Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts.

Lewis rats given total lymphoid irradiation (TLI) accepted bone marrow allografts from AgB-incompatible donors. The chimeras showed no clinical signs of graft-versus-host disease. Skin allografts from the marrow donor strain survived for more than 150 days on the chimeras. However, third-party skin grafts were rejected promptly. Although heart allografts survived more than 300 days in Lewis recipients given TLI and bone marrow allografts, detectable levels of chimerism were not required for permanent survival.

Preparation, characterization, and primate testing of monoclonal antithymocyte globulin.

Monoclonal antibodies specific for T cells from both the human and rhesus primate species were detected by their ability to inhibit T cell rosette formation with sheep erythrocytes. The antibodies were shown by fluorescence techniques to react with all thymocytes and peripheral blood T cells but not to B cells, monocytes, platelets, or erythrocytes. Rosette inhibition titers of these antibodies were 30-fold lower when rhesus, rather than human T, cells were used as the rosette-forming cell in assay. Nonetheless, two monoclonal antibodies, of the IgG3 isotype, termed antithymocyte monoclonal (ATM) e.1 and 2.2, were shown to depress selectively circulating T cells to nondetectable levels following single dose administration to rhesus primates and to prolong skin allograft survival in a rhesus primate given a 6-dose course of treatment. The rhesus primates suffered no ill effects and no peripheral blood cellular component other than T cells was depressed. Monoclonal antibody secreting hybridoma cells are capable of producing ATM 3.1 and 3.2 in quantity when grown as peritoneal tumors in selected mouse hybrids. Purification of ATM 3.1 or 3.2 is easily accomplished by affinity chromatography on protein A. These properties suggest that ATM antibodies may become useful immunosuppressive agents in clinical transplantation.

Use of rabbit antithymocyte globulin in cardiac transplantation. Relationship of serum clearance rates to clinical outcome.

Serum rabbit globulin (RG) clearance rates were determined in 30 consecutive cardiac transplant recipients by radioimmune assay of serum RG levels after completion of an initial postoperative course of rabbit anti-human antithymocyte globulin (RATG). Twenty patients, who exhibited rapid RG elimination rates (average half-life, 1.6 days), had a rejection onset time of 16.2 days rejection frequency of 3.9 episodes/100 patient days, and a 1-year survival rate of 59%, respectively, as compared with 28.3 days, 1.9 episodes/100 patient days, and 80%, respectively, for the 10 patients with more prolonged initial RG elimination rates (average half-life, 11.4 days); Nineteen patients received one or more repeat courses of RATG. In 16 of these a progressive increase in RG half-life during subsequent RATG administration could be demonstrated. A close correlation was observed between total RATG doses given in the initial course and peak serum levels of RG obtained (r = 0.82) and between onset of rejection and initial t1/2 RG (r = 0.69). This latter correlation was improved by the elimination of one of the 30 patients (r = 0.81) or by considering only those patients treated from a single RATG batch (r = 0.85; n = 15). No significant relationship was detected between any of the parameters assayed and (1) total RATG dose, or (2) rosette inhibition titers of RATG administered. Survival and rejection parameters of the first 30 patients receiving RATG were compared with the previous 20 receiving equine antithymocyte globulin; these 50 comprising the entire population in which rejection was confirmed by cardiac biopsy. Rejection onset was 20 versus 12 days, rejection frequency was 3.1 versus 5.0 episodes/100 patient days, and graft survival at 1 year was 66 versus 41% for the RATG-equine antithymocyte globulin-treated patients, respectively. From these data it was concluded that (1) RATG administration favorably affects transplantation outcome; (2) RATG half-life, as reflected by RG clearance rates, was the most important variable governing RATG effectiveness, (3) variation in rosette inhibition titers within RATG batches made in the same fashion from large rabbit pools were of minimal clinical importance; and (4) monitoring of serum RG levels provided a necessary and rational basis for effective modulation of immunosuppressive therapy.

Radioimmune assay of heterologous serum gamma-globulin in patients receiving rabbit antihuman thymocyte globulin.

A radioimmune assay (RIA) method for detecting heterologous serum rabbit gamma-globulin (RG) and antibody to this protein is described. The methodology is used for monitoring serum levels of rabbit globulin in patients receiving rabbit ATG (RATG). In 7 cardiac recipients receiving RATG, maximum serum levels of RG were achieved 1-3 days after administration of final dose. RG half-life subsequent to peak serum levels was rapid (X = 36 hr) in 4 patients and prolonged (X = 18 days) in 3 patients. Patient antibody to rabbit gamma-globulin was detectable only in those patients with short RG half-life. Antirabbit antibody titers in these patients were extremely low and barely detectable by RIA.

Cardiac allograft survival in rhesus primates treated with combined total lymphoid irradiation and rabbit antithymocyte globulin.

Eighteen abdominal heterotopic cardiac allografts were performed in outbred rhesus primates. For immunosuppression seven animals received six 100-rad/day total lymphoid irradiation (TLI) doses the week preceding transplant and three 3-mg/kg i.m. rabbit antithymocyte globulin (RATG) doses on postoperative days -1, 0, and +1; five animals were given this RATG dose but no irradiation; three were given TLI alone; and three were given no immunosuppressive therapy. Circulating T lymphocyte counts were monitored in all animals (rosettes). Graft survival in the combined TLI-RATG therapy group (169 +/- 15 days) was significantly greater than in untreated (11 +/- 1 days), RATG alone (22 +/- 12 days), or TLI alone (38 +/- 6 days) treated animals (P less than 0.001, 0.0001, and 0.001, respectively). The animals receiving combined TLI-RATG therapy also achieved significantly greater and more prolonged T lymphopenia than that obtained in the other three groups. Six of seven cardiac allografts placed in animals receiving TLI-RATG therapy were removed electively before cessation of electrical activity; however, in four of these rejection pathology was noted. Thus, it seems that combined TLI-RATG therapy may be of benefit in the management of transplant recipients, but its use will probably not abolish these patients' requirements for immunosuppressive maintenance measures.

Clinical heart-lung transplantation.

Combined heart and lung transplantation was carried out in thirteen patients at Stanford University between March 1981 and May 1983. The recipients were between 22 and 45 years old. All patients were suffering from end-stage pulmonary hypertension; nine patients had Eisenmenger's syndrome; the remaining four were transplanted for primary pulmonary hypertension. Three patients died within one month of surgery. The remainder are well at between 22 months and three weeks from operation. The duration of stay in the hospital for the surviving patients ranged from 38 to 85 days. The immunosuppressive protocol has been essentially the same for all recipients, and has consisted of cyclosporine with an initial course of rabbit antithymocyte globulin (RATG) with azathioprine given for the first two weeks, and then replaced with prednisone. Rejection, as diagnosed by cardiac biopsy, was treated with pulses of methylprednisolone. Early complications included bleeding that necessitated reexploration (five patients); damage to the vagus, recurrent laryngeal, or phrenic nerves (three patients); and failure of the donor lungs (one patient). Modifications of technique that have developed include removal of the recipient heart and lungs separately, and preservation of the lungs with a modified Collins' solution instead of a cardioplegic solution. The results of this operation are considerably superior to clinical efforts in lung transplantation. The combined operation may be preferable for the following reasons: All diseased tissue is removed, thus eliminating recurrent infection, and also perfusion/ventilation disparity. Transplantation of the entire heart and lung block preserves coronary-tracheal vascular anastomoses and makes airway dehiscence less likely. Diagnosis of rejection by cardiac biopsy seems to be a satisfactory method of diagnosis and treatment of pulmonary rejection.

Survival of primates following orthotopic cardiac transplantation treated with total lymphoid irradiation and chemical immune suppression.

Fractionated total lymphoid irradiation (TLI) has been used for attempts at induction of a donor-specific tolerant-like state in allograft recipients and for immunosuppressive effects. Cyclosporin A (Cy A) has been shown to suppress rejection of organ grafts in many species including man. The present study was designed to test the effectiveness of TLI in combination with either CY A or rabbit anticynomolgus thymocyte globulin (ATG) and azathioprine. Thirty-one orthotopic cardiac allografts were performed using surface cooling and total circulatory arrest in outbred cynomolgus monkeys. TLI was administered preoperatively in fractions of 100 rad until a total of 600 or 1800 rad was achieved. Cy A was administered 17 mg/kg/day. All treatment groups demonstrated extended survival. Myocardial biopsies as early as 4 weeks were consistent with mild rejection in all treatment groups. No significant synergistic effect upon survival could be demonstrated utilizing TLI plus Cy A when compared with using Cy A alone. TLI (1800 rad) plus ATG and azathioprine was associated with a high incidence of early death attributable to leukopenia and infection. Cy A alone or in combination with TLI was associated with the development of lymphoid malignancy.

Heart and lung transplantation: autotransplantation and allotransplantation in primates with extended survival.

Combined heart and lung transplantation was performed in rhesus or cynomolgus monkeys in order to confirm the ability of primates to withstand complete cardiopulmonary denervation, to develop a satisfactory operative method, and to obtain survival of allotransplant recipients using Cyclosporin-A immune suppression. Twenty-seven monkeys weighing 2.6 to 10.1 kg received either autotransplants or allotransplants by two different operative techniques. Seventeen animals were operated upon with hypothermia and circulatory arrest. One autotransplant recipient is alive at 368 days, but all allotransplant recipients (untreated) died within 5 days despite normal breathing patterns. Ten animals operated upon with the aid of cardiopulmonary bypass fared better. Three autotransplant recipients are alive 60, 199, and 312 days postoperatively. Three of seven allotransplant recipients treated with Cyclosporin-A (25 mg/kg, then tapered) and azathioprine (2 mg/kg for 14 days) were long-term survivors. One died at 144 days of lymphoma and two are currently living 156 and 191 days postoperatively. The results suggest that heart and lung transplantation is possible in primates and that allografted recipients can survive for extended periods with Cyclosporin-A used for immune suppression.

Successful retransplantation of the human heart.

Cardiac retransplantation has been performed in five patients at Stanford University Medical Center. Long-term survival and rehabilitation have been achieved in two cases. In the first case retransplantation was performed 57 days after the initial procedure because of persistent acute graft rejection. The second patient underwent retransplantation 27 months postoperatively because of documented accelerated graft atherosclerosis. The major indications for cardiac retransplantation consist of intractable acute rejection and late postoperative graft atherosclerosis. These complications should prompt consideration of cardiac retransplantation in carefully selected cases.

Current expectations in cardiac transplantation.

Survival after cardiac transplantation has improved significantly over the past 3 1/2 years at our Center as compared to previous experience (Fig. 5). Currently, survival rates for 60 patients who have had heart transplantation since late in 1973 (program year mean survival +/- S.E.) are 66 percent (+/- 6.6 S.E.), 63 percent (+/- 7.0) and 58 percent (+/- 8.2) 1, 2, and 3 years after operation, respectively. In this report the major reasons for this successful trend have been summarized. These consist of transvenous endomyocardial biopsy for diagnosis and management of graft rejection, use of RATG, immunologic monitoring for early detection of impending rejection, and cardiac retransplantation in selected cases. The present expectations for survival and rehabilitation after heart transplantation are fully comparable to the current results of renal transplantation from unrelated donors. These considerations support the inclusion of cardiac transplantation as a realistic therapeutic alternative in the management of patients with advanced heart disease irremediable by standard forms of treatment.

Cardiac transplantation in perspective for the future. Survival, complications, rehabilitation, and cost.

Two hundred twenty-seven cardiac transplant procedures have been performed in 206 patients from January, 1968, to April, 1981. Postoperative survival rates, calculated by the actuarial method for program years 1968 to 1973 (66 patients), are 44%, 33%, 27%, 21%, and 18% at 1, 2, 3, 4, and 5 years after transplantation, respectively. Postoperative survival rates for program years 1974 to 1981 (140 patients) are 63%, 55%, 51, 44%, and 39% at 1, 2, 3, 4, and 5 years after transplantation, respectively. This increase results primarily from improvement in survival achieved in the first 3 postoperative months (59% +/- 7%, 1968 to 1973, versus 80% +/- 40%, 1974 to 1980), reflecting improved patient management. Infection remains the primary cause of death following transplantation (76/131 patients, 58%), followed by acute rejection (24/181, 18.3%), graft arteriosclerosis (14/131, 10.7%), and malignancy (6/131, 4.6%). The development of graft arteriosclerosis has been examined in 85 one-year survivors studied by annual coronary arteriograms. Coronary lesions of varying severity have developed in 21 patients. HLA-A2 incompatibility was associated with a higher incidence of graft arteriosclerosis than was apparent for all other A locus incompatibilities (p less than 0.0003). Lymphoma has been shown to be associated with younger recipient age, a primary disease diagnosis of idiopathic cardiomyopathy, and retransplantation. One hundred six patients have survived at least 1 year after transplantation; 97% were in NYHA Class 1 at that time interval and 82% returned to employment or activity of choice. The longest survival time is new 11 years, 3 months. Cardiac transplantation can be considered "reasonable and therapeutic treatment to extend life" in selected individuals.

Aortic valve allografts for mitral valve replacement.

Between May, 1967, and April, 1971, 122 patients underwent mitral valve replacement with fresh aortic valve allografts mounted on rigid support rings. The operative mortality rate was 6.6 percent. Current evaluation was obtained on all patients; the average postoperative follow-up interval for surviving patients is 4.8 years (range, 3.3 to 7.1). Survival rates 1, 2, and 5 years after mitral valve replacement are 89, 86, and 71 percent, respectively. The average functional class of 90 current survivors is 1.6, as compared to 2.9 preoperatively. Thirty-six thromboembolic episodes have occurred in 28 patients, generating a thromboembolism rate of 7 percent per patient year of analysis. Allograft valve dysfunction has occurred in 64 patients, requiring reoperation in 16 and causing death in two. The linearized valve dysfunction rate is 13 percent per patient year. Pathological examination of recovered allograft valves revealed predominantly leaflet fibrosis and calcification, acellular collagenous valve matrix, and infiltration with chronic inflammatory cells. The results of this long-term analysis indicate that mitral valve replacement with fresh aortic allografts provides significant functional improvement and an acceptably low rate of thromboembolism. However, the time-related rate of allograft valve dysfunction is unacceptably high and does not justify further clinical use of this type of bioprosthesis.

Human heart transplantation: current status.

The overall survival rate for 97 heart transplant recipients operated on from 1968 to 1975 has been 49% at one year and 23% at five years. Progressive improvement in one-year survival has been achieved, from 22% in 1968 to 62% in 1974. The major factors responsible for increasing survival are better understanding and management of acute and chronic rejection. Current results suggest that heart transplantation deserves wider application in the treatment of selected patients with end-stage myocardial insufficiency.

Diagnosis and treatment of acute cardiac allograft rejection.

Integration of data derived from immunologic monitoring techniques and endomyocardial biopsy currently permits more precise administration of immunosuppressive medications for the treatment of acute cardiac allograft rejection than was formerly possible, resulting in a substantially improved outlook for long-term survival. Indeed, the probability of survival for 5 years postoperatively of 50% exceeds that for several categories of cardiac patients currently undergoing other more common forms of heart surgery. The continuing demonstration of the potential for cardiac transplantation clearly warrants further application of this procedure for the treatment of appropriate patients with end-stage cardiac disease.

Use of rabbit anti-human thymocyte globulin in cadaver kidney transplantation. Preliminary experiences.

Rabbit anti-human thymocyte globulin was given as an adjunctive immunosuppressive agent to 23 recipients of cadaver kidneys in the immediate 14 day post-transplantation period. A drastic reduction in the number of T-lymphocytes in the peripheral blood was recorded and there was a very low frequency (13%) of early, acute rejection episodes. Two to 8 months after transplantation 13 of 14 patients (93%) below 55 years of age survive with a functioning graft. Among 9 older recipients, the patient and graft survival was 56 and 33% and 4 of the patients died with septic complications. It is concluded that the elderly patients were over-immunosuppressed and we will no longer use RATG in this patient category.