Fachbereich Veterinärmedizin



    Promoter squelching as a tool to downregulate the inflammatory cascade in canine osteoarthritis (2006)

    Rachakonda, S.
    Rai, M. F.
    Manning, K. S.
    Schmidt, M. F. G.
    6th World Congress of the International Cartilage Repair Society
    Prague/ Chech Republic, 07. – 10.12.2006
    Osteoarthritis and cartilage; 14, Suppl. B — S. 135
    ISSN: 1063-4584
    Institut für Immunologie und Molekularbiologie

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    Tel.+49 30 838 - 518 34 Fax.+49 30 838 451 834

    Abstract / Zusammenfassung


    Purpose:<br>Osteoarthritis (OA) characterised by cartilage degradation
    is a severe joint disorder affecting animals and humans
    alike. Canine species provide a useful model to study this disease
    process as it mimics the human form of OA both clinically
    and pathophysiologically. It has been proven that IL-1β and TNFα
    are the inflammatory cytokines that accentuate the disease process.
    The absence of Th2 cells in the inflammatory synovial fluid
    points out that this disorder could be corrected by installing a
    balance between the Th1 and Th2-response. Previous work has
    shown that Interleukin 4 (IL-4) downregulates the inflammatory
    pathway and therefore is a good candidate to combat OA in
    canine species.<br><br
    Methods:<br>Present work focuses on simulating the OA disease
    process in-vitro using canine chondrocytes in monolayer and 3D
    cell culture systems. Our approach is based on dosage regulated
    IL-4 expression under cell culture conditions. To achieve this we
    are using promoter squelching as a method to self regulate
    IL-4 expression involving the canine COX-2 promoter which we
    partially sequenced.
    Results: Our experiments have shown that canine chondrocytes
    can be passaged till the 7th generation without any change in
    morphology. We could also show by qPCR that these cells over
    express inflammatory cytokines such as IL-6, IL-8 and IL-18 in
    comparison to the house keeping gene GAPDH upon exogenous
    stimulation with either human recombinant IL-1β or TNFα.<br><br
    Conclusions: <br>We report on the optimization of our canine invitro
    model for OA therapy which includes IL-1β and TNFα
    synthesis from canine PBMC as well as a reporter assay for
    COX-2 promoter activity for our indigenously designed vector.
    Efforts are on the way to increase expression levels of canine
    IL-4 in cell culture.