Karl E. Herbert

Fragmented fibronectin and other synovial fluid proteins in chronic arthritis: their relation to immune complexes

Fibronectin, an opsonic glycoprotein has been shown to exist in fragmented forms in serum and synovial fluid. Some fragments in synovial fluid appear to be polyethylene glycol (PEG) precipitable, suggesting incorporation into immune complexes (IC). PEG precipitation, SDS-PAGE and immunoblotting were used to determine whether PEG precipitable fragments are real or artefactual. Disease specificity of fragmentation and IC incorporation of fibronectin and other proteins were also studied using these techniques. PEG precipitable fragments do not appear to be artefactual, although some fibronectin fragments are cryoprecipitable. Protein fragments showed similar distributions in whole serum and synovial fluid, disease specific differences being confined to PEG precipitates. Rheumatoid arthritis (RA) synovial fluid PEG precipitates displayed the greatest array of fragmented immunoglobulins and fibronectin. No PEG precipitates contained albumin fragments. Protein fragments in IC may impair their effective removal from RA joints. Accumulated IC could lead to tissue damage via complement activation.

Determination of serum cytidine deaminase activity using ion-pair reversed-phase liquid chromatography

A rapid and sensitive assay for serum cytidine deaminase has been developed utilising ion-pair reversed-phase high-performance liquid chromatography. The addition of 1-octanesulphonic acid (OSA) caused the retention of cytidine and uridine to reverse and uridine, the minor component in the assay, to elute first. Cytidine, uridine and allopurinol (internal standard) were separated on a 5-micron Hypersil ODS column using 100 mM ammonium acetate with 1% (v/v) methanol and 1 mM OSA adjusted to pH 5.0. Detection was at 262 nm. Peak areas were linear from 7 pmol to 6 nmol injected (r = 0.99). Intra-assay variation was 7.8% (n = 10) and the correlation with a colorimetric assay was r = 0.78 (p less than 0.001).

Determination of cytidine deaminase activity in synovial fluid by HPLC

A reversed-phase high-performance liquid chromatographic method for the determination of cytidine deaminase activity in synovial fluid is described. Diluted synovial fluid was incubated for 10 min at 56 degrees C with 0.4 mM cytidine. The protein in 5 vol of incubate was then precipitated using 1 vol of trichloroacetic acid (20% m/v) and the substrate, cytidine, and the product, uridine, were determined in the resultant supernatant. These substances were separated by reversed-phase HPLC using 0.05 M potassium dihydrogen orthophosphate (pH 6.5) containing methanol (3% v/v) and were detected at 280 nm. The enzyme activity was determined by measuring uridine formation. The effects of substrate concentration, pH and reaction temperature on uridine formation are described.

Synovial fluid fibronectin fragments: No evidence for a mitogenic effect on fibroblasts

SummaryFragments of bovine plasma fibronectin produced by cathepsin D digestion are reportedly mitogenic for hamster fibroblasts. Rheumatoid arthritis synovial fluid contains many fibronectin fragments, which may contribute to the proliferation of synovial cells. We have therefore investigated the potential of fibronectin fragments to stimulate proliferation of synovial fibroblast-like cells using human material. Affinity-purified human plasma and synovial fluid fibronectin was digested with cathepsin D at pH 3.5 for 0–18 h and proteolysis stopped with pepstatin. A variety of fragments were produced ranging from 50 to 200 kDa when analysed by SDS-PAGE. The proliferative activity of various test preparations was studied using quiescent human skin and synovial fibroblasts. Tests were applied for 24 h to 104 cells and DNA synthesis measured by tritiated thymidine incorporation. Both undigested and peptides of fibronectin consistently failed to stimulate DNA synthesis in fibroblasts at all concentrations tested, compared with a phosphate-buffered saline control. This was in marked contrast to human synovial fluid from either rheumatoid arthritis or osteoarthritis patients, which stimulated DNA synthesis in the same system (P<0.01). Therefore, our data do not confirm the findings of previous studies in which animal materials were used. We can find no evidence that fibronectin fragments play a role in stimulating synovial proliferation in inflammatory arthritis.

The Effect of Aspirin on Blood Cell Nucleotides in Vivo

Erythrocytes from rheumatoid arthritis (RA) patients have significantly lower levels of ATP compared to normals (our unpublished observations; 1093+182nmol/ml in RA compared to 1371+292nmol/ml for controls). These findings have important implications for cell function in RA and not exclusively for erythrocytes. Severe depletion of ATP has been linked to programmed cell death in resting lymphocytes (1), adenosine deaminase deficient nucleated cells (2), and may also play a vital role in the formation of mucosal injury caused by the nonsteroidal anti-inflammatory drugs (NSAIDs) (3). We considered our original observations may relate to NSAIDs which the patients take regularly; salicylates have been known to inhibit cellular ATP synthesis for over thirty years (4). To further investigate this phenomenon we studied changes to erythrocyte nucleotides in normal subjects taking enteric-coated aspirin daily (900mg) for three weeks.