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In Vitro Prevention Of Cell‐mediated Xeno‐graft Rejection Via The Fas/FasL‐pathway In CrmA‐transducted Porcine Kidney Cells

M. Fujino, X. Li, T. Suda, M. Hashimoto, K. Okabe, H. Yaginuma, K. Mikoshiba, L. Guo, T. Okuyama, S. Enosawa, H. Amemiya, T. Amano, S. Suzuki
Published 2001 · Medicine, Biology

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Abstract: Cell‐mediated cytotoxicity may be involved in delayed and/or chronic xenograft rejection in which apoptosis is induced in the grafted cells via the Fas/Fas‐ligand (FasL) and perforin/granzyme pathways. One barrier to the potential use of xeonogenic grafts for humans may be Fas/FasL‐mediated apoptosis, which would be blocked by the gene expression of cytokine response modifier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptosis in an in vitro model of xenograft rejection, using Fas‐expressing non‐primate cells cultured with a soluble recombinant human FasL (sFasL). A recombinant adenovirus vector expressing CrmA (AxCALNLCrmA) was successfully generated with a Cre‐mediated switching system. PK15 cells, derived from a porcine kidney and infected with AxCALNLCrmA and/or AxCANCre at a multiplicity of infection (MOI) ranging from 0.1 to 100, were cultured with human sFasL derived from KFL74.18, a human FasL‐overexpressed cell line. The gene‐expression level of the PK15 cells was confirmed by CrmA‐immune staining. Approximately 70% of the control PK15 cells showed induced apoptosis when cultured with sFasL. In contrast, the apoptosis was dramatically reduced in crmA‐gene‐transduced PK15 cells. The inhibitory effect of apoptosis increased with an increase in the infection dose of AxCANCre. In addition, the activity of caspases 3 and 8 was significantly inhibited in the crmA‐transduced cells. These results indicate that CrmA is an effective gene product for inhibiting Fas/FasL‐mediated apoptosis, which suggests the potential therapeutic use of its gene transduction to protect against graft damage due to delayed and/or chronic xenograft rejection.
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