Furthermore, the survival of cardiac allografts was moderately prolonged (MST = 27 98 days; group 12, = 6) when the mixture of donor BMCs (1 108/rat) and SPLCs (5 107/rat), combined with a single dose of recombinant CTLA4Ig (05 mg/rat), was given 2 days after cardiac transplantation. while promptly rejecting third-party Stigmastanol skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts. Introduction Currently, organ transplantation is the optimal treatment for patients with end-stage organ failure. Outcomes have improved considerably owing to improvements in immunosuppressive protocols and follow-up care. Nevertheless, strong transplantation tolerance remains an elusive goal. Standard lifelong immunosuppression for transplant recipients often fails to prevent chronic graft rejection and may be accompanied with many severe side-effects, including recurrent opportunistic infections and increased incidence of malignancies and metabolic disorders. Clearly, the alternative approach of deliberately inducing strong donor-specific tolerance while preserving immunocompetence would obviate the unwanted side-effects of standard immunosuppressive therapies and YWHAB provide an alternative to lifelong therapy. Since the early 1990s, T-cell costimulatory blockade has proven sufficient to prolong the survival of various vascularized organs and cell transplants in different rodent and primate models.1C4 CTLA4Ig, a recombinant fusion protein containing the extracellular domain name of cytotoxic T-lymphocyte antigen-4 (CTLA4) fused in-frame with a site-mutated immunoglobulin G1 (IgG1) Fc fragment, efficiently disrupts the reaction of CD28 and B7, resulting in T-cell hyporesponsiveness both and = 5) or day 100 (= 5) immediately after heart transplantation. Irradiated recipients (= 5) without cell transfer were used as controls. Assessment of GVHDRecipient rats were weighed at the time of cardiac transplantation and at 7-day intervals thereafter. Clinical assessment of GVHD was based on the characteristic appearance of GVHD in rats, including: unkempt appearance; hair loss; diarrhoea; rash on paws, snout and skin; and failure to thrive.32 Clinical GVHD was graded as absent, mild or severe. When the rats were killed, Stigmastanol sections of skin and tongue were stained with haematoxylin and eosin (H & E) and examined for the presence of dermal lymphoid infiltration, subepidermal cleft formation, or loss of epidermis, indicative of GVHD.33 Cardiac histologyThe heart graft was excised, fixed in 4% paraformaldehyde until processed, and embedded in paraffin. Tissue sections (5 m) were cut and stained using H & E. Each histology specimen was examined by a single histologist who was blind to Stigmastanol the treatment modality. Statistical analysisResults were expressed as the mean standard deviation (SD). Statistical significance was determined by either the MannCWhitney = 6) in each of groups 1C5, which were not treated with AdCTLA4Ig, rejected DA cardiac allografts within 10 days (Table 1), while LEW recipients (= 6) in group 6 accepted the allografts that survived with a mean survival time (MST) of 457 124 days. When a low dose of either donor BMCs (group 7) or donor SPLCs (group 8) was added to the AdCTLA4Ig protocol, the MST of the cardiac allografts was 482 132 days (= 6) and 468 127 days (= 6), respectively. These MSTs were not statistically different from that of group 6 ( 005). Interestingly, AdCTLA4Ig, combined with a low dose of both donor BMCs (1 108/rat) and SPLCs (5 107/rat), substantially improved cardiac allograft survival in LEW recipients (MST 200 days, 0001; group 9, = 6). However, the MST of cardiac allografts was 1443 246 days (group 10, = 6) when the dose of donor BMCs in the combination was reduced to 5 107/rat, and the MST of cardiac allografts was 1028 372 days (group 11, = 6) when the dose of donor SPLCs in the combination was reduced to 1 1 107/rat. Furthermore, the survival of cardiac allografts was moderately prolonged (MST = 27 98 days; group 12, = 6) when the mixture of donor BMCs (1 108/rat) and SPLCs (5 107/rat), combined with a single dose of.