Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

AbstractMatrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using TNO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable

Continued inhibition of structural damage over 2 years in patients with rheumatoid arthritis treated with rituximab in combination with methotrexate

Background Rituximab inhibited structural damage at 1 year in patients with rheumatoid arthritis (RA) who had had a previous inadequate response to tumour necrosis factor (TNF) inhibitors. Objective To assess structural damage progression through 2 years. Methods Intention-to-treat patients with one post-baseline radiograph (rituximab n = 281; placebo n = 187) received background methotrexate (MTX) and were randomised to rituximab (2 x 1000 mg infusions, 2 weeks apart) or placebo; patients were eligible for rituximab re-treatment every 6 months. By week 104, 82% of the placebo population had received >= 1 dose of rituximab. Radiographic end points included the change in total Sharp score (TSS), erosion and joint space narrowing scores at week 104. Results At week 104, significantly lower changes in TSS (1.14 vs 2.81; p < 0.0001), erosion score (0.72 vs 1.80; p < 0.0001) and joint space narrowing scores (0.42 vs 1.00; p < 0.0009) were observed with rituximab plus MTX vs placebo plus MTX. Within the rituximab group, 87% who had no progression of joint damage at 1 year remained non-progressive at 2 years. Conclusions Rituximab plus MTX demonstrated significant and sustained effects on joint damage progression in patients with RA and a previously inadequate response to TNF inhibitor