Leopold Muller Arthritis Research Centre, Robert Jones and Agnes Hunt Orthopaedic and District Hospital, Oswestry, Shropshire, UK
Correspondence to:
J. Middleton.
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Abstract |
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Methods. The concentrations of 3-B-3 and 7-D-4 in sera were quantitated by immunoassays.
Results. The levels of 3-B-3 and 7-D-4 were significantly lower in RA than in controls (3- to 30-fold, P<0.001). Changes in 3-B-3 and 7-D-4 were apparent with disease duration. At first presentation, the 3-B-3 concentration was lowest and increased at 12 months (3-fold, P<0.001). This increase was transient since by 24 and 36 months the concentrations were not different to those at first presentation. The level of 7-D-4 was also lowest when the patients first presented at clinic and increased with time at 6 months (2-fold, P<0.001). The increase was more prolonged for 7-D-4, remaining elevated at 12, 24 and 36 months. The lack of correlations of serum 3-B-3 and 7-D-4 with clinical measurements showed that these markers were not prognostic for disease severity.
Conclusions. The levels of 3-B-3 and 7-D-4 differed between RA and control sera, and changed with disease duration. These markers were not prognostic in predicting disease outcome.
KEY WORDS: Chondroitin sulphate epitopes, 3-B-3 and 7-D-4, Early rheumatoid arthritis, Sera.
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Introduction |
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Monoclonal antibodies 3-B-3 and 7-D-4 are specific for chondroitin sulphate and recognize neo-epitopes which may reflect pathophysiological events occurring in RA and osteoarthritis (OA) [5]. In dogs, these epitopes are absent or only weakly expressed in mature cartilage, and occur in very high levels in experimental OA cartilage [6, 7]. In addition, 3-B-3 and 7-D-4 occur in elevated levels in human articular cartilage from RA and OA joints, and in synovial fluid from knees following traumatic injury [3, 8]. It has been suggested that the increased expression of these epitopes may reflect an early reparative response to cartilage damage in which chondrocytes re-commence a high level of matrix synthesis similar to that seen in early development [8].
The aims of the present RA study were (1) to examine the levels of serum 3-B-3 and 7-D-4 to find out whether they are different from controls, (2) to find out whether the concentrations of these epitopes change with disease duration in early RA and (3) whether the serum concentrations of 3-B-3 and 7-D-4 in early RA are prognostic for subsequent disease progression.
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Subjects and methods |
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Control serum was obtained from volunteers employed at the Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, and residents in the local area. These subjects had no previous joint symptoms and were not on any current medication (Table 1).
Immunoassays
3-B-3 concentrations were measured in 51 patients and 7-D-4 levels were measured in the same serum samples from 35 patients. The 3-B-3 and 7-D-4 competitive ELISAs were as described by Hazell et al. [8] with the following modifications: in the 7-D-4 assay, bovine tracheal proteoglycan, kindly supplied by Dr M. Bayliss (Royal Veterinary College, London, UK) was used to coat the plates (3 µg/ml) and was the standard antigen (range 0.0466 µg/ml). In both assays, the peroxidase substrate was tetramethyl benzidine (Sigma) diluted 1:4 in distilled water (100 µl). Colour development proceeded for 10 min and was stopped by the addition of 10% (1.8 M) sulphuric acid (50 µl). Absorbances at 450 nm were measured using a Bio-Tek EL 312 microplate reader.
Standards and samples were assayed in triplicate, the concentration of each epitope was calculated from the equation of the linear portion of the plot of inhibition of binding against concentration. Samples with epitope concentrations above the linear range were diluted with saline and reassayed. At least five samples were assayed in a minimum of four assays for the calculation of intra- and interassay variability. The performance characteristics of the assays are summarized in Table 2.
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Statistical analyses
The significance of differences between groups was calculated using the MannWhitney U-test for unpaired variables with Bonferroni correction for multiple tests. Correlations were calculated using the Spearman rank correlation coefficient or linear regression. The Fisher exact test was used to find out whether a significant proportion of patients showed increased epitope levels with time. P values of <0.05 were considered significant.
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Results |
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The serum concentrations of 7-D-4 in controls and RA are shown in Fig. 1B. As with 3-B-3, the control values were higher than the RA patient values at each time point (P<0.001 in each case). The greatest difference in the medians was between the control and zero month RA (30-fold), and the least was between the control and 6 month RA (16-fold).
The lowest 7-D-4 level was evident when the patients first presented at clinic. At 6 months, there was a 2-fold increase in the median value (P<0.001) and an increase was shown by a significant proportion of patients (86%, P<0.005). At 12, 24 and 36 months, 7-D-4 concentrations, although elevated compared to the zero time point (P<0.02), were not different to the 6 month value.
There were no significant effects of age or gender on the amount of 3-B-3 and 7-D-4 in either the control or RA sera. The concentrations of the two epitopes showed no significant correlation to each other whether considered overall or at individual time points.
The association of levels of 3-B-3 and 7-D-4 in RA sera with clinical parameters was examined by regression analysis. There were no significant associations at any time point between the concentration of either antigen and the concurrent clinical parameters. In order to assess the potential usefulness of serum chondroitin sulphate epitopes as prognostic markers in RA, correlations were performed between zero, 6 and 12 month 3-B-3 and 7-D-4 concentrations and clinical variables at subsequent time points (up to 36 months). No significant correlations were found.
Serum creatinine concentrations did not differ significantly between controls and RA samples. In addition, there were no differences in serum creatinine levels between the various RA time points. These data suggest that the changes in the amounts of 3-B-3 and 7-D-4 with disease duration and in controls are not attributable to variation in renal clearance.
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Discussion |
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The large difference in 7-D-4 levels between control and RA sera is in agreement with that of knee synovial fluid where the 7-D-4 concentration is significantly lower in RA than controls [4]. This similarity suggests that the changes demonstrated in the serum reflect changes occurring in the synovial joints and that the 7-D-4 epitope detected in the serum derives, at least in part, from articular cartilage.
The difference between serum 3-B-3 in RA and controls was also evident, albeit smaller than that of 7-D-4 (3- to 12-fold for 3-B-3 as opposed to 16- to 30-fold for 7-D-4). However, in knee synovial fluid, Belcher et al. [4] found no difference between the concentration of 3-B-3 in RA and controls. The disparity between the reported synovial fluid 3-B-3 levels and those found by us in serum may reflect the fact that synovial fluid analysis monitors changes occurring in one joint, whereas the serum value reflects a contribution from all joints.
At the earliest time point, when patients first present at clinic, the levels of serum 3-B-3 and 7-D-4 were at their lowest, and over the next 612 months the concentrations of both markers increased. Although the changes in marker levels showed no significant association with clinical measures of disease activity and joint destruction, the increase corresponded to the time when the patients were administered second-line, `disease-modifying', drugs and thus may reflect the ability of the therapy to enable some attempt at cartilage repair to occur. In this context, anti-inflammatory drugs are reported to modulate proteoglycan synthesis and the development of cartilage erosions [1517].
Some differences were apparent between 7-D-4 and 3-B-3 with time. 7-D-4 levels increased significantly at 6 months, whereas 3-B-3 increased later at 12 months. In addition, the increase in 7-D-4 was more prolonged, remaining elevated at 12, 24 and 36 months. The increase in 3-B-3, however, was more transient since by 24 and 36 months the concentration had decreased to that of the zero time point. Differences are reported in the expression of the two epitopes in proteoglycans extracted from arthritic and normal human articular cartilage [3]. In addition, in synovial fluid, the difference between RA and control 3-B-3 and 7-D-4 levels varies (as mentioned above, [4]) and variation is reported following traumatic injury [8]. Taken together, these data suggest that subtle differences may exist in the expression and/or turnover of the two chondroitin sulphate epitopes.
It should be noted that serum levels of 3-B-3 and 7-D-4 could be affected by clearance from the lymph. Although this has not been documented in the case of chondroitin sulphate epitopes, it is known that some hyaluronan molecules from joint tissues enter the blood circulation via the lymphatic system [18]. Furthermore, the amount of hyaluronan transported via the lymph in RA patients could be elevated [19]. Disease-modifying drugs could also affect lymphatic activity and therefore influence the epitope levels in the blood.
The initial concentrations of serum 3-B-3 and 7-D-4 did not correlate with clinical data at subsequent time points. Therefore, over the time period studied, these markers do not appear to be useful as prognostic indicators of progressive RA. In this respect, other extracellular matrix constituents such as the aggrecan epitope 846, cartilage oligomeric matrix protein (COMP) and hyaluronan may be more effective [2, 20].
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Acknowledgments |
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References |
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