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Objective. To investigate the utility of serum COMP level measurements as a predictor of future damage of the weight-bearing (large) joints in RA patients participating in intensive exercise. Methods. Data of the 281 completers of a 2-yr randomized controlled trial (Rheumatoid Arthritis Patients In Training; RAPIT) comparing the effects of usual care physical therapy with high-intensity weight-bearing exercises were analysed. The primary outcome variable was defined as the change in radiological joint damage (Larsen score) of the large joints. Potential predictors of outcome were defined: baseline and change in serum level of COMP after 3 months, baseline radiological damage of the large and small joints, number of months on glucocorticoids, change in disease activity and in physical capacity (aerobic fitness and muscle strength) after 2 yrs, and participation in the exercise group. Results. In cross-sectional evaluation of baseline data, we found strong association between the high serum COMP level and current damage of the large joints. Serum COMP level at baseline, however, was not associated with an increased rate of radiological joint damage after 2 yrs of follow-up. Furthermore, neither interaction between baseline COMP level and participation in exercises, nor change in COMP level after 3 months of exercising were associated with future damage of the large joints. Conclusion. Neither baseline serum COMP level nor its individual change after 3 months from start of intensive exercise predict longitudinal progression of damage of the large joints in this population. [ABSTRACT FROM AUTHOR]

Objective: To determine whether ascorbic acid might be of benefit for the treatment of spontaneous osteoarthritis (OA) when administered over a long period of time., Methods: We investigated the effects of 8 months' exposure to low, medium, and high doses of ascorbic acid on the in vivo development of histologic knee OA in the male Hartley guinea pig. The low dose represented the minimum amount needed to prevent scurvy. The medium dose was the amount present in standard laboratory guinea pig chow and resulted in plasma levels comparable with those achieved in a person consuming 200 mg/day (5 fruits and vegetables daily). The high dose was the amount shown in a previous study of the guinea pig to slow the progression of surgically induced OA., Results: We found an association between ascorbic acid supplementation and increased cartilage collagen content but, in contrast to findings in a previous study of surgically induced OA in the guinea pig, ascorbic acid worsened the severity of spontaneous OA. Active transforming growth factor beta (TGF beta) was expressed in marginal osteophytes, whose size and number were significantly increased with increasing intake of ascorbic acid. Synovial fluid levels of cartilage oligomeric matrix protein, a biomarker of cartilage turnover, corroborated the histologic findings., Conclusion: Ascorbic acid has been shown to activate latent TGF beta. Prolonged intraarticular exposure to TGF beta has been shown to cause OA-like changes. We found expression of active TGF beta in osteophytes, a prominent feature of the joint histology seen in association with ascorbic acid treatment. Thus, the deleterious effects of prolonged ascorbic acid exposure may be mediated in part by TGF beta. This worsening of OA with ascorbic acid supplementation suggests that ascorbic acid intake should not be supplemented above the currently recommended dietary allowance (90 mg/day for men and 75 mg/day for women).

Objective: Previous descriptions of potential biomarkers of osteoarthritis (OA) have been limited to Caucasians. In the present study, we examined associations between serum levels of cartilage oligomeric matrix protein (COMP) and ethnicity (African American or Caucasian) and sex in the Johnston County Osteoarthritis Project, a population-based study of OA in rural North Carolina., Methods: All African Americans and a randomly selected sample of Caucasians who had available sera and either no radiographic evidence of knee or hip OA according to the Kellgren/Lawrence (K/L) system (K/L grade 0) or radiographic evidence of knee OA (K/L grade 2 or higher) were included. Serum COMP levels were quantified by sandwich enzyme-linked immunosorbent assay, using monoclonal antibodies 16-F12 and 17-C10. Linear regression models were used to assess relationships between serum levels of natural log-transformed COMP (ln COMP) and ethnicity and sex, controlling for age, height, body mass index (BMI), radiographic OA, and the presence of other symptomatic joints. Radiographic OA was defined in separate models as the presence, severity, and laterality of radiographic knee OA, the co-occurrence of radiographic knee and hip OA, and the number of knees and hips with radiographic OA., Results: The 769 subjects in the study sample had a mean +/- SD age of 62 +/- 10.3 years. Levels of ln COMP were associated with age, BMI, and all definitions of radiographic OA (P = 0.0001), and varied by ethnicity and sex. In adjusted models, ln COMP was higher in African American women than in Caucasian women (P = 0.003) and higher in Caucasian men than Caucasian women (P = 0.0001). There were no statistically significant differences in serum ln COMP levels between African American men and women., Conclusion: Serum COMP levels vary by ethnicity and sex. These factors should be considered in the derivation of standards using this, and possibly other, potential biomarkers of OA.

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.

Objective: To examine the cross-sectional relationship between serum cartilage oligomeric matrix protein (COMP) and hip and knee clinical signs and symptoms in a sample of adults without radiographic hip or knee osteoarthritis (OA)., Design: A total of 145 persons with available sera and no evidence of radiographic hip or knee OA (Kellgren-Lawrence grade 0) were randomly selected from the Caucasian participants of the Johnston County Osteoarthritis Project. COMP was quantified by a competitive ELISA assay with a monoclonal antibody 17-C10. Hip and knee clinical signs and symptoms were assessed by physical examination and interview, and their associations with Ln COMP analysed with general linear models., Results: After adjustment for age, gender, body mass index (BMI), and other symptomatic joints, mean Ln COMP was statistically significantly higher among persons with hip-related clinical signs (P=0.018), among those with hip-related symptoms (P=0.046), and among individuals meeting American College of Rheumatology clinical criteria for hip OA (P=0.021). There were no statistically significant associations between any of the knee-related clinical signs and symptoms and Ln COMP., Conclusion: Serum COMP may be useful as a biomarker of pre-radiographic hip joint pathology; its utility as a biomarker of pre-radiographic knee joint pathology is unclear., (Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.)

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.

Damage to the meniscus can lead to posttraumatic osteoarthritis. Early markers of joint injury and tissue disease may be useful in developing and administering clinical treatment. We investigated the effects of total medial meniscectomy on biomarkers measured serially in synovial lavage fluid each month for 3 months. Following meniscectomy in dogs, four biomarkers were evaluated: cartilage oligomeric matrix protein, keratan sulfate epitope (5D4), the 3B3(-) neoepitope of chondroitin-6-sulfate, and the 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate. Meniscectomy led to statistically significant elevations of all four biomarkers, with levels peaking at 4 weeks. By 12 weeks, the level of the 5D4 epitope returned to the preoperative baseline level whereas that of cartilage oligomeric matrix protein, 3B3(-), and 3B3(+) remained above the baseline. Concentrations of these biomarkers in the knees not operated on did not change significantly from the baseline. The levels of cartilage oligomeric matrix protein and 3B3(-) relative to 3B3(+) remained constant in all knees. In contrast, the level of 5D4 relative to 3B3(+) declined over time in the knee operated on but remained constant in the knee not operated on. These results demonstrate a quantitative change in the molecular components of synovial fluid after meniscectomy, as well as a qualitative change evinced by an alteration in the relative proportions of these epitopes. Extensive analyses showed a strong correlation between serum levels of 3B3(-) from the femoral and cephalic veins; however, serum 3B3(-) was not correlated with synovial fluid 3B3(-). These findings support the hypothesis that the concentrations of select cartilage biomarkers in synovial fluid are altered following meniscectomy and are promising tools for objectively monitoring the induction of osteoarthritis in this model system.

Objective: To characterize serum cartilage oligomeric matrix protein (COMP) levels by age and gender for a radiographically defined population free of hip and knee osteoarthritis (OA), and to examine the potential utility of COMP as a diagnostic biomarker for knee OA., Methods: Serum samples and knee and hip radiographs were obtained at a baseline evaluation as part of the Johnston County Osteoarthritis Project, a population-based study of OA in rural North Carolina. A total of 291 Caucasian participants were randomly selected for COMP analysis, 143 patients with radiographic knee OA (Kellgren/Lawrence [K/L] grade > or = 2) and 148 controls with neither hip nor knee OA (K/L grade 0), evenly distributed by age and gender. COMP was quantified by competitive enzyme-linked immunosorbent assay with monoclonal antibody 17-C10. The natural log-transformed COMP data were analyzed using general linear models., Results: Serum COMP levels were significantly elevated (P = 0.0001) in the age > or = 65 group (mean +/- SD 1,302.1 +/- 496.7 ng/ml) versus the age 45-54 and age 55-64 groups (1,058.1 +/- 432.4 and 1,038.6 +/- 313.3, respectively). Serum COMP levels of the OA group were significantly higher than those of the control group (1,208.57 +/- 487.47 ng/ml versus 1,061.83 +/- 370.58 ng/ml; P = 0.0093). Serum COMP levels also increased significantly with knee OA K/L grade (P = 0.0047), knee OA laterality (P = 0.0043), and number of knee and hip joints involved (P = 0.0001). There was no significant difference in serum COMP levels by gender or obesity., Conclusion: We demonstrate that in a population-based sample, serum COMP levels can distinguish an OA-affected subgroup from an unaffected subgroup and can reflect disease severity and multiple joint involvement in OA.

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.

Cartilage oligomeric matrix protein (COMP) is a high-molecular-weight glycoprotein found at a high concentration in articular cartilage. Recent studies have shown that the joint fluid and serum levels of antigenic COMP, measured by an enzyme-linked immunosorbent assay (ELISA) which uses a polyclonal antiserum raised against bovine COMP, provide important information about metabolic changes occurring in the cartilage matrix in joint disease. In this report, we describe the specificity of three monoclonal antibodies (mAbs) to human COMP and their usefulness in quantifying antigenic COMP fragments in body fluids. Two of the mAbs (16-F12 and 18-G3) recognized both oligomeric and monomeric forms of COMP, but the third (17-C10) reacted positively only with the former. Immunoblots of human COMP, predigested with trypsin for up to 6 h, showed that the three mAbs are directed against different epitopes identified on small tryptic fragments of 30 kDa (16-F12), 25 kDa (17-C10), and 40 kDa as well as 30 kDa (18-G3), respectively. The antibodies also recognized a different pattern of fragments in human pathological synovial fluids. This was particularly striking in the case of the medium size fragments (16-F12: 90 and 110 kDa; 17-C10: 70 and 90 kDa; 18-G3: up to five bands from 70 to 130 kDa). Competitive indirect inhibition ELISAs developed with mAbs 16-F12 and 17-C10 revealed further differences in the specificities of these antibodies. Thus, while mAb 16-F12 can be used only to quantify antigenic COMP in human synovial fluid and serum, mAb 17-C10 is useful in addition when analyzing canine and horse synovial fluid as well as canine serum. The results of analyses of synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis provided preliminary evidence in support of the contention that measurement of the different COMP epitopes recognized by these mAbs in body fluids could prove useful in the clinical assessment of patients with joint disease.

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.

Sulfated macromolecules of rat lung tissue were labeled in vivo with 35SO4 and extracted with a solution of 7 M urea containing 0.4% Triton X-100. DEAE-Sephacel chromatography separated sulfated macromolecules into three pools. Pool I consisted of high-molecular-weight, low-density sulfated glycoprotein, probably of mucous secretion origin. Pool II contained a mixture of proteoheparan sulfate and proteodermatan sulfate, together with core protein-free heparan sulfate chains. Pool III was very heterogeneous; its resolution into at least four proteoglycan species was achieved by CsCl density gradient centrifugation. Those included two (high- and low-density) species of proteoheparan sulfate, high-density proteochondroitin sulfate, and medium-density (1.45 less than rho less than 1.55 g/ml) proteodermatan sulfate.

Single intraperitoneal injection of acrylonitrile, administered prior to the start, at the onset, or during oxygen exposure, respectively, in all cases significantly impaired the survival rate of rats exposed to 98% oxygen. Short periods of lung glutathione depletion by acrylonitrile accelerated the manifestation of O2 toxicity regardless of their timing with respect to the start of oxygen exposure, but in dependence on their intensity and duration. However, the effect of acrylonitrile was probably not solely glutathione-depletion-mediated, since O2 toxicity was enhanced even by acrylonitrile injection, given sufficiently in advance to allow the lung glutathione level to recover before the oxygen exposure started.

The review presents a survey of published findings concerning the mechanism of luminol-dependent chemiluminescence in biological systems. The potential of various oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) to react with luminol is discussed. The ability of commonly used enzymes (superoxide dismutase, catalase), inhibitors, and oxygen radical scavengers to discriminate between individual oxygen species is assessed together with the potential of a variety of substances encountered in biological systems to interfere in luminol-dependent chemiluminescence reactions. It is concluded that luminol-dependent chemiluminescence gives at present very little ability to discriminate between individual oxygen or radical species. Furthermore, luminol-dependent chemiluminescence used in biological systems is extremely prone to many interferences, which are very difficult to control.

Sulfated macromolecules occurring in bronchoalveolar lavage fluid supernatant were radiolabelled in vivo by 35SO4, that was insufflated via trachea. DEAE-Sephacel chromatography dissociated sulfated glycoproteins, presumably of tracheobronchial mucus origin, from a minor, but heavily labelled component. Degradative analysis and acetyl cellulose electrophoresis identified this component as a mixture of heparan sulfate and chondroitin sulfate proteoglycans.