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Fachbereich Veterinärmedizin



    Cloning of eq.GM-CSF as an essential factor for equine dendritic cells (2001)

    Mauel, S.
    Commandeur, Uli
    Steinbach, F.
    Jahrestagung der DGfI
    Dresden, 26. – 29.09.2001
    Bandzählung: 204
    Seiten: 317
    ISSN: 0171-2985
    Institut für Virologie

    Robert-von-Ostertag-Str. 7-13
    14163 Berlin
    +49 30 838 51833

    Abstract / Zusammenfassung

    Dendritic cells (DC) play an important role in the modulation of immunity, representing the only antigen presenting cells (APC), able to stimulate naive T cells. DC can be isolated from various tissues or generated from monocytes (MoDC). For generating and in vitro culture, however, eq.GM-CSF and in the case of MoDC eq.IL-4 are necessary cytokines. Both cytokines are considered species-specific, although a recent report indicated hu.GM-CSF to be used in the generation of equine MoDCs. Using degenerated primers, partially delineated from other species, cDNA of the ORFs coding for eq.GM-CSF and eq.IL-4 were generated and sequenced. The coding region of the cytokine was cloned in expressions vectors for bacteria, yeast, mammalian cells and plants. PBM were isolated by adherence from equine blood and stimulated with hu.GM-CSF, eq.GM-CSF and eq.IL-4, respectively. Cloning and sequencing revealed that eq.GM-CSF is distancally related both to hu.GM-CSF and bo.GM-CSF with the homology of 84% for DNA and 82% (with 76% identity) for protein sequence, whereas ungulates among each others have homologies >90%. Interestingly, IL-4 differed significantly both from its homologues and from the sequences published previously. Eq.GM-CSF was expressed in high amounts in bacteria and biologically active in mammalian cells. Using eq.GM-CSF or hu.GM-CSF plus eq.IL-4, we generated cells with the typical morphology and phenotype of dendritic cells from monocytes (MoDC). Eq.GM-CSF was cloned, expressed and characterised. Using eq.GM-CSF or hu.GM-CSF and eq.IL-4, monocyte-derived dendritic cells (MoDC) could be obtained such as described in other species, providing options for immunotherapeutic approaches in the future.