Therapeutic effect of farnesylthiosalicylic acid on adjuvant-induced arthritis through suppressed release of inflammatory cytokines.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by pronounced inflammation and leucocyte infiltration in affected joints. Despite significant therapeutic advances, a new targeted approach is needed. Our objective in this work was to investigate the anti-inflammatory effects of the Ras inhibitor farnesylthiosalicylic acid (FTS) on adjuvant-induced arthritis (AIA) in rats, an experimental model for RA. Following AIA induction in Lewis rats by intradermal injection of heat-killed Mycobacterium tuberculosis, rats were treated with either FTS or dexamethasone and assessed daily for paw swelling. Joints were imaged by magnetic resonance imaging and computerized tomography and analysed histologically. The anti-inflammatory effect of FTS was assessed by serum assay of multiple cytokines. After adjuvant injection rats demonstrated paw swelling, leucocyte infiltration, cytokine secretion and activation of Ras-effector pathways. Upon FTS treatment these changes reverted almost to normal. Histopathological analysis revealed that the synovial hyperplasia and leucocyte infiltration observed in the arthritic rats were alleviated by FTS. Periarticular bony erosions were averted. Efficacy of FTS treatment was also demonstrated by inhibition of CD4(+) and CD8(+) T cell proliferation and of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17 release. The Ras effectors PI3K, protein kinase B (AKT), p38, and extracellular-regulated kinase (ERK) were significantly attenuated and forkhead box protein 3 (FoxP3) transcription factor, a marker of regulatory T cells, was significantly increased. Thus, FTS possesses significant anti-inflammatory and anti-arthritic properties and accordingly shows promise as a potential therapeutic agent for RA. Its effects are apparently mediated, at least in part, by a decrease in proinflammatory cytokines.

Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.

The hepatitis B virus-trimera mouse: a model for human HBV infection and evaluation of anti-HBV therapeutic agents.

Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. The HBV-Trimera model was used to evaluate the therapeutic effects of human polyclonal anti-HBs antibodies (Hepatect) and of two reverse-transcriptase inhibitors, lamivudine (3TC) and beta-L-5-fluoro-2',3'-dideoxycytidine (beta-L-5FddC). Treatment of HBV-Trimera mice with these drugs effectively reduced both the percentage of infected animals and the viral load in their sera. Treatment cessation resulted in rebound of viral load, indicating HBV replication upon drug withdrawal. These results show that the HBV-Trimera model represents a novel experimental tool for simulating human HBV infection and evaluating potential anti-HBV therapeutic agents.

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.