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Background: The role of apoptosis in the development of graft coronary artery disease (GCAD) is poorly understood. We have previously shown that early overexpression of the anti-apoptotic protein Bcl-2 lessens the development of GCAD. We hypothesized that early inhibition of apoptosis with a caspase-3 inhibitor would also lessen the development of GCAD., Methods: Heterotopic heart transplantation was performed in 4 groups of rats. Donor hearts were pretreated with 50 microg DEVD-CHO, a cell-permeable caspase-3 inhibitor, or vehicle. Recipient animals were pretreated with 1.7 mg/kg intraperitoneal DEVD-CHO or vehicle. Animals were treated with 7.5 mg/kg/d cyclosporine for 10 days to prevent acute rejection. On post-operative day 90, the animals were sacrificed and the transplanted hearts were assessed morphometrically for evidence of GCAD., Results: At 90 days, intimal proliferation was significantly higher in vehicle treated animals than in inhibitor treated animals. Moreover, the percentage of vessels with high-grade occlusion (>50%) was also lower in inhibitor treated animals., Conclusions: Early inhibition of caspase-3 activity with cell-permeable DEVD-CHO lessens the development of GCAD. Caspase-3 inhibition may be a useful strategy for prevention of GCAD in clinical transplantation.

Introduction: Reperfusion injury is an inflammatory cell-mediated response that causes tissue damage immediately following transplantation, and has been implicated in the development of acute and chronic rejection. NF-kappaB is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1 following reperfusion. We hypothesized that treatment with sulfasalazine, a potent inhibitor of NF-kappaB, would decrease adhesion molecule expression, decrease reperfusion injury, and prolong allograft survival in rat cardiac transplants., Methods: Heterotopic rat heart transplants were performed. Donor allografts were treated with saline, sulfasalazine (SSA), or lipopolysaccharide (LPS), a potent inducer of NF-kappaB activity. Reperfusion injury was assessed with cardiac edema (percent wet weight), neutrophil infiltration (MPO activity), and histologic damage (contraction band necrosis). Immunohistochemistry was performed to analyze protein expression. Acute rejection was determined by daily palpation., Results: Treatment with a single 100 mg/kg intraperitoneal injection of sulfasalazine decreased reperfusion injury compared to saline controls (MPO activity, saline: 2.1+/-0.3, SSA: 1.2+/-0.31, P < 0.005; % wet weight, saline 77.6+/-1.1%; SSA 75.8+/-1.0%, P < 0.005; contraction band necrosis, saline: 13.1+/-2.5%, SSA: 6.1+/-3.4%, P < 0.001). LPS administration increased all parameters of reperfusion injury. Treatment with sulfasalazine prior to LPS also decreased reperfusion injury compared to LPS and saline groups. Sulfasalazine treatment decreased ICAM-1 and VCAM-1 protein expression. Administration of 500 mg/kg sulfasalazine increased graft survival to 15.4+/-1.8 days compared to saline (6.8+/-1.4 days, P < 0.005)., Conclusion: Treatment with sulfasalazine is an effective method to decrease reperfusion injury and prolong allograft survival in a rat cardiac transplantation model.

Background: We hypothesized that ex vivo donor allograft transfection with antisense oligodeoxynucleotide (AS ODN) would inhibit the expression of intercellular adhesion molecule (ICAM)-1, an important mediator of T-cell adhesion and costimulation, and therefore suppress acute cardiac rejection., Methods: Hearts were transfected ex vivo with AS, reverse AS ODN, or saline by applying 3 atm pressure for 45 min at 4 degrees C. Grafts were then transplanted into allogenic recipients +/- treatment with leukocyte function-associated antigen (LFA)-1 monoclonal antibody (mAb) (1.5 mg/kg intravenously), cyclosporine (2.5 mg/ kg/day p.o.), or rapamycin (0.025 mg/kg/day intraperitoneally). Reperfusion injury was assessed in grafts harvested at early time points using the myeloperoxidase, %wet weight, and %contraction band necrosis assays; transfection efficiency was assessed using fluorescent microscopy; and efficacy of ICAM-1 blockade was assessed using immunohistochemistry. Other grafts were followed until rejection with donor/third-party skin grafting, adoptive transfer, and interleukin 2 infusion studies in selected recipients., Results: Transfection was highly efficient (fluorescein isothiocyanate-ODN in 48+/-5% of total myocardial nuclei), nontoxic, and reduced the ICAM-1-positive area to 53+/-14% versus having no effect on MHC class I expression (n=4). The incidence of survival >60 days after AS ODN + LFA-1 monoclonal antibody was 75%, significantly higher than other regimens., Conclusion: AS ODN hyperbaric transfection proved highly efficient, effective at ICAM-1 blockade, and induced cardiac allograft tolerance when combined with LFA-1 monoclonal antibody. This highly targeted alteration of allograft immunogenicity may have an important role in future immunosuppressive strategies.

With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipient's immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.

Correlation between the effective refractory period of the conduction system and the histopathologic grade of the ventricle was examined in the rat cardiac allograft during acute rejection. Lewis rats were recipients of intraabdominal heart grafts from brown Norway rats (allogeneic group, n = 42) or Lewis rats (syngeneic group, n = 15). No immunosuppressant was given. The effective refractory period of the conduction system was measured by the programmed atrial extrastimulus method (basic cycle length, 150 msec) just before the time of death. Specimens of the transplanted hearts were examined histopathologically and the histopathologic grade of rejection was scored according to the standardized grading system of the International Society for Heart and Lung Transplantation. The effective refractory period of the allogeneic heart was significantly longer starting on the third day after transplantation (p less than 0.01). The effective refractory period of the allogeneic heart was more prolonged as the severity of rejection increased. The correlation between the effective refractory period (Y) and the histopathologic grade (X) of the allogeneic group was statistically significant (r = 0.955; Y = 10.3X + 81.0; p less than 0.01). The effective refractory period of all syngeneic hearts and allogeneic hearts of postoperative day 1 and 2 were statistically equivalent to the period of native rat hearts (81.0 +/- 2.0 msec; n = 6). The histopathologic grade of the conduction system was the same as that of the ventricle. We conclude that the effective refractory period of the conduction system could be a useful measure to predict the histopathologic grade of cardiac allograft rejection.

New monoclonal antibodies directed to membrane molecules unique to lymphocyte subsets have provided the means to alter the immune response to alloantigens in a more selective fashion. This investigation demonstrates that monoclonal antibody-induced depletion of CD4 helper/inducer T lymphocytes before transplantation of a fully mismatched heart allograft allows permanent engraftment in rats without further immunosuppression. Five adult male ACI (RT1a) rats received cell-depleting doses of the mouse anti-rat CD4 monoclonal antibody, MRC Ox-38, for 1 month before undergoing heterotopic abdominal heart transplantation. No other immunosuppression was administered, and immunotherapy was discontinued the day of transplantation. After all five Lewis rat (RT1(1)) hearts were maintained free of rejection for more than 3 months, a second heterotopic transplant was performed, this time to the femoral vessels, using either fresh Lewis heart allografts (n = 3) or third-party, Brown-Norway (RT1n) hearts (n = 2). Histologic evaluation was performed 3 weeks later and revealed severe rejection of the femoral Brown-Norway grafts with no evidence of rejection in any of the femoral or original abdominal Lewis grafts. These results suggested that limited, pretransplant anti-CD4 immunotherapy allowed permanent engraftment of fully mismatched cardiac allografts in rats and conferred donor-specific unresponsiveness.

The ability of a new cyclosporine (Cs) derivative, (Nva2)-Cs (CsG), to suppress rejection of lung and heart allografts in rats was determined and compared with that of CsA. Left lungs were transplanted orthotopically; hearts were transplanted heterotopically into the abdomen. (Nva2)-Cs was used in three experimental protocols: (1) single or three (Nva2)-Cs injections given to lung-transplanted rats, (2) daily oral (Nva2)-Cs treatment at different doses compared with similar CsA treatments in heart allografted rats, and (3) An 11-day (Nva2)-Cs treatment starting at increasing intervals after transplantation of hearts. (Nva2)-Cs was found to be immunosuppressive, and effective even when the treatment started as late as four days after transplantation. However, (Nva2)-Cs was less effective than CsA in suppressing rejection of lung and heart allografts at low doses. Because (Nva2)-Cs is possibly not nephrotoxic, it might be a useful drug if used in higher doses than CsA or in combination with other immunosuppressive agents.

Background: Graft coronary artery disease (GCAD) limits allograft survival after cardiac transplantation. The objective of this study was to correlate GCAD with the level of immunosuppression in an established allogeneic rodent cardiac chronic rejection model to better understand the mechanisms of GCAD in this system.Methods: Donor PVG hearts were transplanted into the abdomen of ACI rats. Six recipient groups received either 10, 7.5 or 5 mg/kg/day of oral cyclosporine (CsA), for 90 (10mg/90d, 7.5mg/90d, 5mg/90d) or 10 days (10mg/10d, 7.5mg/10d, 5mg/10d; n = 10 all groups), and grafts procured on Day 90. GCAD was assessed by histology for percent luminal narrowing (%LN), percent affected vessels (%AV) and intima/media ratio (I/M ratio). Sections were stained for ED1-positive macrophages and MHC Class II-positive cells.Results: The 10mg/90d treatment group showed significantly reduced GCAD compared with the 5mg/10d treatment group (%LN = 4.3 ± 3.1% vs 39 ± 11.9%, p < 0.05). The 7.5mg/90d group had a reduced %LN and I/M ratio compared with the 5mg/10d group (%LN = 8.0 ± 3.5% vs 39 ± 11.9%, p < 0.05; I/M ratio = 0.06 ± 0.02 vs 0.41 ± 0.14, p < 0.05). There was a trend toward reduction of GCAD with both increasing the dose of CsA as well as the duration of treatment. Continuous treatment with CsA reduced perivascular macrophage and MHC II cell infiltration. Macrophage infiltrates correlated strongly with GCAD (R2 > 0.90, p < 0.01), and MHC II infiltrates showed a weak correlation, although not statistically significant (R2 > 0.56, p = NS).Conclusions: This study further defines the effect of cyclosporine on GCAD in this cardiac transplantation model. In this system, higher dose and longer duration of treatment with CsA markedly reduces macrophage and MHC II infiltration, correlating with diminished GCAD. However, increasing dose and duration of CsA did not completely eliminate the development of GCAD. Non-immunologic factors could contribute to this occurrence. [Copyright &y& Elsevier]