RESUMEN
Matrix metalloproteinases (MMPs) are important enzymes found in connective tissues and thought to be involved in cartilage degradation. They are detectable in bovine synovial fluid and may play a destructive role in bovine septic arthritis. The MMP gelatinase enzymes were detected by gelatin zymography using image analysis of the gels. The active gelatinase levels were determined by a gelatin degradation enzyme-linked immunosorbent assay (ELISA). Increased concentrations of MMP-9 activity were found in the synovial fluids of cows with septic arthritis (P < 0.001) in comparison with fluids from normal joints. Using the gelatin degradation ELISA the net active gelatinases were measured, and significant increases were found in gelatinase bioactivities in synovial fluids from septic joint disease cases (P < 0.001). Increased concentrations of MMP-2 activity were found in the synovial fluids of cows with aseptic arthritis, which appeared to be playing an important role in degradation of articular cartilage in joint disease. This finding required further investigation.
Asunto(s)
Artritis Infecciosa/veterinaria , Enfermedades de los Bovinos/enzimología , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Líquido Sinovial/enzimología , Animales , Artritis Infecciosa/enzimología , Bovinos , Electroforesis/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Gelatina/química , Procesamiento de Imagen Asistido por Computador , Estadísticas no ParamétricasRESUMEN
Matrix metalloproteinases (MMPs) are considered important mediators of tissue damage in joint diseases. The levels of MMPs 2 and 9 were measured in samples of synovial fluid from 20 joints in seven dogs with rheumatoid arthritis by gelatin zymography. The results were compared with the actual gelatinolytic activity of the fluid measured in a gelatin-degradation ELISA. The gelatinolytic activity in synovial fluid from arthritic joints was markedly greater than that in fluid from disease-free joints. The zymographic activity attributable to MMP-9 (identified by Western blotting) was absent from synovial fluid from control joints but prominent in fluid from arthritic joints, and in these joints the presence of a 75 kDa form of MMP-9 was correlated with the gelatinolytic activity of the fluid measured by the ELISA (r = 0.81, P < 0.05). Synovial fluid from one dog with rheumatoid arthritis was examined before and after treatment with corticosteroids. After treatment its zymographic pattern had returned to normal.
Asunto(s)
Artritis Reumatoide/veterinaria , Colagenasas/metabolismo , Enfermedades de los Perros/enzimología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Líquido Sinovial/enzimología , Corticoesteroides/uso terapéutico , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Western Blotting , Colagenasas/aislamiento & purificación , Perros , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Gelatina/metabolismo , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la MatrizRESUMEN
Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflammatory cells (polymorph neutrophils [PMN] and peripheral blood monocytes [PBM]). The TIMPs were characterised further using reverse zymography, affinity chromatography and N-terminal amino acid sequencing. Synovial fluid was recovered from horses with articular sepsis and aseptic joint disease (AJD) and compared with that from normal horses (n = 4). TIMP activity was minimal in articular sepsis but significantly increased, albeit a small increase, in AJD when compared to normal (P<0.05). Cell culture supernatants from synovial fibroblasts, chondrocytes and PBMs contained TIMP activity, although supernatants from PMN cell culture did not. Reverse zymography of synovial fluid recovered from normal and AJD horses showed two protein bands, 22 and 28 kDa in size, exhibiting inhibitory activity against MMP-9. Reverse zymography of culture supernatants of synovial fibroblasts and chondrocytes gave similar results whereas the culture supernatants from PMNs and PBMs showed the presence of only the 28 kDa protein. The N-terminal amino acid sequence was obtained for the 22 kDa protein and revealed a 66% homology with human TIMP-2. The identification of TIMPs in equine synovial fluids and cell culture supernatants suggest that they may have a fundamental role in the homeostasis of the normal joint and in the excess proteolysis which occurs in articular disease in the horse.
Asunto(s)
Caballos/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/análisis , Líquido Sinovial/enzimología , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-2/análisis , Secuencia de Aminoácidos , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Fibroblastos/enzimología , Enfermedades de los Caballos/enzimología , Artropatías/enzimología , Artropatías/veterinaria , Metaloendopeptidasas/análisis , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidor Tisular de Metaloproteinasa-2/farmacologíaRESUMEN
A canine gelatinase, with an apparent molecular mass of 62 kDa in non-reducing zymography, is produced by fibroblasts, chondrocytes and a myelomonocytic cell line. The enzyme has similar characteristics to human matrix metalloproteinase (MMP) 2 and cross-reacts in Western blotting analysis with a sheep polyclonal antiserum raised against human MMP-2. The 62 kDa canine protein was purified from cell culture media, and the N-terminal amino acid sequence determined following blotting on to a polyvinylidene difluoride (PVDF) membrane. The sequence was 87% identical to that published for human MMP-2. We therefore consider this enzyme to be canine MMP-2.
Asunto(s)
Perros/fisiología , Gelatinasas/aislamiento & purificación , Metaloendopeptidasas/aislamiento & purificación , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Células Cultivadas , Gelatinasas/química , Humanos , Técnicas In Vitro , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/química , Datos de Secuencia MolecularRESUMEN
A previously unpublished surgical technique for the management of disc-associated wobbler syndrome is described. A series of 17 middle-aged dobermanns (mean age 7.4 years) with this condition were managed by a technique of partial slot fenestration and position screw fixation of the affected disc space (C6/7 in all cases). One patient was lost to follow-up and, of the remaining 16 cases, 13 improved following surgery (81 per cent). The three failures were a consequence of incorrect placement of one of the screws. Careful evaluation of immediate postoperative radiographs allows this problem to be identified; the screw can then be correctly repositioned with an immediate return to surgery.
Asunto(s)
Vértebras Cervicales/anomalías , Perros/cirugía , Compresión de la Médula Espinal/veterinaria , Animales , Tornillos Óseos/veterinaria , Femenino , Masculino , Compresión de la Médula Espinal/cirugía , SíndromeRESUMEN
Matrix metalloproteinases (MMPs) may be important in the destruction of cartilage seen in equine osteoarthritis and may be detectable in synovial fluid. Synovial fluids were obtained from normal equine joints and from joints of horses with aseptic and septic joint diseases. The total MMP gelatinase enzyme activities were measured by gelatin zymography and image analysis of the gels. The bioactivity of gelatinase in synovial fluid was determined by a gelatin degradation ELISA. Potential MMP-2 & MMP-9 monomer enzyme activities were significantly elevated in both septic and aseptic joint disease synovial fluids in comparison to fluids from normal joints. The dimer form of MMP-9 enzyme activity was significantly elevated in fluids from septic joint disease cases in comparison to fluids form normal joints, but not fluids from horses with aseptic joint diseases. MMP-9 monomer and dimer levels in synovial fluids correlated with the synovial fluid white blood cell count. Using the gelatin degradation ELISA to measure net active gelatinases, significant increases in gelatinase bioactivities were seen in synovial fluids from both aseptic and septic joint disease cases. The enzymes in equine joint diseases were present in a bioactive form, in that they were present in the activated form and present in excess of inhibitors, and could therefore be important in the degradation of articular cartilage in joint disease.
Asunto(s)
Colagenasas/análisis , Gelatinasas/análisis , Caballos/metabolismo , Metaloendopeptidasas/análisis , Líquido Sinovial/enzimología , Animales , Biomarcadores/análisis , Cartílago Articular/citología , Cartílago Articular/metabolismo , Colagenasas/metabolismo , Colagenasas/fisiología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Gelatina/metabolismo , Gelatinasas/metabolismo , Gelatinasas/fisiología , Enfermedades de los Caballos/enzimología , Enfermedades de los Caballos/fisiopatología , Procesamiento de Imagen Asistido por Computador , Artropatías/enzimología , Artropatías/fisiopatología , Artropatías/veterinaria , Leucocitos/citología , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/fisiología , Análisis de Regresión , Líquido Sinovial/citología , Líquido Sinovial/metabolismoRESUMEN
The cellular production by resident articular cells and infiltrating inflammatory cells of the gelatinase matrix metalloproteinases (MMP) was investigated by tissue culture methods and analysis of cell supernatants by gelatin zymography. Peripheral blood neutrophils in short term culture produced MMP-9, as did peripheral blood monocytes in culture. Isolated articular chondrocytes in monolayer culture produced both MMP-2 and MMP-9, although articular cartilage maintained as explant culture produced MMP-2 alone. Synovial fibroblasts grown in monolayer culture produced MMP-2 alone, although synovial membrane in explant culture produced both MMP-2 and the active form of MMP-2. Lysis of blood polymorph neutrophils produced large quantities of MMP-9, but lysis of blood monocytes, synovial fibroblasts and articular chondrocytes produced little enzyme indicating that, unlike the other cell types, polymorph neutrophils store MMPs intracellularly. Equine MMP-2 was purified from synovial fibroblast cell culture supernatant, and equine MMP-9 from polymorph neutrophil cell culture supernatant, by gelatin-sepharose affinity chromatography. The 2 enzymes were identified from their molecular weights and by their respective N-terminal amino acid sequences which showed homology with the enzymes from other species. The demonstration that invasive cells and resident articular cells can produce enzymes which are capable of digestion of certain component molecules of the articular cartilage matrix, shows that therapeutic targeting of these enzymes could be a valid proposition in the prevention of cartilage destruction in osteoarthritis.
Asunto(s)
Colagenasas/biosíntesis , Gelatinasas/biosíntesis , Caballos/metabolismo , Articulaciones/enzimología , Metaloendopeptidasas/biosíntesis , Monocitos/enzimología , Neutrófilos/enzimología , Membrana Sinovial/enzimología , Secuencia de Aminoácidos , Animales , Cartílago Articular/citología , Cartílago Articular/enzimología , Células Cultivadas , Quelantes/farmacología , Condrocitos/citología , Condrocitos/enzimología , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/veterinaria , Colagenasas/química , Técnicas de Cultivo , Perros , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida/métodos , Electroforesis en Gel de Poliacrilamida/veterinaria , Fibroblastos/citología , Fibroblastos/enzimología , Gelatinasas/antagonistas & inhibidores , Gelatinasas/química , Humanos , Articulaciones/citología , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Peso Molecular , Monocitos/citología , Neutrófilos/citología , Homología de Secuencia de Aminoácido , Membrana Sinovial/citologíaRESUMEN
A classification scheme for sacral fractures was based on a review of sacral fractures in 34 dogs and 17 cats. They were classified into five categories based on their radiographic appearance on standard lateral and ventrodorsal radiographs of the pelvis. Concomitant pelvic injuries were commonly observed: 32 per cent of dogs had a fracture of an ilium and 65 per cent of cats had union bilateral sacroiliac subluxation. Neurological deficits were common where the fractures traversed the spinal canal or sacral foraminae. Comparison is made between sacral fractures in dogs and cats, and humans.