RESUMO
Synovial tissue from patients with rheumatoid arthritis produces lysis of gels of reconstituted collagen fibrils in culture and releases soluble collagenase when cultured in collagen-free medium. Collagen molecules in solution at neutral pH at 20 degrees and 27 degrees C are cleaved by the synovial enzyme into (3/4) and (1/4) length fragments. In this respect the action of synovial enzyme is similar to that of amphibian collagenase and distinct from that of bacterial collagenase. At 37 degrees C reconstituted collagen fibrils and native fibers are attacked by the enzyme and further degraded to polypeptides of low molecular weight. These polypeptides are produced only after denaturation of the larger fragments, which occurs at temperatures near 37 degrees C.
Assuntos
Artrite Reumatoide/enzimologia , Colagenase Microbiana/metabolismo , Membrana Sinovial/enzimologia , Isótopos de Carbono , Cromatografia em Gel , Colágeno , Técnicas de Cultura , Eletroforese , Glicina , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Peptídeos , Desnaturação Proteica , Soluções , ViscosidadeRESUMO
Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.
Assuntos
Artrite Reumatoide/enzimologia , Colagenase Microbiana/metabolismo , Membrana Sinovial/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Isótopos de Carbono , Cromatografia em Gel , Colágeno/metabolismo , Técnicas de Cultura , Eletroforese , Glicina/metabolismo , Cobaias , Histocitoquímica , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Peso Molecular , Proteínas/farmacologia , Temperatura , Tripsina/farmacologia , ViscosidadeRESUMO
No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.
Assuntos
Colagenase Microbiana/metabolismo , Proteoglicanas/farmacologia , Animais , Relação Dose-Resposta a Droga , Mucosa Gástrica/enzimologia , Granulócitos/enzimologia , Humanos , Cartilagens Laríngeas , Colagenase Microbiana/antagonistas & inibidores , Doenças Reumáticas/enzimologia , Pele/enzimologia , Suínos , Membrana Sinovial/enzimologiaRESUMO
1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.
Assuntos
Gengiva/enzimologia , Colagenase Microbiana/metabolismo , Animais , Proteínas Sanguíneas/fisiologia , Meios de Cultura , Cães , Cinética , Colagenase Microbiana/isolamento & purificação , Peso Molecular , Técnicas de Cultura de Órgãos , Fatores de Tempo , alfa-Macroglobulinas/fisiologiaRESUMO
This article describes a prospective, randomized, controlled trial of screening and treatment for psychiatric disorder in medical in-patients. The study has assessed whether increased recognition of psychiatric disorder among medical in-patients improves clinical outcome and reduces the costs of care, and whether routine involvement of a psychiatrist in the assessment and care of medical in-patients with probable psychiatric disorder is superior to the efforts of the physicians alone. A total of 218 medical in-patients who scored over the screening threshold for psychiatric disorder on the General Health Questionnaire were randomly allocated to one of two intervention groups or a control group. Six months later their mental health, subjective health status, quality of life, and costs of care was reassessed. Mental health and quality of life at 6 months were similar in the two intervention groups and the control group. Patients whose physicians were told the results of the screening test had lower costs for subsequent admissions, but this was probably due to differences between the groups in terms of employment status. Treatments recommended by psychiatrists broke down when patients were discharged home, leading to inadequate treatment of psychiatric disorders. We have not been able to show that routine screening for psychiatric disorder produces any benefit, either in better outcome for patients or reduced costs for the NHS. Further research should: consider examining a more homogeneous group in terms of costs of care; screen only for disorders likely to respond to a specific treatment; and ensure that treatment recommendations are carried out.
Assuntos
Custos de Cuidados de Saúde , Hospitalização/economia , Pacientes Internados/psicologia , Transtornos Mentais/diagnóstico , Transtornos Mentais/terapia , Psiquiatria , Encaminhamento e Consulta , Análise de Variância , Intervalos de Confiança , Análise Custo-Benefício , Feminino , Nível de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Estudos Prospectivos , Qualidade de Vida , Inquéritos e QuestionáriosAssuntos
Colagenase Microbiana/metabolismo , Pele/enzimologia , Aminoácidos/análise , Animais , Cartilagem , Células Cultivadas , Galinhas , Cromatografia em Gel , Cromatografia por Troca Iônica , Colágeno/análise , Eletroforese Descontínua , Temperatura Alta , Humanos , Radioisótopos do Iodo , Peso Molecular , Desnaturação Proteica , Fatores de Tempo , ViscosidadeAssuntos
Hipocalcemia/tratamento farmacológico , Hipocalcemia/etiologia , Glândulas Paratireoides , Hormônio Paratireóideo/farmacologia , Extratos de Tecidos/uso terapêutico , Adolescente , Adulto , Idoso , Cálcio/sangue , Feminino , Humanos , Hiperparatireoidismo/complicações , Hipoparatireoidismo/complicações , Falência Renal Crônica/complicações , Masculino , Pessoa de Meia-Idade , Fosfatos/análise , Raquitismo/complicaçõesAssuntos
Mastócitos/fisiologia , Melanoma/fisiopatologia , Receptores Histamínicos H2/fisiologia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/fisiopatologia , Melanoma/patologia , RatosAssuntos
Osteíte Deformante , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
This report attempts to summarize our present knowledge of rheumatoid synovial collagenase and its natural serum inhibitors, beta1-anticollagenase and alpha2-macroglobulin, in relation to cartilage collagen resorption in the rheumatoid joint. Immunolocalization of collagenase across the cartilage/pannus junction is described, and in the light of the finding of the specific, small molecular weight beta1-anticollagenase we propose a model of cartilage erosion based on the interaction between collagenase and its natural inhibitors.
Assuntos
Artrite Reumatoide/enzimologia , Cartilagem Articular/metabolismo , Colagenase Microbiana/metabolismo , Cartilagem Articular/enzimologia , Imunofluorescência , Colagenase Microbiana/antagonistas & inibidores , Modelos Biológicos , Proteoglicanas/farmacologia , Temperatura , alfa 1-Antitripsina/farmacologia , alfa-Macroglobulinas/farmacologiaRESUMO
It is known that both interleukin-1 alpha (IL-1 alpha) and 12-O-tetradecanoylphorbol 13-acetate (TPA) promote increases in intracellular levels of the glycolytic regulatory metabolite fructose 2,6-bisphosphate [Fru(2,6)P2] and in the production of prostaglandin E (PGE) by subcultured rheumatoid synovial cells (RSC) and human dermal fibroblasts in vitro. We report here that the protein kinase C inhibitor staurosporine enhanced the IL-1 alpha-induced increase in [Fru(2,6)P2] and PGE production by RSC, whereas in similar concentrations (3-30 nM) this inhibitor decreased the TPA-induced stimulation of these parameters. Staurosporine produced a similar enhancement of the response to IL-1 alpha by normal human dermal fibroblasts. The increased PGE production provoked by tumour necrosis factor alpha (TNF alpha) in RSC was also augmented by staurosporine, but, in contrast, the increases in cellular [Fru(2,6)P2] induced by transforming growth factor beta (TGF beta) and interferon-gamma (IFN-gamma) were diminished. Thus the protein kinase C inhibitor staurosporine discriminates not only between the effects produced by IL-1 alpha and TPA, but also between those of IL-1 alpha and two other cytokines (but not between IL-1 alpha and TNF alpha). These findings suggest that IL-1 alpha and probably TNF alpha act via an intracellular mechanism different from that mediating the action of TPA, TGF-beta and IFN-gamma, and provide evidence that staurosporine is capable of amplifying the IL-1 signal.
Assuntos
Alcaloides/farmacologia , Fatores Biológicos/farmacologia , Fibroblastos/metabolismo , Frutosedifosfatos/biossíntese , Hexosedifosfatos/biossíntese , Prostaglandinas E/biossíntese , Proteína Quinase C/antagonistas & inibidores , Células Cultivadas , Citocinas , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Pele/citologia , Estaurosporina , Estimulação Química , Líquido Sinovial/citologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Crescimento Transformadores/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The addition of either recombinant human interleukin 1 (IL1 alpha) or 12-O-tetradecanoyl phorbol-13-acetate (TPA) to cultured rheumatoid synovial cells (RSC) caused dose-related increases in PGE production and cellular fructose 2,6-bisphosphate (Fru-2,6-P2). IL1 consistently produced the greater increases in both parameters. A close association between increases in PGE production and Fru-2,6-P2 was demonstrated for both IL1- and TPA-stimulated cells. The combined addition of IL1 with TPA resulted in an additive increase in both parameters. When IL1 was added together with human recombinant interferon-gamma (IFN-gamma), the resulting Fru-2,6-P2 level was synergistically increased, whilst the combination of IFN-gamma and TPA produced only an additive increase. Thus despite their very similar effects on RSC in culture, the data suggests that IL1 and TPA do not act via an identical intracellular mechanism.
Assuntos
Frutosedifosfatos/biossíntese , Hexosedifosfatos/biossíntese , Interleucina-1/farmacologia , Prostaglandinas E/biossíntese , Doenças Reumáticas/metabolismo , Membrana Sinovial/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Humanos , Interferon gama/farmacologia , Proteínas Recombinantes/farmacologia , Membrana Sinovial/efeitos dos fármacosRESUMO
Interleukin-1 alpha (IL-1 alpha) produced alterations in human dermal fibroblast glucose metabolism in vitro of the type seen in severe sepsis in man. Glycolysis and glucose uptake were increased but the oxidation of glucose within the tricarboxylic acid (TCA) cycle was reduced. The combined addition of tumour necrosis factor alpha (TNF alpha) with interferon-gamma (IFN-gamma) similarly increased the dependency for cellular energy provision from an oxidative to the glycolytic state. These cytokine-induced changes in glucose metabolism were unaffected when prostaglandin production was inhibited with a cyclo-oxygenase inhibitor, but were significantly reduced by the steroid dexamethasone. Thus, the inflammatory cytokines IL-1 and TNF alpha reportedly detected in the circulation during severe sepsis may directly affect not only glucose uptake but also its subsequent metabolism within tissue fibroblasts.
Assuntos
Glucose/metabolismo , Glicólise/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Transporte Biológico/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Indometacina/farmacologia , Oxirredução , Proteínas Recombinantes/farmacologia , Choque Séptico/metabolismoRESUMO
The presence of circulating IgG, IgA and IgM antibodies to native cartilage collagens in some patients with rheumatoid arthritis (RA) suggests that an autoimmune response to cartilage collagens may be involved in the pathogenesis of RA. However, the relevance of such antibodies to the pathological process remains unclear, and it is likely that many humoral and cellular derived factors combined to trigger events leading to the chronicity of the rheumatoid lesion. Since histological and biochemical studies have suggested the involvement of mast cells in the rheumatoid joint, we have studied the frequency of IgE antibodies directed against the cartilage collagens type II, IX and XI in patients with active rheumatoid disease. Of the 91 patients' sera tested, 32 had significant levels of IgE anti-cartilage collagen antibodies when compared with non-arthritic controls. Total serum IgE levels did not correlate with the presence of IgE anti-collagen antibodies, nor were any patients positive for IgE antibodies to fibronectin, a widely distributed extracellular matrix component. These results are consistent with an allergic type I hypersensitivity reaction to cartilage antigens in RA involving mast cell and basophil degranulation.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Cartilagem/imunologia , Colágeno/imunologia , Imunoglobulina E/análise , Western Blotting , Eletroforese em Gel de Poliacrilamida , HumanosRESUMO
Immunolocalization studies of rheumatoid tissues employing specific synovial collagenase antibody have demonstrated immunoreactive enzyme at the cartilage/pannus junction. Collagenase was not detected in chondrocytes or the cartilage matrix remote from the resorbing front, and relatively little enzyme was observed in the hypertrophied synovial membrane itself. These observations directly support the idea that synovial collagenase participates in cartilage erosion in rheumatoid arthritis.
Assuntos
Artrite Reumatoide/enzimologia , Cartilagem Articular/enzimologia , Colagenase Microbiana/metabolismo , Anticorpos , Antígenos , Artrite Reumatoide/patologia , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/farmacologia , Cartilagem Articular/patologia , Eletroforese em Gel de Poliacrilamida , Exsudatos e Transudatos/enzimologia , Humanos , Colagenase Microbiana/antagonistas & inibidores , Colagenase Microbiana/imunologia , Membrana Sinovial/enzimologia , Membrana Sinovial/patologia , alfa-Macroglobulinas/farmacologiaRESUMO
The main features of Paget's disease are described, together with the indications for medical treatment. A brief summary is given of the drugs available for treatment of Paget's disease with particular emphasis on sodium etidronate (EHDP, ethylidene-1-hydroxy-1, 1-diphosphonate). Sodium etidronate, given at doses between 5 and 20 mg per kilogram per day for 3-6 months, causes a progressive reduction in the biochemical abnormalities (raised plasma alkaline phosphatase and urinary hydroxyproline) and in the histological abnormalities of bone. Clinical symptoms also improve. The usual dose is 5 mg per kilogram body weight per day to be given for not longer than 6 months. Higher doses (10 and 20 mg per kilogram per day) may cause impairment of normal bone mineralisation and should be given for short periods only (1-3 months). Sodium etidronate also has a limited place in the treatment of certain disorders of ectopic calcification, notably heterotopic ossification after spinal cord injury or hip surgery. At the present time there is insufficient evidence to justify its use in the treatment of renal stones or in osteoporosis other than that due to immobilisation after spinal cord injury.
Assuntos
Difosfonatos/uso terapêutico , Osteíte Deformante/tratamento farmacológico , Anti-Inflamatórios não Esteroides/uso terapêutico , Calcitonina/uso terapêutico , Difosfonatos/farmacologia , Ácido Etidrônico/efeitos adversos , Ácido Etidrônico/uso terapêutico , Humanos , Osteíte Deformante/diagnóstico , Osteíte Deformante/epidemiologia , Osteíte Deformante/metabolismo , Plicamicina/uso terapêuticoRESUMO
Seventeen patients with Paget's disease of the skull and deafness were followed for nine to 18 months. Patients who received calcitonin treatment showed less deterioration in hearing than untreated patients. Calcitonin treatment may retard the progression of deafness in Paget's disease, and further studies are indicated.
Assuntos
Calcitonina/uso terapêutico , Surdez/tratamento farmacológico , Osteíte Deformante/complicações , Idoso , Fosfatase Alcalina/análise , Surdez/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteíte Deformante/tratamento farmacológicoRESUMO
1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.
Assuntos
Colagenase Microbiana/metabolismo , Pele/enzimologia , Proteínas Sanguíneas , Colágeno/metabolismo , Eletroforese Descontínua , Gelatina/metabolismo , Humanos , Colagenase Microbiana/antagonistas & inibidores , Colagenase Microbiana/isolamento & purificação , Microscopia Eletrônica , Peso Molecular , Peptídeo Hidrolases/metabolismoRESUMO
Specimens of cartilage with contiguous bone and overlying synovial pannus were obtained from 22 rheumatoid knee joints and examined histologically using specific histochemical staining techniques. All showed significant erosions of cartilage by synovial cells, but seven specimens also showed substantial cartilage erosion by cells from the subchondral bone region. This bidirectional attack on rheumatoid knee cartilage did not represent an 'underpinning' of cartilage by synovial pannus, as judged by serial sectioning and the identification of specific cells. Whereas cartilage-pannus junctions had mainly macrophagic or fibroblastic cells, cartilage-bone lesions were usually characterised by chondroclasts and blood vessels. Lymphocytes were generally absent from all sites of cartilage erosion. The bidirectional attack on articular knee cartilage suggests that changes have occurred within the cartilage that make it vulnerable to cellular invasion and erosion. Such changes might reflect a deficiency in 'anti-invasion factors', or the exposure of hidden epitopes and subsequent immunogenicity, or a combination of both.