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Background: FK778 is a malononitrilamide, a class of immune suppressive compounds with antiviral features and experimental activity in chronic rejection, a potentially interesting combination for organ transplantation. The goal of this project was to study the tolerability, immune suppressive efficacy, and anti-cytomegalovirus (CMV) activity of FK778 and to assess the in vivo relevance of its previously described inhibition of de novo pyrimidine synthesis., Methods: Heart transplants were performed in rats (Brown Norway [BN] to Lewis) and treated with varying doses of FK778 or leflunomide for 28 days. At 28 days, at the time of rejection or at the death of the animal, the allograft and other vital organs were obtained for study by light microscopy and immunohistochemistry. In separate experiments, Lewis rats were given sublethal irradiation, inoculated with rat CMV (Maastricht strain), and treated with varying doses of FK778 and leflunomide. In both the transplant and CMV studies, IP uridine was given at 250 mg/kg to cohorts or animals receiving FK778 and leflunomide., Results: FK778 controls acute rejection and inhibits CMV replication at 20 mg/kg but is toxic at 25 mg/kg. Toxicity is manifested as anemia, changes in hepatic and intestinal histology, and mortality. The toxicity but not the immune suppressive or antiviral efficacy, is reduced significantly by exogenous uridine administration., Conclusion: FK778 has both immune suppressive and antiviral activities, neither of which is entirely dependent on inhibition of pyrimidine synthesis. These, and other published observations, suggest that the antiviral activity and a considerable part of the efficacy of the malononitrilamide family of drugs is attributable to activities other than drug induced pyrimidine deficiency.

Background: In the experiment described here, we investigated the effects of the immunosuppressants FK506 and leflunomide (Lef) on the survival of hamster hearts and liver xenografts in Lewis rats., Methods: Lewis rats were used as recipients of hamster heart or liver grafts using different regimens of FK506 and Lef. Donor-matched heart grafts were transplanted into long-term surviving Lewis rat recipients of hamster xenografts to test donor-specific prolongation of xenograft survival. Hyperimmune, late xenograft rejection, and naive sera were transferred into long-term surviving Lewis rat recipients of hamster heart xenografts to determine whether these sera could inhibit the efficacy of donor-specific long-term survival. Anti-donor-specific antibodies were analyzed by flow cytometry., Results: After a short induction with FK506 plus Lef, maintenance treatment with FK506 alone was sufficient to prolong survival of hamster xenografts. All hamster heart and four of six hamster liver xenografts survived for more than 3 months. Second hamster hearts were permanently accepted by Lewis rats bearing long-term surviving hamster heart xenografts when rats were treated with FK506 monotherapy (mean survival time >60 days, n=4). Long-term surviving hamster heart grafts were rejected after transfer of hyperimmune serum but not late xenograft rejection serum or naive serum. Lef and FK506 significantly reduced the production of anti-donor-specific antibodies in Lewis rats transplanted with hamster liver and heart xenografts., Conclusion: Long-term survival of hamster liver and heart xenografts in Lewis rats could be induced by a regimen of short-term FK506 in combination with Lef followed by FK506 monotherapy. The acquired sensitivity of late xenoreactivity to FK506 reflects primarily a modification in the host immune response to the hamster graft.

Background: Vigorous antibody-mediated responses prevent the successful engraftment of hamster hearts transplanted into Lewis rats. Early antibody responses mediating acute rejection of the xenograft are T cell-independent and resistant to the T-cell immunosuppressant, cyclosporine (CsA). Immunosuppression with the combination of leflunomide plus CsA completely prevents xenograft rejection, but when such immunosuppression is stopped the hamster heart is rejected by a process that we term late xenograft rejection. We report here on some of the immunological features of late xenograft rejection., Methods: Lewis rats transplanted with hamster hearts were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Serum was harvested and the functional activities of the xenoreactive antibodies were quantitated by in vivo passive transfer of sera, flow cytometry, in vitro C3 deposition assays, and Western blotting., Results: CsA alone prevented late xenograft rejection and the accompanying production of xenoreactive antibodies. The xenoreactive antibodies accompanying acute or late xenograft rejection were predominantly IgM, but only serum from rats undergoing acute xenograft rejection was able to induce hyperacute rejection. The ability of serum to induce hyperacute rejection correlated with its ability to induce C3 deposition on hamster lymphocytes in vitro. The repertoire of hamster antigens recognized by IgM in the serum of rats undergoing late xenograft rejection is more restricted than that of IgM in the serum of rats undergoing acute xenograft rejection. We additionally demonstrate that long-term graft survival is not dependent on graft accommodation., Conclusions: These studies demonstrate that a brief treatment with the combination of leflunomide and CsA profoundly modifies the humoral xenoreactivity in the recipient, converting it from a T-independent into a T cell-dependent response. Differences in functional activity of sera from acute or late xenograft rejection suggest that antigenic specificity defines the ability of IgM to induce complement activation and hyperacute rejection.