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Antiinflammatory Effect of Locally Synthetized scFv-CH2CH3 Anti-C5 Induced By Transfection of Specific Gene

Posted on: Thursday, 17 June 2004, 06:00 CDT

We have recently obtained evidence for an important role played by TCC in the development of antigen-induced arthritis in rat and for the therapeutic effect of a human scFv anti-C5 that recognized the cleavage site of the C5 convertase on C5 from human and animal source including rat. The aim of this study was to evaluate the intraarticolar (i.a.) synthesis of a human ricombinant antibody against C5 after transfection with liposome-plasmid DNA complexes and to evaluate the protective role of the locally synthesized antibodies in the control of antigen-induced arthritis. To this end, a recombinant antibody form (minibody) was produced by genetic fusion of antiC5 scFv to rat IgG2b CH2-CH3 domains in pcDNA3 expression vector. This construct was transfectecl with liposomes (DMRIE-C) injected i.a. to a final concentration of 6g of DNA/50l. Initially, we evaluated the time of persistence of the cDNA and its product into the synovial tissue and joint cavity in a group of 6 healthy Wistar male rats using PCR, ISH and ELISA. Control rats received liposomes alone. In rats receiving plasmid DNA-liposomes, scFv-CH2-CH3 DNA and protein presence were revealed in the synovial membrane and in the synovial washing respectively up to 7 days following liposome injection peaking at the 3rd day. We were unable to detect measurable levels of the minibody anti-C5 in the sera of treated rats and failed to reveal DNA in lung, liver and kidney. The effects of the anti-C5 transfection was then evaluated in a model of BSA-incluced arthritis. Rats received DNA-liposomes in the right knee joint and liposomes alone in the controlateral joint three days before the i.a injection of the antigen and sacrificed 60 hours after the onset of arthritis. As expected, DNA was detected in synovial membrane of the right knee. Conversely, we failed to detect free minibodies able to bind to C5 in the synovial washing by ELISA. The minibodies were revealed instead by Western Blotting, suggesting their presence in the joint cavity as immunecomplexes. Locally produced anti-C5 minibodies was associated with a significantly reduced number of PMN both in the synovial membrane and in synovial washing fluid and also with marked decrease in the level of TNF-a. Considering that liposomes preferentially localize in inflamed joints, our data indicate that liposomal transfection may represent an alternative strategy to treat arthritis promoting local production of anti-C5.

F. Fischetti1,2, P. Macor1, F. Ziller2, P. Durigutto1, D. Sblattero3, R. Marzari3, F. Tedesco1

1 Department of Physiology and Pathology, University of Trieste, Trieste, Italy; 2 Department of Medicine and Neurology, University of Trieste, Trieste, Italy; 3 Department of Biology, University of Trieste, Trieste, Italy

Copyright Edizioni Minerva Medica Mar 2004

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