An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes.

The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701).

The proliferation of rabbit synovial cells, 3T3 cells, or simian virus-transformed 3T3 cells in cell culture was inhibited by the addition of hyaluronate to the culture medium. This effect was markedly dependent on the molecular weight and concentration of the hyaluronate. At the molecular weight and concentration of hyaluronate present in normal synovial fluid, proliferation was inhibited. At lower molecular weights or concentrations, as found in rheumatoid synovial fluid, hyaluronate was significantly less inhibitory. Thus, the changes in synovial fluid hyaluronate that are associated with arthropathies may contribute to a favorable environment for rheumatoid pannus expansion.The viscosity of synovial fluid from the rheumatoid joint is usually considerably less than that of normal synovial fluid, because of a decrease either in the degree of polymerization or in the concentration of hyaluronate (HA) in rheumatoid synovial fluid (1-4). A recent study suggests that the more important of these 2 factors is the decreased concentration of HA (4). Synovial cells, which are normally quiescent, are in constant contact with the HA of synovial fluid. In rheumatoid arthritis, these cells proliferate and con- In this study, we examined the effect of HA on proliferation of synovial and other cells in culture and found it to be inhibitory. However, our results indicate that the effect of HA on cell proliferation is markedly dependent on both the molecular weight and the concentration of HA in the cell culture medium. High molecular weight HA at high concentrations, as found in normal synovial fluid, is a potent inhibitor of cell division, whereas at lower molecular weights or at lower concentrations, as found in rheumatoid synovial fluid, it is less inhibitory and, in some cases, is even stimulatory . Therefore, alterations in either the concentration or the degree of polymerization of the HA of synovial fluid may contribute to the pathophysiology of rheumatoid arthritis by facilitating proliferation of the pannus. MATERIALS AND METHODSCells and culture procedures. Rabbit synovial fibroblast lines were obtained from Dr. C. Biswas (Tufts University School of Medicine, Boston, MA). These cells had been passaged in culture for approximately 2 years and had retained a constant morphology and the ability to produce large amounts of hyaluronate. The simian virus 40-transformed 3T3 (SV-3T3) and 3T3 cell lines were those used previously (5). All cells were maintained in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Gibco), 100 units of penicillin per ml, 100 pg of streptomycin per ml, and 2.5 p g of fungizone per ml. The cells were grown and maintained in

Plasma levels of hyaluronate (HA) in patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA), measured by enzyme‐linked immunosorbent assay, were compared with levels in a healthy, age‐matched non‐arthritic control group, in a retrospective study. Compared with the controls, the mean level of plasma HA was sevenfold higher in the RA group and twofold higher in the OA group. There was no statistically significant correlation between HA levels and 7 other clinical and biochemical parameters in patients with RA. In the OA group, however, plasma HA levels were found to correlate with an objective functional capacity score and with an articular index based on the total amount of cartilage in involved joints. In a retrospective longitudinal study of 6 patients with RA, plasma levels of HA did not show a significant correlation with plasma levels of elastase or with the erythrocyte sedimentation rate. These data support in part the contention that plasma HA may be unique as a marker, in that it may be a reflection of synovial involvement and inflammation, rather than only of inflammation, in arthritis.