Objective. To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands.Methods. TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescenceactivated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-␤ (IFN␤), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays.Results. TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers.

Objective. To study possible mechanisms that mediate induction of the recently described adipocytokine pre-B cell colony-enhancing factor (PBEF) in joints of patients with rheumatoid arthritis (RA), and to analyze whether levels of PBEF correlate with disease severity and whether PBEF itself has the potential to act as a proinflammatory and destructive mediator in RA.Methods. RA synovial fibroblasts (RASFs) and monocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and recombinant human PBEF or were transfected with PBEF expression constructs or with PBEF-specific small interfering RNA. Production of interleukin-6 (IL-6), IL-8, and tumor necrosis factor ␣ (TNF␣) was measured by enzymelinked immunosorbent assay, and expression of matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. PBEF expression in synovial tissue, synovial fluid, serum, and SFs was assessed by immunohistochemistry, in situ hybridization, Western blotting, and enzyme immunoassays. Results. In RASFs, PBEF was up-regulated by

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Objective To evaluate the decrease of cartilage destruction by a novel orally active and specifi c matrix metalloproteinase 13 (MMP-13) inhibitor in three different animal models of rheumatoid arthritis (RA). Materials and methods The SCID mouse co-implantation model of RA, the collagen-induced arthritis (CIA) model in mice and the antigen-induced arthritis model (AIA) in rabbits were used. Results In the SCID mouse co-implantation model, the MMP-13 inhibitor reduced cartilage destruction by 75%. In the CIA model of RA, the MMP-13 inhibitor resulted in a signifi cant and dose-dependent decrease in clinical symptoms as well as of cartilage erosion by 38% (30 mg/kg), 28% (10 mg/kg) and 21% (3 mg/kg). No signifi cant effects were seen in the AIA model. No toxic effects were seen in all three animal models. Conclusion Although several MMPs in concert with other proteinases have a role in the process of cartilage destruction, there is a need for highly selective MMP inhibitors to reduce severe side effects that occur with non-specifi c inhibitors. Signifi cant inhibition of MMP-13 reduced cartilage erosions in two of three tested animal models of RA. These results strongly support the development of this class of drugs to reduce or halt joint destruction in patients with RA.

The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.Despite the existence of efficient vaccines, chronic hepatitis B virus (HBV) infection continues to be a major public health problem worldwide, with more than 350 million chronic carriers. These individuals are at high risk of developing cirrhosis and hepatocellular carcinoma (28). Interferon alpha therapy is only moderately effective and often is limited by dose-dependent side effects (20). The discovery that certain nucleoside inhibitors of human immunodeficiency virus reverse transcriptase, such as lamivudine, also inhibit HBV polymerase has led to the development of these agents for the treatment of HBV infection. Lamivudine has been shown to be highly effective in inhibiting HBV replication (10, 25) and has recently been licensed in many countries for the therapy of chronic hepatitis B. However, analysis of the kinetics of viral clearance during lamivudine therapy revealed that since lamivudine does not completely inhibit viral replication and the rate of clearance of infected cells is slow, prolonged therapy is required for elimination of virus (38). The initial reactions required for the conversion of the incoming relaxed circular (RC) DNA into covalently closed circular (CCC DNA) are still not elucidated, but it can be hypothesized that HBV polymerase (23) and cellular enzymes (2) may be required for this process. CCC DNA serves as the template for viral transcription (46), and its production is regulated and amplified by an intracellular pa...