Objective. To investigate the efficacy of oral type I1 collagen in the treatment of early rheumatoid arthritis (RA).Meth&. Ninety patients with RA (disease duration 5 3 years) were treated for 12 weeks with oral bovine type II collagen at 1 mglday (n = 30) or 10 mglday (n = 30) or with placebo (n = 30), in a double-blind randomized study.Results. There was no significant difference between the 3 groups in terms of response to treatment. However, we observed a higher prevalence of responders in the type I1 collagen-treated groups: 7 responders in the 10-mg type I1 collagen group and 6 in the l-mg group, versus 4 in the placebo group. Furthermore, 3 patients in the 10-mg type I1 collagen group and 1 patient in the 1-mg type I1 collagen group, but no patients in the placebo group, had very good response.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase.Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase.The progelatinase -TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.The tissue inhibitor of metalloproteinases (TIMP-1) is a glycoprotein with a molecular mass of about 30 kDa, purified initially from culture medium conditioned by normal skin fibroblasts, and later from various human sources, including plasma, amniotic fluid and synovial fluid [l -71.The TIMP-1 gene has been localized to the x chromosome [8]. The secreted protein consists of 184 amino acids and possesses six disulfide bonds and two glycosylation sites containing N-linked oligosaccharides [5, 91.TIMP-1 specifically inhibits the extracellular-matrix-degrading metalloproteinases such as collagenase, gelatinase and stromelysin. It has no activity on mammalian membrane metalloproteinase, procollagen peptidase, or on bacterial metalloproteinases such as thermolysin [lo].Originally it was thought that TIMP-1 only binds to active enzymes and not to latent ones. However, it was reported recently, that the 72-kDa progelatinase secreted by several cell types, such as Simian-virus-40-transformed human lung fibroblasts, Harvey murine sarcoma virus-transformed human bronchial epithelial cells, human A2058 melanoma cells and normal skin fibroblasts, exists in a stable, but noncovalent 1 : 1 stoichiometric complex with a 24-kDa inhibitor of metalloproteinases called TIMP-2 [ l l , 121. In addition, a 92-kDa progelatinase -TIMP-1 complex is secreted by human lung fibroblasts [13]. This proteinase is normally produced by macrophages and polymorphonuclear leukocytes (PMNL). PMNL do not produce a tissue inhibitor of metalloproteinases, which may account for its 10-fold-higher specific activity 1131.We found human rheumatoid synovial cells to secrete not only the 72-kDa-progelatinase -TIMP-2 complex and TIMP-1, but in addition free 72-kDa progelatinase. In this communication, we show that TIMP-2, although complexed with the progelatinase, still remains an inhibitor of metalloproteinases. MATERIAL AND METHODS Cell cultureHuman rheumatoid synovium obtained by surgery was washed in Hank's solution and freed from fat and cartilage, cut into pieces (approximately 1 mm3 in size), washed with Hank's solution again, and distributed into 75-cmZ culture flasks. 8 ml Dulbecco's modified Eagle's medium containing ...

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