Analysis of cartilage oligomeric matrix protein and matrix metalloproteinase-9 in cerebrospinal fluid of miniature dachshund with intervertebral disc herniation

We evaluated whether the cerebrospinal fluid (CSF) concentration of cartilage oligomeric matrix protein (COMP) is related to disease severity, prognosis and matrix metalloproteinase (MMP)-9 activity of the CSF in miniature dachshund with intervertebral disc herniation. Samples were obtained from 23 patients and 6 normal dogs, and all patients received hemilaminectomy. Twenty dogs recovered successfully and 3 of 11 dogs without deep nociception had MMP-9 activity in the CSF and an unsuccessful outcome. The COMP levels from patients were significantly higher than those from normal dogs. MMP-9 activity and neurological severity were not related to the COMP levels. However, the COMP levels from 3 unsuccessful cases that had MMP-9 activity were significantly lower than those from all recovered cases and/or successful cases without deep nociception. Concerning severe cases, increased proteolytic activity might affect the COMP concentration and prognosis due to MMP-9 associated deleterious effects.     

Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.     

Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.     

Effects of treatment with polysulfated glycosaminoglycan on serum cartilage oligomeric matrix protein and C-reactive protein concentrations, serum matrix metalloproteinase-2 and -9 activities, and lameness in dogs with osteoarthritis

OBJECTIVE: To investigate the effects of polysulfated glycosaminoglycan (PSGAG) treatment on serum cartilage oligomeric matrix protein (COMP) concentration, matrix metal-loproteinase-2 (MMP-2) and -9 (MMP-9) activities, C-reactive protein (CRP) concentration, and lameness scores in dogs with osteoarthritis. ANIMALS: 16 dogs with osteoarthritis and 5 clinically normal dogs. PROCEDURES: Dogs with osteoarthritis had a history of chronic lameness, and osteophytes were observed on radiographic evaluation of the affected joint. Polysulfated glycosaminoglycan was administered IM twice a week for a total of 8 treatments to all dogs with osteoarthritis and to clinically normal control dogs. RESULTS: Lameness scores after PSGAG treatment in osteoarthritic dogs improved in 12 of the 16 dogs. Serum COMP concentrations in osteoarthritic dogs were significantly higher than in control dogs before treatment. Lameness scores in osteoarthritic dogs decreased significantly after treatment, compared with before treatment. Lameness scores of 9 dogs with hind limb lameness improved significantly after treatment; these dogs had corresponding decreases in serum COMP concentrations. After treatment, serum COMP concentrations and lameness scores of 7 dogs with forelimb lameness remained high and were significantly higher than those of dogs with hind limb lameness. Serum MMP-9 activities of dogs with forelimb lameness were significantly higher than in dogs with hind limb lameness after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: IM administration of PSGAG inhibited COMP degradation in dogs with osteoarthritis. Results indicate that decreases in serum COMP concentrations might be related to improvement in lameness after PSGAG treatment.     

Matrix metalloproteinase-9 activity in the cerebrospinal fluid and serum of dogs with acute spinal cord trauma from intervertebral disk disease

OBJECTIVE: To detect matrix metalloproteinase (MMP)-9 in serum and CSF and determine relationships between MMP activity and severity of disease, duration of clinical signs, and duration of hospitalization in dogs with acute intervertebral disk disease (IVDD). ANIMALS: 35 dogs with acute IVDD and 8 clinically normal control dogs. PROCEDURE: CSF and serum were collected from affected and control dogs. Zymography was used to detect MMP-9. RESULTS: Activity of MMP-9 in CSF was detected in 6 of 35 dogs with IVDD; activity was significantly more common in dogs with duration of signs < 24 hours. Paraplegic dogs were more likely to have MMP-9 activity in the CSF than non-paraplegic dogs. No significant difference in hospitalization time was detected in dogs with IVDD between those with and without activity of MMP-9 in the CSF. Serum MMP-9 was detected more frequently in dogs with IVDD than in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Data were consistent with results of experimental rodent spinal cord injury studies that indicate that MMP-9 is expressed early during secondary injury.     

Canine cerebral leishmaniasis: Potential role of matrix metalloproteinase-2 in the development of neurological disease

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.     

Characterization of matrix metalloproteinase-2 and -9 in cerebrospinal fluid of clinically normal dogs

OBJECTIVE: To characterize matrix metalloproteinase (MMP)-2 and -9 in CSF of clinically normal dogs. SAMPLE POPULATION: Samples of CSF collected from 23 dogs. PROCEDURE: Dogs were anesthetized, CSF samples were collected, and dogs were then euthanatized. Each CSF sample was evaluated immediately for RBC count, WBC count, and protein and glucose concentrations, and cytologic examination also was performed. Samples were considered normal when protein concentration was < 25 mg/dL and CSF contained < 6WBCs/microL and < 25 RBCs/microL. Samples were stored at -70 degrees C. Sections of brain tissue were collected and processed for histologic examination. The MMPs were evaluated by use of gelatin zymography and a polyclonal antibody-based sandwich ELISA. RESULTS: Mean WBC count for CSF samples was < 1 WBC/microL (range, 0 to 3 WBCs/mL). Mean protein concentration was 12 mg/dL (range, 8 to 17 mg/dL). Mean RBC count was 3.65 RBCs/microL (range, 0 to 21 RBCs/microL). All CSF samples generated a clear band on zymography gels that corresponded to the human commercial standard of proenzyme MMP-2. Other major clear bands were not detected on zymography gels. Bands correlating to MMP-9 were not detected in any samples. The ELISA results revealed a mean +/- SD proenzyme MMP-2 concentration of 5.61 +/- 1.92 ng/mL (range, 3.36 to 10.83 ng/mL). CONCLUSIONS AND CLINICAL RELEVANCE: The proenzyme form of MMP-2 is detectable in CSF of clinically normal dogs, whereas MMP-9 is not detectable. Additional investigation of MMPs in CSF from dogs with various diseases of the nervous system is indicated.     

Synovial Fluid Studies in Navicular Disease

The purpose of this study was to investigate biochemical changes in synovial fluid in navicular disease, and to establish if synovial fluid from the distal interphalangeal joint (DIP) could be used diagnostically to assess alterations in the synovial fluid of the navicular bursa. Cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan (HA), metalloproteinases 2 and -9 (MMP-2 and MMP-9) and total protein (TP) levels were determined in synovial fluids obtained from 18 navicular bursae and 35 DIP -joints from animals suffering from navicular disease, and the same synovial structures in 16 joints of horses with no evidence of abnormalities involving the foot. To avoid dilution effects, GAG/COMP, HA/COMP, MMP-2/ COMP and MMP-9/COMP ratios were also calculated for different synovial cavities. There was a good correlation, for COMP, GAG, HA, MMP-2 and TP levels, between synovial fluid from the navicular bursa and fluid from the DIP -joint in healthy animals. However, in animals with navicular disease, only COMP levels showed no difference between the navicular bursal fluid and the DIP-joint fluid concentration. Thus, enabling the use of COMP to standardise other biochemical concentration measurements from the synovial joint fluids. In horses with navicular disease, there was a significantly lower absolute concentration of GAG, and a significantly lower GAG/COMP ratio, in the synovial fluid of the navicular bursa and the DIP-joint compared to synovial fluid from the same joints from healthy horses. In contrast, the absolute HA concentration and HA/ COMP, MMP-2/COMP and MMP-9/COMP ratios were higher in synovial fluid from the DIP-joint of horses with navicular disease, and MMP-2 and MMP-9 relative activity levels and MMP-2/COMP and MMP-9/ COMP ratios were increased in fluid from navicular bursae in horses with navicular disease when compared to a control group.     

Concentrations of cartilage oligomeric matrix protein in dogs with naturally developing and experimentally induced arthropathy

OBJECTIVE: To assay concentrations of cartilage oligomeric matrix protein (COMP) in canine sera and synovial fluid (SF), to compare COMP concentrations in clinically normal dogs and dogs with joint disease, and to analyze changes in COMP concentrations in dogs with experimentally induced acute synovitis. ANIMALS: 69 control dogs without joint disease, 23 dogs with naturally occurring aseptic arthropathy, and 6 dogs with experimentally induced synovitis. PROCEDURE: Serum (n = 69) and SF (36) were obtained from control dogs. Samples of serum (n = 23) and SF (13) were obtained from dogs with naturally occurring aseptic arthropathy with or without radiographic features of osteoarthritis (OA). Serum and SF were obtained before and 1, 2, 3, and 7 days after induction of synovitis. The COMP concentrations were determined by use of an inhibition ELISA that had canine cartilage COMP and monoclonal antibody against human COMP. RESULTS: Concentrations of COMP in serum and SF of control dogs were 31.3+/-15.3 and 298.7+/-124.7 microg/ml, respectively. In naturally occurring OA, COMP concentrations in serum (44.9+/-177 microg/ml) and SF (401.7+/-74.3 microg/ml) were significantly higher than corresponding concentrations in control dogs. The COMP concentration in SF peaked 24 and 48 hours after induction of synovitis, whereas concentration in serum peaked on day 3. CONCLUSIONS AND CLINICAL RELEVANCE: These results supported the hypothesis that COMP concentration in serum and SF of dogs may be altered after cartilage degradation or synovitis. Measurement of COMP concentrations can be useful when differentiating arthropathies in dogs.     

Biochemical characterisation of navicular hyaline cartilage,navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease

The study hypothesis was that navicular disease is a process analogous to degenerative joint disease, which leads to changes in navicular fibrocartilage and in deep digital flexor tendon (DDFT) matrix composition and that the process extends to the adjacent distal interphalangeal joint. The objectives were to compare the biochemical composition of the navicular articular and palmar cartilages from 18 horses with navicular disease with 49 horses with no history of front limb lameness, and to compare navicular fibrocartilage with medial meniscus of the stifle and collateral cartilage of the hoof. Cartilage oligomeric matrix protein (COMP), deoxyribonucleic acid (DNA), total glycosaminoglycan (GAG), metalloproteinases MMP-2 and MMP-9 and water content in tissues were measured. Hyaline cartilage had the highest content of COMP and COMP content in hyaline cartilage and tendon was higher in lame horses than in sound horses (p<0.05). The concentration of MMP-2 amount in hyaline cartilage was higher in lame horses than in sound horses. The MMP-2 amounts were significantly higher in tendons compared to other tissue types. Overall, 79% of the lame horses with lesions had MMP-9 in their tendons and the amount was higher than in sound horses (p<0.05). In horses with navicular disease there were matrix changes in navicular hyaline and fibrocartilage as well as the DDFT with potential implications for the pathogenesis and management of the condition.     

Evaluation of matrix metalloproteinase concentrations in precorneal tear film from dogs with Pseudomonas aeruginosa-associated keratitis

OBJECTIVE: To evaluate the changes in concentrations of matrix metalloproteinase (MMP)-2 and MMP-9 in the precorneal tear film of dogs with Pseudomonas aeruginosa-associated keratitis during corneal healing and stromal remodeling. ANIMALS: 10 dogs with unilateral P aeruginosa-associated keratitis and 10 clinically normal dogs. PROCEDURES: Precorneal tear film samples were collected from both eyes of 10 dogs with unilateral P aeruginosa-associated keratitis on the day of admission to the hospital and then at various time points until complete healing of the cornea was achieved. Precorneal tear film samples were also collected from both eyes of 10 clinically normal adult dogs (control group). Concentrations of MMP-2 and MMP-9 in precorneal tear film samples from each group were determined via gelatin zymography for comparison. RESULTS: The proteolytic processes in the ulcerated eyes decreased as corneal healing progressed. On the day of admission, concentrations of latent and active forms of MMP-2 and MMP-9 in ulcerated eyes were significantly higher than values in the contralateral unaffected eyes in dogs with P aeruginosa-associated keratitis; concentrations of latent MMP-2 and MMP-9 were also greater than control group values. Concentrations of latent and active forms of MMP-2 and MMP-9 in the healed eyes of dogs with P aeruginosa-associated keratitis were significantly lower than concentrations in the ulcerated eyes on the day of admission. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that reduction of precorneal tear film concentrations of MMPs by use of proteinase inhibitors may be effective in the treatment of dogs with P aeruginosa-associated keratitis.     

Association of cerebrospinal fluid analysis findings with clinical signs and outcome in acute nonambulatory thoracolumbar disc disease in dogs

Background: Cerebrospinal fluid (CSF) pleocytosis recently was associated with the severity of neurologic signs in dogs with intervertebral disc disease (IVDD). Hypothesis/Objectives: To look for an association among CSF cell counts, total protein concentration, and severity of neurologic signs at presentation with outcome in dogs with acute thoracolumbar IVDD. Our hypothesis was that CSF total nucleated cell count (TNCC) and percentage cell types would be associated with the severity of spinal cord damage and therefore with both the presenting clinical signs and the prognosis of affected dogs. Animals: Fifty‐four dogs with acute nonambulatory thoracolumbar IVDD were evaluated. Methods: Retrospective study. Signalment, neurologic grade, CSF TNCC, protein concentration, red blood cells count and differential cell percentages, and short‐ and long‐term outcomes were evaluated. Results: CSF pleocytosis (>5 cells/μL) was present in 54% of dogs and was positively associated with neurologic grade at presentation and with postoperative time to regaining ambulation. Neutrophils were observed most frequently. The percentage of CSF macrophages and macrophage to monocyte ratio were higher (P= .001, for both) in dogs presented without deep pain sensation (DPS) that did not regain ambulation. Receiver operator characteristics curve analysis yielded a cut‐off point of 13% macrophages with a sensitivity and specificity of 100 and 83%, respectively, for prediction of a negative outcome. Conclusions and Clinical Importance: CSF pleocytosis is positively associated with the severity of spinal cord damage in dogs with thoracolumbar IVDD. The percentage of CSF macrophages can be used as a prognostic indicator for regaining ambulation in dogs that have lost DPS.     

An immunohistochemical study of the role of matrix metalloproteinases and their tissue inhibitors in chronic mitral valvular disease (valvular endocardiosis) in dogs

While the pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, alterations in the activity of specific metalloproteinase enzymes and their inhibitors within the valve stroma are suspected of having a role. This study describes the immunohistochemical distribution pattern of matrix metalloproteinase (MMP) types 2, 9 and 14 and their tissue inhibitors, termed tissue inhibitors of metalloproteinase (TIMP), types 2 and 3, in normal canine mitral valves (MVs) (n=10) and in dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal MVs, MMP-2 and -14, and TIMP-2 were expressed in isolated stromal cells. Tissue inhibitor of metalloproteinase-3 exhibited moderate intracellular and mild extracellular expression. With increasing severity of CVD, the expression of MMP-2 decreased. The number of stromal cells expressing MMP-14 increased, predominantly in the margins of the nodular lesions. Tissue inhibitor of metalloproteinase-2 and -3 expression increased both intra- and extracellularly. Matrix metalloproteinase-9 was not detected in normal or diseased valves. In conclusion, CVD was characterised by alterations in the distribution and intensity of valvular MMP and TIMP expression, suggesting that depressed catabolism and the accumulation of extracellular matrix components within affected valves contributes to their structural alteration and consequent loss of function.     

Marked MMP-2 transcriptional up-regulation in mononuclear leukocytes invading the subarachnoidal space in aseptic suppurative Steroid-Responsive Meningitis–Arteritis in dogs

Canine Steroid-Responsive Meningitis–Arteritis (SRMA) is a suitable animal model for studies on the development of neutrophilic pleocytosis in aseptic meningitis. Samples of dogs in the acute phase of SRMA (n = 16) were examined for gene expression of matrix metalloproteinases (MMP)-2 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2. Results were compared to those of dogs under glucocorticosteroid treatment for SRMA (n = 16) and dogs with other inflammatory and neoplastic diseases of the central nervous system (CNS) (n = 19). Samples included mononuclear (PBMCs) and polymorphonuclear cells (PBPMNs) of peripheral blood and cerebrospinal fluid white blood cells (CSF WBCs). In the acute phase of SRMA CSF WBCs showed mRNA expression for MMP-2 and -9 and TIMP-1 and -2, highlighting a contribution of these cells to the overall content of MMPs and TIMPs in CSF. MMP-2 mRNA levels in CSF WBCs were significantly up-regulated in comparison to PBMC expression levels, suggesting that MMP-2 is relevant for PBMC invasion into the subarachnoidal space and that the expression is influenced by migratory activity through the blood–CSF-barrier.     

Serum level of cartilage oligomeric matrix protein (COMP) in equine osteoarthritis

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.     

Changes to plasminogen activators and matrix metalloproteinase-9 in dogs with toxocarosis

The relationship between proteolytic enzymes and hematological response to infection was studied in five 1-month-old dogs inoculated experimentally with 2000 eggs of Toxocara canis. Moderate leukocytosis and marked eosinophilia occurred 14 days post-infection with T. canis. Plasminogen activators (PAs) and matrix metalloproteinase-9 (MMP-9) activity in serum was significantly different in dogs infected with T. canis, compared with controls. Urokinase-type PA (uPA) activity was positively correlated with eosinophilia, and tissue-type PA (tPA) and MMP-9 activity was negatively correlated with eosinophilia. However, there was no correlation between inflammation and MMP-2. The use of uPA, tPA or MMP-9 proteolytic enzymes as laboratory reference markers for toxocarosis requires further study.     

Cartilage oligomeric matrix protein and hyaluronan levels in synovial fluid from horses with osteoarthritis of the tarsometatarsal joint compared to a control population

REASONS FOR PERFORMING STUDY: Quantification of cartilage oligomeric matrix protein (COMP) levels within synovial fluid from the tarsometatarsal joint has not previously been reported and an effective synovial fluid marker would allow monitoring of disease progression and treatment. OBJECTIVES: To quantify levels of COMP and hyaluronan (HA) in synovial fluid from the tarsometatarsal joint, identify differences in levels from horses with osteoarthritis (OA) of the tarsometatarsal joint compared to a control population and to correlate levels with radiographic changes in horses with OA. METHODS: Synovial fluid was collected from the tarsometatarsal joint of 25 horses without hindlimb lameness (controls) and 25 lame horses, subjected to analgesia of the joint. COMP concentrations were measured using a homologous inhibition ELISA. Immunoblots of synovial fluid from 3 lame horses and 3 controls were performed to identify fragmentation of COMP. Hyaluronan (HA) concentration in synovial fluid was determined using a competition ELISA. Radiographs of the lame horses with OA were scored and correlated with levels of COMP and HA. RESULTS: Concentrations of COMP in OA of the tarsometatarsal joint were significantly lower than in the control samples. An additional fragment band of COMP (approximately 30 kDa) was identified on the immunoblots of the horses with OA and this fragment was not identified in controls. No significant difference was identified in the HA or HA:COMP ratio between lame and control horses. There was no correlation between levels of synovial fluid COMP and HA, and radiographic changes. CONCLUSIONS AND POTENTIAL RELEVANCE: Lowered levels of COMP in synovial fluid of tarsometatarsal joints correlates with the presence of osteoarthritis. However, a single value cannot be used to stage the disease process. Levels of HA may not be a useful marker for this disease. Decreased, rather than increased COMP levels, may reflect significant loss of cartilage in established osteoarthritis. A specific assay for the COMP fragment generated with osteoarthritis may allow the earlier detection of clinical cases.     

Gelatinase activity in synovial fluid and synovium obtained from healthy and osteoarthritic joints of dogs

OBJECTIVE: To determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions. SAMPLE POPULATION: 35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII). PROCEDURE: MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints. RESULTS: Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD.     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Increased cartilage oligomeric matrix protein concentrations in equine digital flexor tendon sheath synovial fluid predicts intrathecal tendon damage

Objectives: To evaluate digital flexor tendon sheath (DFTS) synovial fluid cartilage oligomeric matrix protein (COMP) concentrations as a molecular marker for intrathecal pathology. Study Design: Case control study. Animals: Horses (n=46) with DFTS tenosynovitis; 23 fresh cadaver horses. Methods: DFTS synovial fluid samples were collected from clinical cases with noninfected DFTS tenosynovitis and from control DFTS. Clinical and surgical findings were recorded, and dissection of control limbs was performed to confirm the DFTS to be grossly normal. Synovial fluid COMP was quantified using a homologous competitive inhibition ELISA. Results: Abnormalities were identified tenoscopically: intrathecal tendon/ligament tearing was identified in 37 cases and 9 had other lesions. In control horses, synovial fluid COMP was higher in younger horses. Clinical cases with intrathecal tendon/ligament tearing had higher synovial fluid COMP than either clinical cases with other lesions, or controls. In horses ≥5 years old, the sensitivity and specificity of the assay was high for diagnosing intrathecal tendon/ligament tearing. Conclusions: COMP concentrations in DFTS synovial fluid were significantly greater than those in normal horses with noninfected tenosynovitis caused by intrathecal tendon/ligament tearing, but not by other lesions.