animals, although the effect was much less than that observed with the prophylactic dosing protocol.
Assuntos
Artrite Experimental/tratamento farmacológico , Moduladores de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Densidade Óssea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estrogênios/farmacologia , Feminino , Pé/patologia , Interleucina-6/metabolismo , Masculino , Ratos , Ratos Endogâmicos Lew , Tamoxifeno/farmacologiaRESUMO
The effects of hymenialdisine (SK&F 108752) were evaluated on interleukin-1 (IL-1)-induced proteoglycan (PG) degradation, PG synthesis, nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) gene expression in bovine articular cartilage (BAC) and/or cartilage-derived chondrocytes. Cartilage disks from 0- to 3-month-old calves were treated with IL-1alpha or retinoic acid. PG release was determined by measuring glycosaminoglycan release, and nitrite production was measured as a readout for NO. Inhibition of iNOS gene expression was measured by Northern blot analysis in IL-1alpha-stimulated, cartilage-derived chondrocytes. To measure PG synthesis, chondrocytes were established in alginate beads and treated with hymenialdisine, and then [(35)S]sulfate incorporation into PGs was determined. Hymenialdisine inhibited IL-1alpha-stimulated PG breakdown in BAC in a dose-related manner with an IC(50) of approximately 0.6 microM. Herbimycin, a protein tyrosine kinase inhibitor, also inhibited PG breakdown, whereas RO 32-0432, a protein kinase C inhibitor, had no effect. Both hymenialdisine and herbimycin also were able to inhibit retinoic acid-stimulated PG release. IL-1alpha-stimulated NO production in BAC was inhibited by hymenialdisine and herbimycin at similar concentrations. The effect on iNOS gene expression was determined by Northern blot analysis in chondrocytes grown in monolayer, and inhibition by hymenialdisine was observed with an IC(50) of approximately 0.8 microM. In chondrocytes cultured in alginate beads, IL-1alpha inhibited PG synthesis, whereas hymenialdisine stimulated synthesis at low concentrations (0.6 and 1.25 microM), and higher doses (2.5 microM) were not stimulatory. Compounds with this profile may have utility in the treatment of osteoarthritis.
Assuntos
Azepinas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Proteoglicanas/metabolismo , Pirróis/farmacologia , Animais , Benzoquinonas , Northern Blotting , Cartilagem Articular/citologia , Bovinos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Indicadores e Reagentes , Interleucina-1/farmacologia , Lactamas Macrocíclicas , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinonas/farmacologia , RNA/antagonistas & inibidores , Rifabutina/análogos & derivados , Sulfatos/metabolismo , Tretinoína/farmacologiaRESUMO
Azaspiranes are novel macrophage-targeting agents with activity in preclinical animal models of autoimmune disease and transplantation. The purpose of this work was to determine the effects of atiprimod (SK&F 106615), an azaspirane being developed for the treatment of rheumatoid arthritis, on rat pulmonary alveolar macrophage (AM) function and immunocompetance in Candida-infected mice. AM from rats treated with 20 mg/kg/day of atiprimod for 15 days demonstrated enhanced killing of Candida albicans ex vivo. Concentration-dependent increases in candidacidal activity were also observed as early as one hour after exposure in vitro in AM from untreated normal rats. Treatment of AM with atiprimod in vitro did not increase particulate-stimulated superoxide production or phagocytosis of Candida but decreased their ability to concentrate acridine orange, indicating an increase in lysosomal pH. Increased candidacidal activity was inhibited by superoxide dismutase and catalase, suggesting a role for reactive oxygen intermediates (ROI). Atiprimod also increased free radical-mediated killing of Candida in the presence of H2O2, iron and iodide in a cell-free system. These findings indicated that treatment with atiprimod increased the candidacidal activity of rat AM in a free radical-dependent manner. The data also suggested that atiprimod did not increase ROI production by AM, but rather increased the efficiency of radical-mediated killing. This increase may be caused by cyclization of atiprimod, facilitating electron transfer and peroxidation of lipid membranes. In vivo studies in Candida-infected CBA mice showed that atiprimod (10 mg/kg/day), did not compromise immune function in the infected mice and could be differentiated from prototypical immunosuppressive compounds used for treatment of autoimmune diseases.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antifúngicos/farmacologia , Candidíase/imunologia , Imunossupressores/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Compostos de Espiro/farmacologia , Laranja de Acridina , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Candidíase/tratamento farmacológico , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Corantes Fluorescentes , Lisossomos , Macrófagos Alveolares/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Ratos , Ratos Endogâmicos LewRESUMO
The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on mRNA expression and macromolecular synthesis of aggrecan and type-II collagen. Bovine cartilage explants were exposed to different magnitudes and durations of applied mechanical compression, and levels of aggrecan and type-IIa collagen mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase were measured and quantified by Northern blot analysis. Synthesis of aggrecan and type-II collagen protein was measured by radiolabel incorporation of [35S]sulfate and [3H]proline into macromolecules. The results showed a dose-dependent decrease in mRNA levels for aggrecan and type-II collagen, with increasing compression relative to physiological cut thickness applied for 24 hours. Radiolabel incorporation into glycosaminoglycans and collagen also decreased with increasing compression in a dose-related manner similar to the changes seen in mRNA expression. The modulation of aggrecan and type-II collagen mRNA and protein synthesis were dependent on the duration of the compression. Aggrecan and type-II collagen mRNA expression increased during the initial 0.5 hours of static compression; however, 4-24 hours after compression was applied total mRNA levels had significantly decreased. The synthesis of aggrecan and collagen protein decreased more rapidly than did mRNA levels after the application of a step compression. Together, these results suggest that mechanical compression rapidly alters chondrocyte aggrecan and type-II collagen gene expression on application of load. However, our results indicate that the observed decreases in biosynthesis may not be related solely to changes in mRNA expression. The mechanisms by which mechanical forces affect different segments of the biosynthetic pathways remain to be determined.
Assuntos
Condrócitos/metabolismo , Colágeno/genética , Proteínas da Matriz Extracelular , Regulação da Expressão Gênica , Proteoglicanas/genética , Agrecanas , Animais , Bovinos , Regulação para Baixo , Lectinas Tipo C , Prolina/metabolismo , Estresse Mecânico , Sulfatos/metabolismo , Fatores de TempoRESUMO
The ability of the second generation phosphodiesterase 4 inhibitor SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P <.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors.