大肠杆菌及脂多糖对奶牛乳腺上皮细胞基质金属蛋白酶表达的影响

为研究大肠杆菌(E.coli)及脂多糖(LPS)对奶牛乳腺上皮细胞(BMECs)基质金属蛋白酶(MMPs)表达的影响,以及MMPs与基质金属蛋白酶组织抑制因子(TIMPs)、尿激酶型纤溶酶原激活物(uPA)系统在调控细胞外基质(ECM)代谢中的作用,分别以106 CF U/mL热灭活E.coli菌液、7.5μg/mL ...     

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.     

Short- and long-term effects of platelet-rich plasma upon healthy equine joints: Clinical and laboratory aspects

This study aimed to verify whether transient inflammatory reactions incited by the administration of intra-articular platelet-rich plasma (PRP) affected joint components through short- and long-term in vivo evaluation of inflammatory biomarkers and extracellular matrix degradation products in synovial fluid. The effects of PRP were analyzed in a short phase protocol (SPP) and in a prolonged phase protocol (PPP), using saline-injected joints as controls. In the SPP, higher white blood cell counts and prostaglandin E₂ and total protein concentrations were observed in the synovial fluid of PRP-treated joints (P < 0.05). There were no differences between the interleukin-1β, interleukin-1 receptor antagonist protein, tumor necrosis factor-α, chondroitin sulfate, or hyaluronic acid concentrations between PRP and saline injected joints. In the PPP, there were no differences in evaluated parameters between groups. PRP injection elicits a mild and self-limiting inflammatory response shortly after administration, without long-term deleterious effects on joint homeostasis.     

Regenerative therapy for tendon and ligament disorders in horses. Terminology, production, biologic potential and in vitro effects

Conventional treatments of equine tendon injuries lead to an unsatisfactory healing process that usually results in a relatively high recurrence rate. Therefore, in recent years so-called regenerative therapeutics were studied scientifically in vitro and in laboratory animals. These include substances that ideally lead to the formation of replacement tissue, which in contrast to the low quality scar, has similar functional properties as the original intact tendon. Currently, a plethora of different substrates is either commercially available or can be produced in practice with the help of kits. The current knowledge on the production and the regenerative potential of nucleated cells like stem cells from bone marrow and fat tissue, of the blood products PRP (platelet rich plasma), ACP (autologous conditioned plasma), ACS (autologous conditioned serum) and of the scaffold substance UBM (urinary bladder matrix) are presented. Finally, the potential of some growth factors and of gene therapy is considered. Currently, it is assumed that the regeneration of tendon tissue is promoted by a complex interaction of scaffolds, growth factors and cells. At present, only very few studies are available which allow a comparison between these substances. Studies on the effect of regenerative substrates on tendons in live horses are presented elsewhere.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.     

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.     

Matrix metalloproteinase expression and localization in turkey (Meleagris gallopavo) during the endochondral ossification process

Vertebrate long bones are formed by endochondral ossification, a process accompanied by changes in extracellular matrix synthesis and remodeling, performed mainly by the matrix metalloproteinases (MMP). The temporal/spatial expression patterns of 5 members of the MMP family known to be important for endochondral ossification were studied, for the first time, in the turkey growth plate during embryonic and juvenile stages. The expression of MMP-2 was detected in the proliferative zone, MMP-3, MMP-9, and MMP-13 in cells lining the blood vessels; MMP-13 was also detected in hypertrophic chondrocytes. The MMP-16 expression was detected in the reserve zone of the growth plate. These results present a detailed survey of turkey MMP, serving as a data source (atlas) for further studies in this subject.     

Arthroscopic diagnosis and treatment of intra-articular insertional injuries of the suspensory ligament branches in 18 horses

REASONS FOR PERFORMING STUDY: Clinical association between the branches of insertion of the suspensory ligament (SL) and metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints has been reported. However, there has been no assessment of the lengths of the SL branches which are subsynovial with respect to the joints or reports of involvement of the MCP/MTP joints in injuries of the SL branches. OBJECTIVES: To establish proportions of SL branches subsynovial with respect to the MCP/MTP joints and report clinical and arthroscopic findings in horses with desmitis of SL branches identified as having an articular component to the lesion. HYPOTHESIS: Arthroscopic surgery enables identification and potential treatment of intra-articular injuries of SL branches. METHODS: Twelve forelimbs and 13 hindlimbs were dissected and the total and subsynovial lengths of the SL branches recorded. Case records of horses with intra-articular injuries of the SL branches were reviewed and 18 animals identified. Diagnostic information and arthroscopic findings were recorded and results of treatment determined by telephone follow-up. RESULTS: Of SL branches, 28.45% in the forelimb and 29.56% in the hindlimb were subsynovial to the MCP and MTP joints. All animals with intra-articular lesions of the SL branch were lame and had distension of the affected MCP/MTP joint. In 16 horses (17/22 branches), there was palpable thickening of the affected SL branch. Disrupted infrastructure was evident ultrasonographically in 15/17 branches and involvement of the dorsal articular surface of the ligament was predicted in 12/17 branches. Following arthroscopic intervention, 13 horses returned to work at a level equal to or greater than that achieved prior to injury and 2 returned to work at a lower level. Three horses incurred separate injuries and were retired or subjected to euthanasia. CONCLUSIONS: Articular involvement should be considered in animals with injuries of an SL branch and concurrent distension of the MCP/MTP joint. Arthroscopy is necessary to identify such lesions confidently and to direct case management. POTENTIAL RELEVANCE: Arthroscopy of the MCP/MTP joints can make a positive contribution to the assessment and management of some SL branch injuries.     

The effect of collection and extension on tarsal flexion and fetlock extension at trot

A recent epidemiological study indicated that various factors may be related to injury in dressage horses, but the mechanism by which these injuries occur has yet to be determined. The suspensory ligament (SL) is a frequent site of injury, and it is assumed that greatest strain is placed on this structure in collected trot; this has yet to be proved conclusively. The study aimed to investigate the effect of collected and extended trot on the hindlimb movement pattern. Four dressage horses were fitted with markers and inertial motion sensors (IMS). High‐speed video was obtained for 2 strides on each rein in collected and extended trot on 3 different surfaces: waxed outdoor; sand/plastic granules; and waxed indoor. Maximal tarsal flexion during stance and distal metatarsal coronary band ratio (MTCR), representing fetlock extension, were determined. Inertial motion sensor data determined stride duration, speed and stride length. Data were compared between collection and extension within horses on each surface, and compared between surfaces. Collected trot had significantly lower speed and stride length but longer stride duration than extended trot on all surfaces. All horses had less tarsal flexion and fetlock extension in collected compared with extended trot (P<0.05), which is likely to increase SL loading. The study findings indicate that extended trot may increase SL strain, providing a possible explanation for the high incidence of SL injury in horses trained for extravagant movement. It is possible that substantial use of extended trot could be a risk factor for development of suspensory desmitis, which might be one contributory factor in the prevalence of suspensory desmitis in young horses repeatedly undertaking extravagant movement.     

Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.     

Determination of MMP-2 and -9 activities in synovial fluid of horses with osteoarthritic and arthritic joint diseases using gelatin zymography and immunocapture activity assays

REASON FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs)-2 and -9 activities have been found elevated in synovial fluid from various joint diseases in man. However, in the horse few data are available. OBJECTIVES: To explore the clinical significance of MMP-2 and -9 activities in synovial fluid of horses with different forms of joint diseases. METHODS: Gelatin zymography and MMP-2 and -9 immunocapture activity assays were applied on synovial fluids from control joints and joints with aseptic joint disease (AJD) and septic arthritis (SA). Additionally, MMP-2 and -9 activities were measured in samples from SA to monitor the disease process. RESULTS: Zymographic analysis revealed that samples from AJD and SA contained significantly increased latent MMP-2 activity compared to controls. Samples from SA showed significantly increased monomeric latent MMP-9 activity compared with all other affected joints and controls. Trace amounts of MMP-9 activity, due to the active and dimer form, were detected in samples from SA; however, these bands were absent in samples from AJD and controls. Using immunocapture activity assays, MMP-2 and -9 activities were found to be significantly elevated in joints from SA compared to controls and AJD samples. MMP-2 activity in samples from AJD was significantly increased compared to controls. Both MMP activities decreased in the joints from SA in the course of successful therapy. CONCLUSIONS: Data from zymographic analysis confirmed that MMP-2 and -9 were elevated in equine joint diseases. Immunocapture activity assays have been shown to be suitable for the quantitative determination of MMP-2 and -9 activities in synovial fluid of horses. Both MMP-2 and -9 activities seem to be useful to indicate SA, and MMP-2 activity might be a suitable marker for AJD. POTENTIAL RELEVANCE: These findings encourage the potential use of MMP-2 and -9 as additional aids to clinical investigation. Further work is required to validate the clinical significance of MMP activities in the progress of different joint diseases in horses.     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.     

Analysis of cartilage oligomeric matrix protein (COMP) degradation and synthesis in equine joint disease

REASONS FOR PERFORMING STUDY: Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. OBJECTIVES: To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. HYPOTHESIS: A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragmentation. METHODS: A mAb (clone14G4) was generated against equine cartilage COMP. The NH2-terminal sequence of enzyme-cut COMP fragments recognised by 14G4 was determined, as was the efficiency of binding to COMP (using a generated COMP peptide). COMP concentration and fragmentation were analysed in synovial fluid (SF) from normal horses and those with OA. RESULTS: The mAb 14G4 had a higher affinity for the smaller fragments of equine COMP, compared with a mAb (clone 12C4) generated against human COMP. The 14G4 epitope was identified as between C134 and F147. The COMP values in OA (mean +/- s.d. 205.8 +/- 90.9 microg/ml) were significantly higher than in the normal (133.1 +/- 31.5 microg/ml) SF. On the immunoblots of OA sample, the proportions of intact COMP were significantly lower, while smaller fragments ranging from 75 to 290 kDa were higher compared with the normal SF. CONCLUSIONS AND POTENTIAL RELEVANCE: The mAb 14G4 reliably detects COMP degradation as well as synthesis, and fragmentation analysis combined with quantification in SF could be useful to study equine OA.     

Platelet-Rich Products and Their Application to Osteoarthritis

Autologous platelet-rich plasma (PRP) is a biological preparation made from the patient’s own plasma that contains a platelet concentration above the whole blood baseline. Owing to the release of growth factors and other cytokines after degranulation, platelets have a central role in inflammation and in different stages of the healing process. For this reason, PRP-derived products have been used to enhance healing of musculoskeletal injuries and modulate progression of inflammatory processes, including osteoarthritis (OA). Osteoarthritis is one of the main causes of musculoskeletal disabilities in horses, and currently, there is no effective treatment for this disease. Treatments that focus on the modulation of inflammation and disease progression offer new hope for OA. Platelet-rich plasma provides a more practical and accessible option of therapy compared to other forms of biological treatment (i.e., stem cell therapies) and is believed to induce the production of functional matrix. However, several factors related to PRP production, including methods of preparation and application, and intraindividual variability, lead to an inconsistent product, precluding reliable conclusions about its efficacy for clinical use. The aim of this study was to review the benefits related to the clinical use of PRP in OA as well as factors that influence its use, the limitations of this treatment, and future directions of PRP research and therapy.     

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.     

Plasma matrix metalloproteinase‐9 activity in cats with lymphoma

In this study, plasma MMP‐9 activity was evaluated in cats with lymphoma. Plasma samples were obtained from 26 cats with lymphoma before treatment. From 13 of the included 26 cats, plasma samples were obtained 4 weeks after the initiation of treatment. Plasma samples were also obtained from 10 healthy cats as a control. Plasma MMP‐9 activity was examined by gelatin zymography and semi‐quantitative value (arbitrary unit; a.u.) for each sample was calculated. Relatively high levels of MMP‐9 were observed in cats with lymphoma compared with those in healthy control cats. MMP‐9 quantification through zymography showed significantly higher activity in cats with lymphoma (median, 0.63 a.u.; range, 0.23–3.24 a.u.) than in healthy controls (0.22 a.u.; 0.12–0.46 a.u.; P < 0.01). MMP‐9 activities were significantly different before (0.73 a.u.; 0.30–3.24 a.u.) and after treatment (0.50 a.u.; 0.14–1.32 a.u.; P = 0.017). Measuring plasma MMP‐9 activity in cats with lymphoma may become an appropriate monitoring tool for feline lymphoma.     

Effects of glucosamine and chondroitin sulfate on mediators of osteoarthritis in cultured equine chondrocytes stimulated by use of recombinant equine interleukin-1beta

OBJECTIVE: To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. SAMPLE POPULATION: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. PROCEDURE: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 to 50.0 microg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor kappaB; and c-Jun-N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay. RESULTS: Glucosamine at a concentration of 10 microg/mL significantly reduced reIL-1beta-induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking.     

Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase-9 and haptoglobin released by bovine granulocytes in vitro

OBJECTIVE: To characterize and purify covalent complexes of matrix metalloproteinase-9 (MMP-9) and haptoglobin released by bovine granulocytes in vitro. SAMPLE POPULATION: Blood samples obtained from healthy cows and cows with acute and chronic inflammation to obtain WBCs and sera. PROCEDURES: WBCs were isolated by differential centrifugation, hypotonic lysis of RBCs, and degranulated by stimulation with phorbol ester (20 ng/mL). Cell-conditioned medium was subjected to affinity and gel chromatography and purified proteins subjected to SDS- PAGE gelatin zymography, western blot analysis, Coomassie blue staining, and peptide mass spectrometry for protein identification. Sera of cows hospitalized for acute and chronic septic conditions and of clinically normal cows were analyzed with similar methods. RESULTS: Matrix metalloproteinase-9 was released from neutrophils in vitro and migrated to a molecular mass of approximately 220 kd (prodimer), approximately 105 kd (promonomer), and > 220 kd (high-molecular mass complexes). These high-molecular mass complexes were composed of alpha- and beta-haptoglobin and MMP-9 (ratio13:13:1). Complexes of MMP-9 and haptoglobin had biochemical properties of both its protein constituents (i.e., enzymatic activity toward gelatin and hemoglobin binding). Complexes of MMP-9 and haptoglobin were also detected in sera of cows with acute inflammation, but not in clinically normal cows or cows with chronic disease. CONCLUSIONS AND CLINICAL RELEVANCE: A fraction of neutrophil MMP-9 is released in complex with haptoglobin. The complex is present in granules and retains biological activity of its components. Detection of the complex in serum may provide an indicator of acute inflammation.     

Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes

OBJECTIVE: To characterize potential mechanisms of action of glucosamine inhibition of matrix metalloproteinase (MMP) expression and activity in lipopolysaccharide (LPS)-stimulated equine chondrocytes. SAMPLE POPULATION: Chondrocytes cultured from samples of metacarpophalangeal articular cartilage collected from cadaveric limbs of horses. PROCEDURE: The effect of glucosamine on MMP activity in conditioned medium from LPS-stimulated cartilage explants was determined by a colorimetric assay with azocoll substrate. Treatments consisted of negative and positive controls, glucose (50 mM), and glucosamine (50, 25, 6.25, 3, and 1.5 mM). The influence of glucosamine on MMP synthesis was determined in chondrocytes in pellet culture incubated with LPS (20 microg/mL). Concentration of MMP-13 was quantified in spent medium via ELISA; nonspecific MMP activity was determined via azocoll digestion in organomercurial-activated medium. Effects of glucosamine on MMP mRNA concentration in similarly treated chondrocytes were determined by northern blot hybridization with MMP-1, -3, and -13 probes. Statistical analyses were performed with 2-way ANOVA. RESULTS: Glucosamine had no effect on activated MMP activity but inhibited MMP protein expression, as determined by azocoll digestion (glucosamine, 3 to 50 mM) and MMP-13 ELISA (glucosamine, 1.5 to 50 mM). Resting mRNA concentrations for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed to glucosamine at concentrations of 50 and 25 mM than those of positive controls. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine appears capable of pretranslational, and possibly also translational, regulation of MMP expression; data suggest a potential mechanism of action for chondroprotective effects of this aminomonosaccharide.