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Improved survival of heart transplants
Improved survival of heart transplants Graft and/or donor pretreatment with antilymphoblast globulin, cyclophosphamide, or methylprednisolone Survival of heart allografts flushed with antilymphoblast globulin (ALC) was significantly prolonged when the grafts were transplanted into minimally immunosuppressed recipients. When no immunosuppression was given, there was no prolongation of survival of ALC-flushed grafts. Donor pretreatment with cyclophosphamide, methylprednisolone, or ALC had no influence in the prolongation of survival of cardiac allografts. We do not have a clear explanation for the prolonged survival of ALG-flushed grafts transplanted into minimally immunosuppressed recipients.
Luis H. Toledo-Pereyra, M.D., Ph.D., and Richard M. Condie, Detroit, Mich., and Minneapolis, Minn.
In 1973, Callender and his associates 1 demonstrated that the immunogenicity of canine renal allografts could be diminished by perfusion with antilymphoblast globulin (ALG) before transplantation into minimally immunosuppressed recipients. Whether the mechanism of action involved opsonization of the passenger leukocytes or the masking of histocompatibility antigens by nonspecific binding of goat globulin to kidney cells was unknown. Nevertheless, his results suggested that antigen modification might supplement immunosuppression in the prolongation of kidney allograft survival. Toledo-Pereyra," 3 Simmons," and Nowygrod," and their associates obtained similar findings on minimally immunosuppressed recipients that received canine kidneys flushed with concanavalin A (ConA), phytohemagglutinin-M, or chondroitin sulfate. Furthermore, Toledo-Pereyra and his group'":" confirmed the beneficial effect of graft flushing or perfusion with ALG on small bowel, liver, and lung canine allografts that also were minimally immunosuppressed. In this work, From the Department of Surgery, Section of Surgical Research and Transplantation, Henry Ford Hospital, Detroit, Mich., and the University of Minnesota, Minneapolis, Minn. Partially supported by Ford Foundation Grant No. 730-0786. Received for publication Sept. 19, 1977. Accepted for publication Nov. 3, 1977. Address for reprints: Dr. L. H. Toledo-Pereyra, Department of Surgery, Section of Surgical Research and Transplantation, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, Mich. 48202.
536
we further study the modification of graft antigenicity on canine heart allografts flushed with ALG before transplantation, and on canine donor hearts pretreated with ALG, cyclophosphamide, or methylprednisolone prior to transplantation into minimally immunosuppressed recipients.
Materials and methods Adult mongrel dogs of either sex, the donors weighing between 11 and 18 kilograms and the recipients between 17 and 25 kilograms, were anesthetized with sodium methohexital for induction and halothane (0.5 to 2.0 percent) for maintenance. Oxygen was continuously given with ventilation through an endotracheal tube. Through a median sternotomy, the heart was excised in a routine fashion from the donor animal. Before the heart was excised, 1,500 U. of heparin was administered intravenously. The donor dog received 500 ml. of Ringer's lactate during the procedure. The dissection technique for removal of the heart basically consisted of the following: The superior and inferior venae cavae were identified; the azygos vein was ligated with 2-0 Tevdek sutures and divided; the branches of the aorta were also ligated with 2-0 Tevdek sutures and divided. The pulmonary artery was identified and was dissected all the way into the lung parenchyma. As soon as the heart was dissected out completely and good control was obtained from the structures to be ligated, the heart was fibrillated with a direct current of 30 to 60 mAmp. Then, the aorta was
0022-5223/78/0475-0536$00.60/0 © 1978 The C. V. Mosby Co.
Volume 75 Number
Survival of heart transplants
4
537
April,1978
Donor's Heart
Recipient's Heart Ringer's or CPP With/Without
ALG
With/Without Donor Pretreotment
Aorta
Methylprednisolone Cyclophosphamide
ALG
Abdominal Cardiac Transplantation
Groft Flushing
Follow
up/
-Cllnlcol -EKG -Histoloqy
Fig. I. Experimental design of canine heart transplantation. Graft and/or donor pretreatment with antilyrnphoblast globulin (ALG), methylprednisolone, or cyclophosphamide. The heart was flushed withRinger's lactateor cryoprecipitated plasma at 4° C. prior to transplantation into minimally immunosuppressed recipients. cross-clamped in its descending portion and divided. The pulmonary artery was cross-clamped close to the lung hilum and divided. The venae cavae and all the rest of the structures -were divided. The pulmonary veins were ligated individually and divided. No warm ischemia was involved in these experiments. The heart was removed and flushed with 500 to 750 ml. of either Ringer's lactate or cryoprecipitated plasma (CPP) at 4°C. The recipient dog was prepared by means of a median laparotomy incision. A long segment of the abdominal aorta and vena cava were prepared for the vascular anastomoses. The pulmonary artery and aorta of the donor's heart were carefully prepared for the anastomoses, with an adequate segment of both vessels being left in order to avoid any kinking or outflow obstruction after transplantation. Both the aorta-toaorta anastomosis and the pulmonary artery-vena cava anastomosis were performed with 5-0 Prolene sutures. A No. 18 needle was placed in the left ventricular chamber to aspirate all the accumulated air before clamp release. As soon as the vascular clamps were opened, the heart started contracting slowly, and if necessary 1 to 2 C.c. of 10 percent calcium chloride was injected into the left ventricle. If beating did not start spontaneously after 3 minutes of massage or after calcium chloride injection, the heart was defibrillated with direct current (20 to 60 watt-seconds). In some other cases it was necessary to instill diluted (1 : 10) epinephrine (l mg.) directly into the left ventricle to induce
cardiac contractility. The abdomen and heart were checked for hemostasis, the viscera were returned to the normal position, and the abdomen was closed in a routine fashion (Fig. 1). Ringer's lactate, 1,000 c.c. in the first 12 hours, was given intravenously. Water and food were resumed on the second postoperative day. Procaine penicillin, 1,200,000 D., was given intramuscularly each day for 3 days. No blood was transfused into the recipient before, during, or after the operation. Immunosuppression included azathioprine, 5 mg. per kilogram per day for 3 days and then 2.5 mg. per kilogram per day until death or sacrifice. The heart grafts were followed by daily clinical examination with palpation of the graft in the abdominal cavity, daily electrocardiograms, and histologic study when the specimen was obtained. Impending rejection was most accurately diagnosed by palpation of the heart. Electrocardiographically, the R wave progressively decreased in voltage and there were numerous atrial arrhythmias associated with a decrease in voltage. If the daily transplant heartbeat or the electrocardiogram showed either terminal or no activity, the dog was put to death and the heart was removed for gross and histologic examination. The ALG was prepared by immunizing goats with biweekly injections of 108 dog lymph node lymphocytes over a total period of 3 months. After absorption with red blood cells, the antilymphocyte serum initially was purified by precipitation with 40 percent (NH 4 h-
The Journal of Thoracic and Cardiovascular Surgery
53 8 Toledo-Pereyra and Condie
1- RinQers
n-cpp
m-
Donor Methylprednisolone + Cyclophosphamide, CPP m:- Donor ALG + Methylprednisolone,
Cpp
3l- CPP + ALG ::5ZI- CPP + ALG No Recipient Immunosuppression JZJI- Donor ALG + Methylprednisolone, CPP + ALG
100
75
0
>
.~
-~
(/)
c ~ 0:>!!
50
25 0
0
5
10
15
20
25
30
40
35
45
Days Post-Transplantation
Fig, 2. Survival in days of all different groups of heart allografts. The best group was the one flushed with ALG prior to transplantation into minimally immunosuppressed recipients.
Table I. Characteristics of all experimental heart transplant groups Donor pretreatment
Group
I II III
IV V VI VII
5 6 6 5 7
5 6
None None Methylprednisolone + cyclophosphamide ALG + methylprednisolone None None ALG + methylprednisolone
S04 and then was purified over a DEAE-cellulose column. The ALG was sterile and free of pyrogens. Doses of 50 mg. per kilogram of ALG prolonged the life of skin grafts in dogs up to 18 days. Lymphocytotoxicity titers against blood lymphocytes from peripheral vessels ranged from I : 1,000 to I : 54,000. Hemagglutination titers were less than I: 64. The microscopic specimens were classified in four main groups according to the previous classification of Gonzalez-Lavin and his associates": Type I, no vascular involvement and minimal cell polymorphonuclear infiltration; Type II, mononuclear and polymorphonuclear cell infiltration of the myocardium and perivascular accumulation of lymphocytes and monocytes; Type III, mild intimal cell infiltration, intimal thickening of the coronary vessels, fibrinoid necrosis of the media, and severe cellular infiltrate; Type IV, severe endothe-
Graft flush and pretreatment
Temperature
Recipient immunosuppression
Ringer's CPP CPP
40 C. 40 C. 40 C.
Azathioprine Azathioprine Azathioprine
cpp CPP CPP CPP
40 40 40 40
Azathi oprine Azathi oprine None Azathioprine
+ + +
ALG ALG ALG
C. C. C. C.
lial damage of the coronary arterioles, disruption of the myocardial fibers associated with dense infiltration of lymphocytes, lymphoblasts, and plasma cells, and extensive myocardial damage. Seven groups of dogs were included in this study (Table I). Group I (five dogs): The donor animal was not pretreated and the heart was flushed with Ringer's lactate. Group II (six dogs): The donor was not pretreated and the graft was flushed with CPP. Group III (six dogs): The donor was pretreated with methylprednisolone (30 mg. per kilogram) and cyclophosphamide (50 mg. per kilogram) 5 hours before excision of the heart and the graft was flushed with CPP. Group IV (five dogs): The donor was pretreated with ALG (30 mg. per kilogram) and methylprednisolone
Volume 75
Survival of heart transplants
Number 4
539
April, 1978
(30 mg. per kilogram) 5 hours before graft excision and the heart was flushed with CPP. Group V (seven dogs): The donor was not pretreated and the graft was flushed with CPP plus 750 mg. per liter of ALG. Group VI (five dogs): The donor was not pretreated and the heart was flushed as in Group V, but the recipient did not receive immunosuppression. Group VII (six dogs): The donor was pretreated with ALG and methylprednisolone at the same doses as described for the other groups, and the graft was flushed with CPP plus ALG at the same dosage as in Group V. All recipient animals received minimal immunosuppression with azathioprine except those in Group VI, which received no immunosuppression. Four dogs were eliminated from the study because they were considered technical failures and died within the first 2 days after the surgical procedure. One dog of Group I died of severe intussusception 36 hours after the operation; one dog of Group III died with intra-abdominal hemorrhage several hours after the operation; one dog of Group VI died in the immediate postoperative period due to anesthetic complication; and the last dog of Group VII died 48 hours after the operation owing to severe distemper. All the parameters of the experimental group of dogs were statistically compared by the method of analysis of variance and the a posteriori Newman-Keuls method. 10
Table II. Causes offailure (death) of all cardiac transplants Groups (No. of dogs) Cause of graftfailure Mechanical Sepsis Arterial thrombosis Venous thrombosis Rejection Hemorrhagic necrosis Unknown
VIl
0 I 0 0 3 I 0
0 0 0 I
3 I I
I I
0 0
0 0 3 I 0
I I
3 0 0
0
I
I I I I I
0 0 1 2
2
0
I
0 0 I I
2 0 2
Table III. Histologic response of all cardiac transplants Groups (No. of dogs) Severity of lesion I. No vascular lesion, minimal cellular infiltrate II. Minimal vascular lesion, moderate cellular infiltrate III. Moderate vascular changes, severe cellular infiltrate IV. Severe vascular changes, severe cellular damage
I
2
VIl
2
2
2
2
3
3
3
2
2
2
o
o
Results
Fig. 2 shows the duration of survival of all heart allografts. Hearts flushed with CPP plus ALG and transplanted into minimally immunosuppressed recipients survived significantly longer than those not so treated (p < 0.01). Donor pretreatment with ALG or methylprednisolone did not contribute to any further prolongation in survival of ALG-flushed grafts (p < 0.05). Donor pretreatment by itself with methylprednisolone, cyclophosphamide, or ALG had no effect on the survival of minimally immunosuppressed cardiac allografts (p < O. I). Flushing the heart with Ringer's solution or CPP alone also resulted in no prolongation of survival. Graft flushing with CPP plus ALG did not prolong the survival of transplanted hearts which were not given minimal doses of immunosuppression (p = 0.5). Table II shows the causes of failure of all cardiac transplants. The main cause of failure was rejection or vascular thrombosis. The rejection phenomenon varied in intensity in the different groups of animals. It was more apparent in the animals that received hearts that
were not flushed with ALG (Groups I, II, III, and IV). The group of hearts that were flushed with ALG but had no immunosuppression had a similar rejection response. Only in the group of hearts flushed with ALG before being transplanted into minimally immunosuppressed recipients was the appearance of the rejection changes delayed. VVhen these changes occurred they were not as severe as the ones observed in the remaining groups. Table III shows the histologic response of all cardiac transplants. The group of hearts flushed with ALG before transplantation into minimally immunosuppressed recipients had moderate cellular infiltrate with minimal vascular lesions, whereas the other group had severe cellular infiltrate with moderate-to-severe vascular changes. Discussion
Our experiments demonstrate that flushing of canine hearts with ALG allowed the grafts to survive for long periods of time in minimally immunosuppressed recipi-
The Journal of
540 Toledo-Pereyra and Condie
Thoracic and Cardiovascular Surgery
ents. Flushing of the graft with ALG in dogs that were not immunosuppressed did not prolong survival. Thus the combination of minimal immunosuppression with graft pretreatment was important in prolonging survival. On the other hand, we were unable to demonstrate any beneficial effect of donor pretreatment with methylprednisolone, cyclophosphamide, or ALG in the survival of canine heart allografts. Antilymphocyte serum has been utilized both to pretreat the donor and to pretreat the graft itself. Guttman and his associates!' pretreated rat donors with rabbit anti-mouse-thymus IgG; survival of kidneys from these rats was prolonged. Cerilli and his co-workers" were unable to confirm Guttman's results with dogs receiving kidneys from donors pretreated with horse antidog ALG. However, confirmatory reports have been made with skin grafts, tumor grafts, corneal allografts, and rat heart allografts." In addition, Callender' in our group was able to obtain significant prolongation of survival (average 66.4 days) of canine kidneys that were perfused with ALG and CPP before being transplanted into minimally immunosuppressed recipients. He explained these findings through either opsonization of the passenger leukocytes or masking of histocompatibility antigens by nonspecific binding of goat globulin to kidney cells. Although our results were not as good as the ones reported by Callender and his associates,' we were able to confirm his observations, not only in hearts, but also in small bowel, liver, and lung allografts.v" Other investigators, however, have obtained different results. For instance, Iwahashi and his associates':' were unable to prolong the survival of canine lungs perfused in situ with antilymphocyte serum and subsequently transplanted into recipients immunosuppressed with horse antidog lymphocyte serum. We failed to obtain prolonged survival of donor allografts pretreated with high doses of cyclophosphamide, methylprednisolone, or ALG. It is possible that the type of animal utilized (mongrel dogs) could account for certain variability of the results, as well as the time and dosage of pretreatment and some other characteristics of the animal before transplantation. All of these factors must be taken into consideration in the final analysis of this study. Guttman and his associates!': 15 have been particularly interested in this area. Zincke and Woods'" 17 have demonstrated that donor pretreatment with methylprednisolone, cyclophosphamide, and other cytotoxic agents will prolong renal allograft survival in animals and men, presumably owing to the reduction of organ antigenicity. The initial studies on donor pretreatment with cytotoxic agents
were confirmed with heart'" and dog kidney allografts, but could not be reproduced with a skin graft from pretreated donors. We firmly believe that donor pretreatment has a definite role in transplantation. There are several factors that have to be better understood, such as dosage and timing of administration of the cytotoxic drugs. Recently, Dienst" has shown that, to achieve maximal depletion of lymphocytes in the donor, pretreatment should be started 24 hours before organ removal. This might be one of the most significant factors in prolonging survival after donor pretreatment. We do not have an explanation for the prolongation of survival of hearts flushed with ALG before transplantation into minimally immunosuppressed recipients. It is possible that ALG nonspecifically binding the graft antigens or interfering with the perception of the antigen by the host. We do not think that elimination of passenger leukocytes is the only factor or most important factor accounting for this prolongation of survival. In summary, we have shown that flushing heart allografts with ALG before transplantation can prolong the survival of recipients that have been minimally immunosuppressed. When no immunosuppression was given, no prolongation of survival was observed. Donor pretreatment had no significant influence on the prolongation of survival. REFERENCES Callender CO, Simmons RL, Toledo-Pereyra LH, Santiago-Delpin EA, Najarian JS: Prolongation of kidney allografts perfused by antilymphocyte globulin in vitro. Transplantation 16:377-378, 1973 2 Toledo-Pereyra LH, Ray PK, CallenderCO, Najarian JS, Simmons RL: Renal allograft prolongation using phytomitogens to mask graft antigens. Surgery 76: 121-128, 1974
3 Toledo-Pereyra LH, Simmons RL, Najarian JS: Modification of immunogenicity of kidney allografts treated with acid mucopolysaccharides. Surg Forum 26:331-332, 1975
4 Simmons RL, Toledo-Pereyra LH, Nowygrod R, Najarian JS: Masking of kidney graft antigens with concanavalin A. Transplant Proc 7:677-680, 1975 5 Nowygrod R, Dombrovskis S, Olson LC, Toledo-Pereyra LH, Simmons RL: Modulating renal alloantigens with concanavalin A. Mixed lymphocyte culture studies. Surg Forum 26:325-328, 1975 6 Toledo-Pereyra LH, Simmons RL, Najarian JS: Modification of graft antigenicity by perfusion with ALG and cytoxan on canine small bowel allografts (abstr). Fourth Ann. Clin. Dyal. Transpl. Forum, Nov. 16-17,1974 7 Toledo-Pereyra LH, Condie RM, CallenderCO, SharpH, Buselmeier TJ, Simmons RL, Najarian JS: Prolonged
Volume 75
Survival of heart transplants
Number 4 April,1978
8
9
10
II
12
survival of canine liver allografts perfused with antilymphocyte globulin (ALG) (abstr). Trans Am Soc Artif Intern Organs 5:81, 1976 Toledo-Pereyra LH, Condie RM, Hau T, Simmons RL, Najarian JS: Lung transplantation. Improved survival of canine lung allografts perfused with antilymphocyte globulin (ATG). Bol Asoc Med PR 68:234-238, 1976 Gonzalez-Lavin L, O'Connell TX, Sinclair R, Porter KA, Mowbray JF: Clearance of passenger blood cells as an adjunct in decreasing rejection in canine heterotopic heart transplants. J. THORAC CARDIOVASC SURG 67:466-473, 1976 Weiner BJ: Statistical Principles in Experimental Design, ed. 2, New York, 1971, McGraw-HilI Book Company Inc., p. 191 Guttman RD, Carpenter CB, Lindquist RR, Merrill JP: Renal transplantation in the inbred rat. III. A study of heterologous anti-thymocyte sera. J Exp Med 126: 10991126, 1967 Cerilli J, Groth C, Taylor P, Daloze, PM: The effect of
13
14
15
16 17
18
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donor pretreatment with heterologous anti-lymphocyte globulin on kidney homograft survival. Transplantation 5:1334-1335, 1967 Freeman JS, Chamberlain EC, Reemtsma K, et al: Prolongation of rat allografts by donor pretreatment with immunosuppressive agents. Transplant Proc 3:580-582, 1971 Iwahashi H, Nagaya H, Sealy We: Effect of in situ perfusion of donor lung on the survival of canine lung allograft. Transplantation 13:183-186, 1972 Guttmann RD, Beaudoin JG, Morehouse DD: Reduction of immunogenicity of human cadaver renal allografts by donor pretreatment. Transplant Proc 5:663-665, 1973 Zincke H, Woods JE: Donor pretreatment in cadaver renal transplantation. Surg Gynecol Obstet 145: 183-185, 1977 Zincke H, Woods JE: Attempted immunological alteration of canine renal allograft donor. Transplantation 18:480-486, 1974 Dienst SG: Personal communication
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January I, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: "The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published. " Authors will be consulted, when possible, regarding republication of their material.
Luis H. Toledo-Pereyra, M.D., Ph.D., and Richard M. Condie, Detroit, Mich., and Minneapolis, Minn.
In 1973, Callender and his associates 1 demonstrated that the immunogenicity of canine renal allografts could be diminished by perfusion with antilymphoblast globulin (ALG) before transplantation into minimally immunosuppressed recipients. Whether the mechanism of action involved opsonization of the passenger leukocytes or the masking of histocompatibility antigens by nonspecific binding of goat globulin to kidney cells was unknown. Nevertheless, his results suggested that antigen modification might supplement immunosuppression in the prolongation of kidney allograft survival. Toledo-Pereyra," 3 Simmons," and Nowygrod," and their associates obtained similar findings on minimally immunosuppressed recipients that received canine kidneys flushed with concanavalin A (ConA), phytohemagglutinin-M, or chondroitin sulfate. Furthermore, Toledo-Pereyra and his group'":" confirmed the beneficial effect of graft flushing or perfusion with ALG on small bowel, liver, and lung canine allografts that also were minimally immunosuppressed. In this work, From the Department of Surgery, Section of Surgical Research and Transplantation, Henry Ford Hospital, Detroit, Mich., and the University of Minnesota, Minneapolis, Minn. Partially supported by Ford Foundation Grant No. 730-0786. Received for publication Sept. 19, 1977. Accepted for publication Nov. 3, 1977. Address for reprints: Dr. L. H. Toledo-Pereyra, Department of Surgery, Section of Surgical Research and Transplantation, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, Mich. 48202.
536
we further study the modification of graft antigenicity on canine heart allografts flushed with ALG before transplantation, and on canine donor hearts pretreated with ALG, cyclophosphamide, or methylprednisolone prior to transplantation into minimally immunosuppressed recipients.
Materials and methods Adult mongrel dogs of either sex, the donors weighing between 11 and 18 kilograms and the recipients between 17 and 25 kilograms, were anesthetized with sodium methohexital for induction and halothane (0.5 to 2.0 percent) for maintenance. Oxygen was continuously given with ventilation through an endotracheal tube. Through a median sternotomy, the heart was excised in a routine fashion from the donor animal. Before the heart was excised, 1,500 U. of heparin was administered intravenously. The donor dog received 500 ml. of Ringer's lactate during the procedure. The dissection technique for removal of the heart basically consisted of the following: The superior and inferior venae cavae were identified; the azygos vein was ligated with 2-0 Tevdek sutures and divided; the branches of the aorta were also ligated with 2-0 Tevdek sutures and divided. The pulmonary artery was identified and was dissected all the way into the lung parenchyma. As soon as the heart was dissected out completely and good control was obtained from the structures to be ligated, the heart was fibrillated with a direct current of 30 to 60 mAmp. Then, the aorta was
0022-5223/78/0475-0536$00.60/0 © 1978 The C. V. Mosby Co.
Volume 75 Number
Survival of heart transplants
4
537
April,1978
Donor's Heart
Recipient's Heart Ringer's or CPP With/Without
ALG
With/Without Donor Pretreotment
Aorta
Methylprednisolone Cyclophosphamide
ALG
Abdominal Cardiac Transplantation
Groft Flushing
Follow
up/
-Cllnlcol -EKG -Histoloqy
Fig. I. Experimental design of canine heart transplantation. Graft and/or donor pretreatment with antilyrnphoblast globulin (ALG), methylprednisolone, or cyclophosphamide. The heart was flushed withRinger's lactateor cryoprecipitated plasma at 4° C. prior to transplantation into minimally immunosuppressed recipients. cross-clamped in its descending portion and divided. The pulmonary artery was cross-clamped close to the lung hilum and divided. The venae cavae and all the rest of the structures -were divided. The pulmonary veins were ligated individually and divided. No warm ischemia was involved in these experiments. The heart was removed and flushed with 500 to 750 ml. of either Ringer's lactate or cryoprecipitated plasma (CPP) at 4°C. The recipient dog was prepared by means of a median laparotomy incision. A long segment of the abdominal aorta and vena cava were prepared for the vascular anastomoses. The pulmonary artery and aorta of the donor's heart were carefully prepared for the anastomoses, with an adequate segment of both vessels being left in order to avoid any kinking or outflow obstruction after transplantation. Both the aorta-toaorta anastomosis and the pulmonary artery-vena cava anastomosis were performed with 5-0 Prolene sutures. A No. 18 needle was placed in the left ventricular chamber to aspirate all the accumulated air before clamp release. As soon as the vascular clamps were opened, the heart started contracting slowly, and if necessary 1 to 2 C.c. of 10 percent calcium chloride was injected into the left ventricle. If beating did not start spontaneously after 3 minutes of massage or after calcium chloride injection, the heart was defibrillated with direct current (20 to 60 watt-seconds). In some other cases it was necessary to instill diluted (1 : 10) epinephrine (l mg.) directly into the left ventricle to induce
cardiac contractility. The abdomen and heart were checked for hemostasis, the viscera were returned to the normal position, and the abdomen was closed in a routine fashion (Fig. 1). Ringer's lactate, 1,000 c.c. in the first 12 hours, was given intravenously. Water and food were resumed on the second postoperative day. Procaine penicillin, 1,200,000 D., was given intramuscularly each day for 3 days. No blood was transfused into the recipient before, during, or after the operation. Immunosuppression included azathioprine, 5 mg. per kilogram per day for 3 days and then 2.5 mg. per kilogram per day until death or sacrifice. The heart grafts were followed by daily clinical examination with palpation of the graft in the abdominal cavity, daily electrocardiograms, and histologic study when the specimen was obtained. Impending rejection was most accurately diagnosed by palpation of the heart. Electrocardiographically, the R wave progressively decreased in voltage and there were numerous atrial arrhythmias associated with a decrease in voltage. If the daily transplant heartbeat or the electrocardiogram showed either terminal or no activity, the dog was put to death and the heart was removed for gross and histologic examination. The ALG was prepared by immunizing goats with biweekly injections of 108 dog lymph node lymphocytes over a total period of 3 months. After absorption with red blood cells, the antilymphocyte serum initially was purified by precipitation with 40 percent (NH 4 h-
The Journal of Thoracic and Cardiovascular Surgery
53 8 Toledo-Pereyra and Condie
1- RinQers
n-cpp
m-
Donor Methylprednisolone + Cyclophosphamide, CPP m:- Donor ALG + Methylprednisolone,
Cpp
3l- CPP + ALG ::5ZI- CPP + ALG No Recipient Immunosuppression JZJI- Donor ALG + Methylprednisolone, CPP + ALG
100
75
0
>
.~
-~
(/)
c ~ 0:>!!
50
25 0
0
5
10
15
20
25
30
40
35
45
Days Post-Transplantation
Fig, 2. Survival in days of all different groups of heart allografts. The best group was the one flushed with ALG prior to transplantation into minimally immunosuppressed recipients.
Table I. Characteristics of all experimental heart transplant groups Donor pretreatment
Group
I II III
IV V VI VII
5 6 6 5 7
5 6
None None Methylprednisolone + cyclophosphamide ALG + methylprednisolone None None ALG + methylprednisolone
S04 and then was purified over a DEAE-cellulose column. The ALG was sterile and free of pyrogens. Doses of 50 mg. per kilogram of ALG prolonged the life of skin grafts in dogs up to 18 days. Lymphocytotoxicity titers against blood lymphocytes from peripheral vessels ranged from I : 1,000 to I : 54,000. Hemagglutination titers were less than I: 64. The microscopic specimens were classified in four main groups according to the previous classification of Gonzalez-Lavin and his associates": Type I, no vascular involvement and minimal cell polymorphonuclear infiltration; Type II, mononuclear and polymorphonuclear cell infiltration of the myocardium and perivascular accumulation of lymphocytes and monocytes; Type III, mild intimal cell infiltration, intimal thickening of the coronary vessels, fibrinoid necrosis of the media, and severe cellular infiltrate; Type IV, severe endothe-
Graft flush and pretreatment
Temperature
Recipient immunosuppression
Ringer's CPP CPP
40 C. 40 C. 40 C.
Azathioprine Azathioprine Azathioprine
cpp CPP CPP CPP
40 40 40 40
Azathi oprine Azathi oprine None Azathioprine
+ + +
ALG ALG ALG
C. C. C. C.
lial damage of the coronary arterioles, disruption of the myocardial fibers associated with dense infiltration of lymphocytes, lymphoblasts, and plasma cells, and extensive myocardial damage. Seven groups of dogs were included in this study (Table I). Group I (five dogs): The donor animal was not pretreated and the heart was flushed with Ringer's lactate. Group II (six dogs): The donor was not pretreated and the graft was flushed with CPP. Group III (six dogs): The donor was pretreated with methylprednisolone (30 mg. per kilogram) and cyclophosphamide (50 mg. per kilogram) 5 hours before excision of the heart and the graft was flushed with CPP. Group IV (five dogs): The donor was pretreated with ALG (30 mg. per kilogram) and methylprednisolone
Volume 75
Survival of heart transplants
Number 4
539
April, 1978
(30 mg. per kilogram) 5 hours before graft excision and the heart was flushed with CPP. Group V (seven dogs): The donor was not pretreated and the graft was flushed with CPP plus 750 mg. per liter of ALG. Group VI (five dogs): The donor was not pretreated and the heart was flushed as in Group V, but the recipient did not receive immunosuppression. Group VII (six dogs): The donor was pretreated with ALG and methylprednisolone at the same doses as described for the other groups, and the graft was flushed with CPP plus ALG at the same dosage as in Group V. All recipient animals received minimal immunosuppression with azathioprine except those in Group VI, which received no immunosuppression. Four dogs were eliminated from the study because they were considered technical failures and died within the first 2 days after the surgical procedure. One dog of Group I died of severe intussusception 36 hours after the operation; one dog of Group III died with intra-abdominal hemorrhage several hours after the operation; one dog of Group VI died in the immediate postoperative period due to anesthetic complication; and the last dog of Group VII died 48 hours after the operation owing to severe distemper. All the parameters of the experimental group of dogs were statistically compared by the method of analysis of variance and the a posteriori Newman-Keuls method. 10
Table II. Causes offailure (death) of all cardiac transplants Groups (No. of dogs) Cause of graftfailure Mechanical Sepsis Arterial thrombosis Venous thrombosis Rejection Hemorrhagic necrosis Unknown
VIl
0 I 0 0 3 I 0
0 0 0 I
3 I I
I I
0 0
0 0 3 I 0
I I
3 0 0
0
I
I I I I I
0 0 1 2
2
0
I
0 0 I I
2 0 2
Table III. Histologic response of all cardiac transplants Groups (No. of dogs) Severity of lesion I. No vascular lesion, minimal cellular infiltrate II. Minimal vascular lesion, moderate cellular infiltrate III. Moderate vascular changes, severe cellular infiltrate IV. Severe vascular changes, severe cellular damage
I
2
VIl
2
2
2
2
3
3
3
2
2
2
o
o
Results
Fig. 2 shows the duration of survival of all heart allografts. Hearts flushed with CPP plus ALG and transplanted into minimally immunosuppressed recipients survived significantly longer than those not so treated (p < 0.01). Donor pretreatment with ALG or methylprednisolone did not contribute to any further prolongation in survival of ALG-flushed grafts (p < 0.05). Donor pretreatment by itself with methylprednisolone, cyclophosphamide, or ALG had no effect on the survival of minimally immunosuppressed cardiac allografts (p < O. I). Flushing the heart with Ringer's solution or CPP alone also resulted in no prolongation of survival. Graft flushing with CPP plus ALG did not prolong the survival of transplanted hearts which were not given minimal doses of immunosuppression (p = 0.5). Table II shows the causes of failure of all cardiac transplants. The main cause of failure was rejection or vascular thrombosis. The rejection phenomenon varied in intensity in the different groups of animals. It was more apparent in the animals that received hearts that
were not flushed with ALG (Groups I, II, III, and IV). The group of hearts that were flushed with ALG but had no immunosuppression had a similar rejection response. Only in the group of hearts flushed with ALG before being transplanted into minimally immunosuppressed recipients was the appearance of the rejection changes delayed. VVhen these changes occurred they were not as severe as the ones observed in the remaining groups. Table III shows the histologic response of all cardiac transplants. The group of hearts flushed with ALG before transplantation into minimally immunosuppressed recipients had moderate cellular infiltrate with minimal vascular lesions, whereas the other group had severe cellular infiltrate with moderate-to-severe vascular changes. Discussion
Our experiments demonstrate that flushing of canine hearts with ALG allowed the grafts to survive for long periods of time in minimally immunosuppressed recipi-
The Journal of
540 Toledo-Pereyra and Condie
Thoracic and Cardiovascular Surgery
ents. Flushing of the graft with ALG in dogs that were not immunosuppressed did not prolong survival. Thus the combination of minimal immunosuppression with graft pretreatment was important in prolonging survival. On the other hand, we were unable to demonstrate any beneficial effect of donor pretreatment with methylprednisolone, cyclophosphamide, or ALG in the survival of canine heart allografts. Antilymphocyte serum has been utilized both to pretreat the donor and to pretreat the graft itself. Guttman and his associates!' pretreated rat donors with rabbit anti-mouse-thymus IgG; survival of kidneys from these rats was prolonged. Cerilli and his co-workers" were unable to confirm Guttman's results with dogs receiving kidneys from donors pretreated with horse antidog ALG. However, confirmatory reports have been made with skin grafts, tumor grafts, corneal allografts, and rat heart allografts." In addition, Callender' in our group was able to obtain significant prolongation of survival (average 66.4 days) of canine kidneys that were perfused with ALG and CPP before being transplanted into minimally immunosuppressed recipients. He explained these findings through either opsonization of the passenger leukocytes or masking of histocompatibility antigens by nonspecific binding of goat globulin to kidney cells. Although our results were not as good as the ones reported by Callender and his associates,' we were able to confirm his observations, not only in hearts, but also in small bowel, liver, and lung allografts.v" Other investigators, however, have obtained different results. For instance, Iwahashi and his associates':' were unable to prolong the survival of canine lungs perfused in situ with antilymphocyte serum and subsequently transplanted into recipients immunosuppressed with horse antidog lymphocyte serum. We failed to obtain prolonged survival of donor allografts pretreated with high doses of cyclophosphamide, methylprednisolone, or ALG. It is possible that the type of animal utilized (mongrel dogs) could account for certain variability of the results, as well as the time and dosage of pretreatment and some other characteristics of the animal before transplantation. All of these factors must be taken into consideration in the final analysis of this study. Guttman and his associates!': 15 have been particularly interested in this area. Zincke and Woods'" 17 have demonstrated that donor pretreatment with methylprednisolone, cyclophosphamide, and other cytotoxic agents will prolong renal allograft survival in animals and men, presumably owing to the reduction of organ antigenicity. The initial studies on donor pretreatment with cytotoxic agents
were confirmed with heart'" and dog kidney allografts, but could not be reproduced with a skin graft from pretreated donors. We firmly believe that donor pretreatment has a definite role in transplantation. There are several factors that have to be better understood, such as dosage and timing of administration of the cytotoxic drugs. Recently, Dienst" has shown that, to achieve maximal depletion of lymphocytes in the donor, pretreatment should be started 24 hours before organ removal. This might be one of the most significant factors in prolonging survival after donor pretreatment. We do not have an explanation for the prolongation of survival of hearts flushed with ALG before transplantation into minimally immunosuppressed recipients. It is possible that ALG nonspecifically binding the graft antigens or interfering with the perception of the antigen by the host. We do not think that elimination of passenger leukocytes is the only factor or most important factor accounting for this prolongation of survival. In summary, we have shown that flushing heart allografts with ALG before transplantation can prolong the survival of recipients that have been minimally immunosuppressed. When no immunosuppression was given, no prolongation of survival was observed. Donor pretreatment had no significant influence on the prolongation of survival. REFERENCES Callender CO, Simmons RL, Toledo-Pereyra LH, Santiago-Delpin EA, Najarian JS: Prolongation of kidney allografts perfused by antilymphocyte globulin in vitro. Transplantation 16:377-378, 1973 2 Toledo-Pereyra LH, Ray PK, CallenderCO, Najarian JS, Simmons RL: Renal allograft prolongation using phytomitogens to mask graft antigens. Surgery 76: 121-128, 1974
3 Toledo-Pereyra LH, Simmons RL, Najarian JS: Modification of immunogenicity of kidney allografts treated with acid mucopolysaccharides. Surg Forum 26:331-332, 1975
4 Simmons RL, Toledo-Pereyra LH, Nowygrod R, Najarian JS: Masking of kidney graft antigens with concanavalin A. Transplant Proc 7:677-680, 1975 5 Nowygrod R, Dombrovskis S, Olson LC, Toledo-Pereyra LH, Simmons RL: Modulating renal alloantigens with concanavalin A. Mixed lymphocyte culture studies. Surg Forum 26:325-328, 1975 6 Toledo-Pereyra LH, Simmons RL, Najarian JS: Modification of graft antigenicity by perfusion with ALG and cytoxan on canine small bowel allografts (abstr). Fourth Ann. Clin. Dyal. Transpl. Forum, Nov. 16-17,1974 7 Toledo-Pereyra LH, Condie RM, CallenderCO, SharpH, Buselmeier TJ, Simmons RL, Najarian JS: Prolonged
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Survival of heart transplants
Number 4 April,1978
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survival of canine liver allografts perfused with antilymphocyte globulin (ALG) (abstr). Trans Am Soc Artif Intern Organs 5:81, 1976 Toledo-Pereyra LH, Condie RM, Hau T, Simmons RL, Najarian JS: Lung transplantation. Improved survival of canine lung allografts perfused with antilymphocyte globulin (ATG). Bol Asoc Med PR 68:234-238, 1976 Gonzalez-Lavin L, O'Connell TX, Sinclair R, Porter KA, Mowbray JF: Clearance of passenger blood cells as an adjunct in decreasing rejection in canine heterotopic heart transplants. J. THORAC CARDIOVASC SURG 67:466-473, 1976 Weiner BJ: Statistical Principles in Experimental Design, ed. 2, New York, 1971, McGraw-HilI Book Company Inc., p. 191 Guttman RD, Carpenter CB, Lindquist RR, Merrill JP: Renal transplantation in the inbred rat. III. A study of heterologous anti-thymocyte sera. J Exp Med 126: 10991126, 1967 Cerilli J, Groth C, Taylor P, Daloze, PM: The effect of
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donor pretreatment with heterologous anti-lymphocyte globulin on kidney homograft survival. Transplantation 5:1334-1335, 1967 Freeman JS, Chamberlain EC, Reemtsma K, et al: Prolongation of rat allografts by donor pretreatment with immunosuppressive agents. Transplant Proc 3:580-582, 1971 Iwahashi H, Nagaya H, Sealy We: Effect of in situ perfusion of donor lung on the survival of canine lung allograft. Transplantation 13:183-186, 1972 Guttmann RD, Beaudoin JG, Morehouse DD: Reduction of immunogenicity of human cadaver renal allografts by donor pretreatment. Transplant Proc 5:663-665, 1973 Zincke H, Woods JE: Donor pretreatment in cadaver renal transplantation. Surg Gynecol Obstet 145: 183-185, 1977 Zincke H, Woods JE: Attempted immunological alteration of canine renal allograft donor. Transplantation 18:480-486, 1974 Dienst SG: Personal communication
Information for authors Most of the provisions of the Copyright Act of 1976 became effective on January I, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: "The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published. " Authors will be consulted, when possible, regarding republication of their material.