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Improved survival after donor pretreatment with anti-lymphoblast globulin (ALG) in perfused kidneys: Experimental findings
JOURNAL
OF SURGICAL
RESEARCH
31, 172-177 (1981)
Improved Survival after Donor Pretreatment with Anti-Lymphoblast Globulin (ALG) in Perfused Kidneys: Experimental Findings LUIS H. TOLEDO-PEREYRA, M.D., PH.D.,’ S. TAIYEBB, M.D., GERALD H. MCKENZIE,
DEBRA A. GORDON,MS., AND G. S. SY, M.D. Department
of Surgery,
Section of Transplantation and Surgical Research, Mount Carmel Mercy Hospital, and Henry Ford Hospital, Detroit, Michigan 48235
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Although donor pretreatment has been utilized both experimentally and clinically, its role in conjunction with subsequent hypothermic pulsatile perfusion of renal allografts prior to transplantation has not been clearly defined. This study tests two modes of donor pretreatment and their compatibility with perfusion. Donor dogs were either (1) untreated, (2) pretreated with cyclophosphamide (CY) and methylprednisolone (MP), or (3) pretreated with equine anti-canine antilymphoblast globulin (ALG). Survival and rejection of perfused and nonperfused kidney allografts were compared in an unrelated mongrel dog population using CY and MP or ALG donor pretreatment. Results indicate that the beneficial effects of donor pretreatment with CY and MP are abrogated when kidneys are perfused for 24 hr prior to transplantation (CY + MP, nonperfused, survival = 17.23 ? 4.75, CY + MP, perfused 24 hr, survival = 12.83 rt 3.94days). Dogs receiving kidneys from donors pretreated with ALG in conjunction with 24-hr pulsatile perfusion, however, demonstrated prolonged renal allograft survival (24.20 f 10.46 days) (nonpretreated controls, survival = 12.60 5 3.79 days) (P < O.OOl),and the onset of rejection was also significantly delayed (ALG + 24hr perfusion, graft rejection = 17.87 f 7.72 days) (non-pretreated controls, graft rejection = 7.87 f 2.59 days) (P < 0.001). Donor pretreatment of renal allografts with ALG, therefore, annears to be an attractive modality for reduction of graft immunogenicity when perfusion is also -_ utilized prior to transplant.
Improvement of renal allograft survival lies importantly in the modification of the allograft rejection response. One modality that has been used in an attempt to accomplish this goal is donor pretreatment. The beneficial effects of cyclophosphamide (CY) and methylprednisolone (MP) as experimental and clinical donor pretreatment have been shown. However, no definite evidence has demonstrated favorable effects of using donor pretreatment in conjunction with organ perfusion. Our study attempts to define the role of donor pretreatment not only with CY and MP but also with anti-lymphoblast globulin (ALG) on the prolongation of perfused canine renal allografts. 1 To whom requests for reprints should be sent at: Department of Surgery, Mt. Carmel Mercy Hospital, 6071 W. Outer Dr., Detroit, Mich. 48235. 0022-4804/81/080172-06$01.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Adult mongrel dogs of either sex weighing between 15 and 25 kg were anesthetized with sodium thyamilal for induction and fluothane (0.5- 1.5%) for maintenance. The donor animals were either (1) nonpretreated, (2) pretreated with cyclosphosphamide (70-100 mg/kg) 12-24 hr before donor nephrectomy and methylprednisolone (30 mg/kg) 2-4 hr before excision, or (3) pretreated with anti-lymphoblast globulin (30 mg/kg) given 1 hr before nephrectomy. The kidney was excised through a flank incision and flushed with Ringer’s lactate (4°C) containing 10,000 units of heparin. Kidney transplantation was performed in a nonrelated animal utilizing the routine technique for implantation in the right iliac fossa. During the operative procedure, 172
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
173
nypothrrmtc PUmatIm Rrfuion 24 Hr. -pmin Ptavnanat* \
\ \ \
\
\
\
\
GROUPS I 8 II -No donor pr.tmtmmt \ GROuPSmaa -Donor prmwtm~d with cyelopno9phamid~ and atihyt(gndni#olw GROUP P -Dow pntmatmmnt
with
ALG
FIG. 1. Experimental design to evaluate the effects of donor pretreatment with MP + CY or ALG and 24-hr hypothermic pulsatile perfusion on kidney rejection and survival.
intravenous fluids consisted of 100 cclhr. kidneys obtained from pretreated donors D5W and 0.5 normal saline. Intravenous with CY + MP and perfused for 24 hr as fluids were continued at an approximate rate Group II, and Group V (n = 15) transof 70 ml/hr for 2 days, then oral fluids were planted with kidneys obtained from prestarted. Azathioprine was used for recipient treated donors with ALG and perfused immunosuppression (5.0 mg/kg for 3 days thereafter for 24 hr prior to transplantation. followed by 2.5 mg/kg/day until death). Biopsies were obtained from all kidneys at Daily serum creatinine values were mon- death for histological examination. Statisitored for 10 days and then every other day tical comparison included Student’s t test or every third day until death (Fig. 1). The for all experimental studies. onset of rejection was defined as the day that Equine anti-canine anti-lymphoblast globthe serum creatinine increased above ulin obtained from the University of Min2.0 mg%. nesota was used. ALG was prepared by Five groups of dogs were studied (Table 1). immunizing horses with log puppy thymoAnimals were randomly assigned to the cytes in Freund’s- complete adjuvant on experimental groups and the different treat- Days 0 and 14 by multiple subcutaneous ments were studied simultaneously. Group neck injections. On Day 21, the horses were I (n = 15) transplanted with fresh control subjected to plasmapheresis and bled every kidneys obtained from nonpretreated donors, other day for a total of four times. Blood Group II (n = 14) transplanted with control was replaced with red cells after each kidneys obtained from nonpretreated donors plasmapheresis. The pool of plasma was but perfused for 24 hr under hypothermic treated with SiO, to remove proteolytic pulsatile perfusion (MOX-100) (7°C) with a enzymes and lipids after absorption to plasmanate-albumin type of perftrsate, Group remove red blood cells. The plasma fraction III (n = 13) transplanted with kidneys was then run over a QAE-A50 Sephadex obtained from donors pretreated with CY column at a pH of 7.1. The first peak con+MP, Group IV (n = 12) transplanted with sisting of 100% IgG was purified and pyro-
174
JOURNAL OF SURGICAL RESEARCH: VOL. 31, NO. 2, AUGUST 1981 TABLE 1 EFFECT OF DONOR PRETREATMENT ON PERFUSED CANINE KIDNEY ALLOGRAFTS Donor pretreatment
Group
Number of animals
I II III IV V’,”
1.5 14 13 12 15
CY + MP
ALG
Perfusion (24 hr)
+ + + -
+
+ +
Graft rejection (M f SE, days) 7.81 + 11.7 + 12.08 + 9.58 2 17.87 t
0.669 0.991 0.895 0.450 1.99
Significance’ (0
survival (M 2 SE, days)
Significance” (P)
co.01 co.01 NS co.001
12.60 f 0.978 17.0 f 1.56 17.23 -c 1.31 12.83 + 1.01 24.20 f 2.70
NS” co.05 NS
” Statistical significance (Student’s f test) compared with Group I. b NS = no significant difference. c Group II vs Group V (graft rejection, P c 0.01). d Group IV vs Group V (graft rejection, P < 0.05).
gens were removed. Lymphocytotoxicity titers against peripheral blood lymphocytes were measured to be 1: 1200 and hemagglutinin titers were 1:16. Significant prolongation of canine renal allograft survival was also demonstrated using this preparation. RESULTS
Table 1 shows the effects of donor pretreatment and perfusion on graft rejection and survival. Comparative values for graft rejection and animal survival (mean f SEM) are displayed, along with the results of the statistical comparison of Group I with Groups II through IV using Student’s t test. In addition, the mean graft rejection of Group V has been statistically compared to that of Groups II and IV using Student’s f test. Figures 2 and 3 show percentage survival and percentage graft rejection, respectively, of all groups post-transplantation. A significant delay in the appearance of rejection and improved survival was observed in the non-pretreated perfused grafts (Group II). Donor pretreatment alone with CY + MP also improved graft survival in nonperfused kidneys (Group III). However, the independent beneficial effect of perfusion and donor pretreatment with CY + MP was not seen when these two modalities were used together (Group IV). ALG donor pretreatment used in conjunc-
tion with 24 hr of perfusion demonstrated the best results of all groups tested, where a significant delay in graft rejection and the longest survival was seen. Histologically, there were no significant differences between pretreated and nonpretreated allografts except for the clinical differences noted before in the onset of rejection and survival time. Since all biopsies were obtained at death when rejection was already established, no subtle histological differences could be appreciated. DISCUSSION
Donor pretreatment has been pursued in recent years as a means of modifying the recipient’s immune response to transplanted organs. Although much experimental work has been done in this area, the results have often been very different from those obtained from clinical application in the transplant setting. In 1969, Guttman and Lindquist [6] found that pretreatment of donors with cytotoxic drugs such as cyclophosphamide, methotrexate, or procarbazine could reduce the amount of kidney rejection observed after renal transplantation in rats. Freeman and co-workers [3, 41 later confirmed Guttman’s observations and demonstrated that donor pretreatment with irradiation, cyclophosphamide or anti-lymphocyte serum could prolong rat
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
.‘*“‘~‘**,.GROUP I --GROUP II GROUP III .***.*- GROUP Ip -.GROUP Y
Fresh Control 24 Hour Perfusion CY + MP CY + MP + 24 Hour Perfusion ALG + 24 Hour Perfusion
..y 2 ;i.W. sv+ 1. :.ii I :I...’ :::: i.-.-. ;3...i s ii. ! D << .Ol
DAYS POST-TRANSPLANTATION FIG. 2. Effect of donor pretreatment with CY + MP or ALG and hypothermic pulsatile perfusion on the survival of canine kidney allografts. The best results were observed in the ALG donor pretreated kidneys that were perfused for 24 hr prior to transplantation. Statistical significance (P values) are results of comparison between Groups III and V and versus Group I controls using Student’s r test.
‘~~~~~~~~~*~~~ GROUP I --GROUP II GROUP m ..-.... GROUP IS2 -m- GROUP Y
Fresh Control 24 Hour Perfusion CY + MP CY + MP + 24 Hour Perfusion ALG + 24 Hour Perfusion
.ol < .Ol
DAYS POST-TRANSPLANTATION FIG. 3. Effect of donor pretreatment with CY + MP or ALG and hypothermic pulsatile perfusion on the onset of rejection of canine kidney allografts. Groups II, III, and V showed a significant delay in the onset of rejection compared with Group I controls (Student’s r test).
175
176
JOURNAL OF SURGICAL
RESEARCH: VOL. 31, NO. 2, AUGUST 1981
heart allograft survival. However, when a potent immunosuppressive drug when Chassot and his group [2] attempted to apply used before and after transplantation and donor pretreatment to the canine renal allo- has been successfully used in graft pretreatgraft model, they were unable to prolong ment of kidney allografts to prolong graft graft survival when kidneys were perfused survival [l]. We chose the 24-hr perfusion after pretreatment. Graft survival improved period to test the efficacy of ALG because in only when cryoprecipitated plasma ob- human cadaver renal transplantation pertained from pretreated donors was used for fusion times of this length are common. kidney perfusion after donor pretreatment. Therefore, for ALG to be clinically useful, Woods and his associates [lo] then tried it would have to be able to modify the perfusion with various immunosuppressive system without being affected by 24-hr drugs for 24-48 hr and were not able to perfusion periods. Our present data demonprolong the survival of canine renal allo- strate that the effect of ALG when used as grafts. Zincke and his group [ 111,in contrast a donor pretreatment is not abrogated when to previous findings, found no detrimental the kidney is subsequently perfused for 24 hr effects on clinical cadaver renal transplanta- prior to transplantation. The onset of retion when cyclophosphamide and methyl- jection is delayed and graft survival is prednisolone were used together for donor significantly improved by this form of donor pretreatment with cryoprecipitated plasma pretreatment. and pulsatile perfusion for 11 hr. Although the exact mechanism for the acOur present study indicates that pro- tion of ALG in donor pretreatment is not longed graft survival of canine nonperfused known, one of the targets for ALG is bekidney allografts can be obtained using lieved to be the “passenger leukocytes” cyclophosphamide and methylprednisolone in the allograft. Stuart and co-workers [9] donor pretreatment. However, when kidney identified these cells as a “drug-sensitive, allografts are perfused in addition to donor highly immunogenic donor-cell population pretreatment for 24 hr rather than 11 hr as that was of nonparenchymal origin.” Their utilized by Zincke et al. [ 111,graft rejection work confirmed previous experimental data and survival are statistically comparable to by Snell [8] suggesting that donor leufresh non-pretreated nonperfused controls. kocytes present in the allograft might conProlonged perfusion after cyclophosphamide tribute an immunogenic stimulus for the and methylprednisolone donor pretreat- host’s response to the allograft. The lymment, therefore, appears to remove the phocytotoxic effects of anti-lymphocyte beneficial effects of either treatment alone. sera have been demonstrated by other inSince hypothermic pulsatile perfusion for vestigators [7]. Evidence has also been pre24 hr did not damage the allografts, we sented by Guttman et a/. [5] suggesting that would have to assume that long-term recir- leukocytes in the transplanted kidney may culation of CY and MP is harmful to the be important immunogenic agents in the kidneys or that damage induced by donor etiology of rejection. Experiments done by pretreatment is augmented by 24 hr of hypo- his group demonstrated that kidneys from donors pretreated with anti-thymocyte serum thermic pulsatile perfusion. Preliminary data indicating this effect led were not rejected as rapidly as controls. us to utilize a new modality of graft Thus, donor pretreatment with ALG might pretreatment in conjunction with pulsatile affect the primary cellular response to the kidney allograft . perfusion of the renal grafts. Anti-lymphoAnti-lymphoblast globulin appears to offer blast globulin (ALG) was chosen for several reasons. ALG can be administered to the a practical means of donor pretreatment donor in a shorter amount of time prior to which could be tested clinically; whereas, nephrectomy (1 hr). It has been proven to be cyclophosphamide should be administered
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
approximately more than 8 to 24 hr prior to nephrectomy, followed by methylprednisolone 2 hr before excision; ALG can be given to the donor 1 hr before the kidneys are removed. In many instances, this would be of significant value since it is often difficult to maintain a potential donor in a stable condition long enough to adhere to the longterm donor pretreatment protocol necessary for utilization of cyclophosphamide and methylprednisolone. In summary, our study indicates that ALG is a feasible alternative to cyclophosphamide and methylprednisolone as a means of donor pretreatment and can be successfully used to prolong canine renal allograft survival after pulsatile perfusion for up to 24 hr. Further experimental work in assessing the various doses and type of ALG, the time of administration, and the combination with other cytochemicals is necessary in order to make final recommendations as to the best use of this potentially beneficial modality of treatment. REFERENCES 1. Callender, C. O., Simmons, R. L., Toledo-Pereyra, L. H. ef al. Prolongation of kidney allografts perfused by antilymphocyte globulin in vitro. Transplantation 16: 377, 1973. 2. Chassot, P., Beaudoin, J., and Guttman, R. D.
177
Prolongation of kidney allograft survival in dogs with donor pretreatment. Surg. Forum 26: 314, 1973. 3. Freeman, J. S., Chamberlain, E., Reemtsma, K. et al. Prolongation of rat heart allograII by donor pretreatment with immunosuppressive agents. TrAnSplAnt Proc. k 580, 1971. 4. Freeman, J. S., Chamberlain, E., Reemtsma, K. et al. Rat heart allograft survival with donor pretreatment. Circulation 43(Suppl. 1): 120, 1971. 5. Guttman, R. D., Carpenter, C. B., Lindquist, R. R. et al. Renal transplantation in the inbred rat. III. A study of heterologous anti-thymocyte sera. .I. Exp. Med. 126: 1099, 1967. 6. Guttman, R. D., and Lindquist, R. R. Renal transplantation in the inbred rat. XI. Reduction of allograft immunogenicity by cytotoxic drug pretreatment of donors. Transplantation 8: 490, 1969. 7. Monaco, A. P., Wood, M. L., Gray, J. G. et al. Studies on heterologous anti-lymphocyte serum in mice. II. Effect on the immune response. J. Immunol. 96: 229, 1%6. 8. Snell, G. D. The homograft reaction. Annu. Rev. Microbial. 11: 439, 1957. 9. Stuart, F. P., Larrick, T., Holter, A. et al. Delayed rejection of renal allografts in the rat and dog by reduction of passenger leucocytes. Surgery 70: 128, 1971. 10. Woods, J. E., Cooperman, A. M., and Holley, K. E. Attempted antigenic alteration of canine kidneys by 24 or 48 hours perfusion with immunosuppressive agents. TrAnSplAntAtiOn 13: 336,1972. 11. Zincke, H., Woods, J. E., Khan, A. V. et al. Immunological donor pretreatment in combination with pulsatile preservation in cadaver renal transplantation. Transplantation 26: 207, 1978.
OF SURGICAL
RESEARCH
31, 172-177 (1981)
Improved Survival after Donor Pretreatment with Anti-Lymphoblast Globulin (ALG) in Perfused Kidneys: Experimental Findings LUIS H. TOLEDO-PEREYRA, M.D., PH.D.,’ S. TAIYEBB, M.D., GERALD H. MCKENZIE,
DEBRA A. GORDON,MS., AND G. S. SY, M.D. Department
of Surgery,
Section of Transplantation and Surgical Research, Mount Carmel Mercy Hospital, and Henry Ford Hospital, Detroit, Michigan 48235
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Although donor pretreatment has been utilized both experimentally and clinically, its role in conjunction with subsequent hypothermic pulsatile perfusion of renal allografts prior to transplantation has not been clearly defined. This study tests two modes of donor pretreatment and their compatibility with perfusion. Donor dogs were either (1) untreated, (2) pretreated with cyclophosphamide (CY) and methylprednisolone (MP), or (3) pretreated with equine anti-canine antilymphoblast globulin (ALG). Survival and rejection of perfused and nonperfused kidney allografts were compared in an unrelated mongrel dog population using CY and MP or ALG donor pretreatment. Results indicate that the beneficial effects of donor pretreatment with CY and MP are abrogated when kidneys are perfused for 24 hr prior to transplantation (CY + MP, nonperfused, survival = 17.23 ? 4.75, CY + MP, perfused 24 hr, survival = 12.83 rt 3.94days). Dogs receiving kidneys from donors pretreated with ALG in conjunction with 24-hr pulsatile perfusion, however, demonstrated prolonged renal allograft survival (24.20 f 10.46 days) (nonpretreated controls, survival = 12.60 5 3.79 days) (P < O.OOl),and the onset of rejection was also significantly delayed (ALG + 24hr perfusion, graft rejection = 17.87 f 7.72 days) (non-pretreated controls, graft rejection = 7.87 f 2.59 days) (P < 0.001). Donor pretreatment of renal allografts with ALG, therefore, annears to be an attractive modality for reduction of graft immunogenicity when perfusion is also -_ utilized prior to transplant.
Improvement of renal allograft survival lies importantly in the modification of the allograft rejection response. One modality that has been used in an attempt to accomplish this goal is donor pretreatment. The beneficial effects of cyclophosphamide (CY) and methylprednisolone (MP) as experimental and clinical donor pretreatment have been shown. However, no definite evidence has demonstrated favorable effects of using donor pretreatment in conjunction with organ perfusion. Our study attempts to define the role of donor pretreatment not only with CY and MP but also with anti-lymphoblast globulin (ALG) on the prolongation of perfused canine renal allografts. 1 To whom requests for reprints should be sent at: Department of Surgery, Mt. Carmel Mercy Hospital, 6071 W. Outer Dr., Detroit, Mich. 48235. 0022-4804/81/080172-06$01.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Adult mongrel dogs of either sex weighing between 15 and 25 kg were anesthetized with sodium thyamilal for induction and fluothane (0.5- 1.5%) for maintenance. The donor animals were either (1) nonpretreated, (2) pretreated with cyclosphosphamide (70-100 mg/kg) 12-24 hr before donor nephrectomy and methylprednisolone (30 mg/kg) 2-4 hr before excision, or (3) pretreated with anti-lymphoblast globulin (30 mg/kg) given 1 hr before nephrectomy. The kidney was excised through a flank incision and flushed with Ringer’s lactate (4°C) containing 10,000 units of heparin. Kidney transplantation was performed in a nonrelated animal utilizing the routine technique for implantation in the right iliac fossa. During the operative procedure, 172
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
173
nypothrrmtc PUmatIm Rrfuion 24 Hr. -pmin Ptavnanat* \
\ \ \
\
\
\
\
GROUPS I 8 II -No donor pr.tmtmmt \ GROuPSmaa -Donor prmwtm~d with cyelopno9phamid~ and atihyt(gndni#olw GROUP P -Dow pntmatmmnt
with
ALG
FIG. 1. Experimental design to evaluate the effects of donor pretreatment with MP + CY or ALG and 24-hr hypothermic pulsatile perfusion on kidney rejection and survival.
intravenous fluids consisted of 100 cclhr. kidneys obtained from pretreated donors D5W and 0.5 normal saline. Intravenous with CY + MP and perfused for 24 hr as fluids were continued at an approximate rate Group II, and Group V (n = 15) transof 70 ml/hr for 2 days, then oral fluids were planted with kidneys obtained from prestarted. Azathioprine was used for recipient treated donors with ALG and perfused immunosuppression (5.0 mg/kg for 3 days thereafter for 24 hr prior to transplantation. followed by 2.5 mg/kg/day until death). Biopsies were obtained from all kidneys at Daily serum creatinine values were mon- death for histological examination. Statisitored for 10 days and then every other day tical comparison included Student’s t test or every third day until death (Fig. 1). The for all experimental studies. onset of rejection was defined as the day that Equine anti-canine anti-lymphoblast globthe serum creatinine increased above ulin obtained from the University of Min2.0 mg%. nesota was used. ALG was prepared by Five groups of dogs were studied (Table 1). immunizing horses with log puppy thymoAnimals were randomly assigned to the cytes in Freund’s- complete adjuvant on experimental groups and the different treat- Days 0 and 14 by multiple subcutaneous ments were studied simultaneously. Group neck injections. On Day 21, the horses were I (n = 15) transplanted with fresh control subjected to plasmapheresis and bled every kidneys obtained from nonpretreated donors, other day for a total of four times. Blood Group II (n = 14) transplanted with control was replaced with red cells after each kidneys obtained from nonpretreated donors plasmapheresis. The pool of plasma was but perfused for 24 hr under hypothermic treated with SiO, to remove proteolytic pulsatile perfusion (MOX-100) (7°C) with a enzymes and lipids after absorption to plasmanate-albumin type of perftrsate, Group remove red blood cells. The plasma fraction III (n = 13) transplanted with kidneys was then run over a QAE-A50 Sephadex obtained from donors pretreated with CY column at a pH of 7.1. The first peak con+MP, Group IV (n = 12) transplanted with sisting of 100% IgG was purified and pyro-
174
JOURNAL OF SURGICAL RESEARCH: VOL. 31, NO. 2, AUGUST 1981 TABLE 1 EFFECT OF DONOR PRETREATMENT ON PERFUSED CANINE KIDNEY ALLOGRAFTS Donor pretreatment
Group
Number of animals
I II III IV V’,”
1.5 14 13 12 15
CY + MP
ALG
Perfusion (24 hr)
+ + + -
+
+ +
Graft rejection (M f SE, days) 7.81 + 11.7 + 12.08 + 9.58 2 17.87 t
0.669 0.991 0.895 0.450 1.99
Significance’ (0
survival (M 2 SE, days)
Significance” (P)
co.01 co.01 NS co.001
12.60 f 0.978 17.0 f 1.56 17.23 -c 1.31 12.83 + 1.01 24.20 f 2.70
NS” co.05 NS
” Statistical significance (Student’s f test) compared with Group I. b NS = no significant difference. c Group II vs Group V (graft rejection, P c 0.01). d Group IV vs Group V (graft rejection, P < 0.05).
gens were removed. Lymphocytotoxicity titers against peripheral blood lymphocytes were measured to be 1: 1200 and hemagglutinin titers were 1:16. Significant prolongation of canine renal allograft survival was also demonstrated using this preparation. RESULTS
Table 1 shows the effects of donor pretreatment and perfusion on graft rejection and survival. Comparative values for graft rejection and animal survival (mean f SEM) are displayed, along with the results of the statistical comparison of Group I with Groups II through IV using Student’s t test. In addition, the mean graft rejection of Group V has been statistically compared to that of Groups II and IV using Student’s f test. Figures 2 and 3 show percentage survival and percentage graft rejection, respectively, of all groups post-transplantation. A significant delay in the appearance of rejection and improved survival was observed in the non-pretreated perfused grafts (Group II). Donor pretreatment alone with CY + MP also improved graft survival in nonperfused kidneys (Group III). However, the independent beneficial effect of perfusion and donor pretreatment with CY + MP was not seen when these two modalities were used together (Group IV). ALG donor pretreatment used in conjunc-
tion with 24 hr of perfusion demonstrated the best results of all groups tested, where a significant delay in graft rejection and the longest survival was seen. Histologically, there were no significant differences between pretreated and nonpretreated allografts except for the clinical differences noted before in the onset of rejection and survival time. Since all biopsies were obtained at death when rejection was already established, no subtle histological differences could be appreciated. DISCUSSION
Donor pretreatment has been pursued in recent years as a means of modifying the recipient’s immune response to transplanted organs. Although much experimental work has been done in this area, the results have often been very different from those obtained from clinical application in the transplant setting. In 1969, Guttman and Lindquist [6] found that pretreatment of donors with cytotoxic drugs such as cyclophosphamide, methotrexate, or procarbazine could reduce the amount of kidney rejection observed after renal transplantation in rats. Freeman and co-workers [3, 41 later confirmed Guttman’s observations and demonstrated that donor pretreatment with irradiation, cyclophosphamide or anti-lymphocyte serum could prolong rat
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
.‘*“‘~‘**,.GROUP I --GROUP II GROUP III .***.*- GROUP Ip -.GROUP Y
Fresh Control 24 Hour Perfusion CY + MP CY + MP + 24 Hour Perfusion ALG + 24 Hour Perfusion
..y 2 ;i.W. sv+ 1. :.ii I :I...’ :::: i.-.-. ;3...i s ii. ! D << .Ol
DAYS POST-TRANSPLANTATION FIG. 2. Effect of donor pretreatment with CY + MP or ALG and hypothermic pulsatile perfusion on the survival of canine kidney allografts. The best results were observed in the ALG donor pretreated kidneys that were perfused for 24 hr prior to transplantation. Statistical significance (P values) are results of comparison between Groups III and V and versus Group I controls using Student’s r test.
‘~~~~~~~~~*~~~ GROUP I --GROUP II GROUP m ..-.... GROUP IS2 -m- GROUP Y
Fresh Control 24 Hour Perfusion CY + MP CY + MP + 24 Hour Perfusion ALG + 24 Hour Perfusion
.ol < .Ol
DAYS POST-TRANSPLANTATION FIG. 3. Effect of donor pretreatment with CY + MP or ALG and hypothermic pulsatile perfusion on the onset of rejection of canine kidney allografts. Groups II, III, and V showed a significant delay in the onset of rejection compared with Group I controls (Student’s r test).
175
176
JOURNAL OF SURGICAL
RESEARCH: VOL. 31, NO. 2, AUGUST 1981
heart allograft survival. However, when a potent immunosuppressive drug when Chassot and his group [2] attempted to apply used before and after transplantation and donor pretreatment to the canine renal allo- has been successfully used in graft pretreatgraft model, they were unable to prolong ment of kidney allografts to prolong graft graft survival when kidneys were perfused survival [l]. We chose the 24-hr perfusion after pretreatment. Graft survival improved period to test the efficacy of ALG because in only when cryoprecipitated plasma ob- human cadaver renal transplantation pertained from pretreated donors was used for fusion times of this length are common. kidney perfusion after donor pretreatment. Therefore, for ALG to be clinically useful, Woods and his associates [lo] then tried it would have to be able to modify the perfusion with various immunosuppressive system without being affected by 24-hr drugs for 24-48 hr and were not able to perfusion periods. Our present data demonprolong the survival of canine renal allo- strate that the effect of ALG when used as grafts. Zincke and his group [ 111,in contrast a donor pretreatment is not abrogated when to previous findings, found no detrimental the kidney is subsequently perfused for 24 hr effects on clinical cadaver renal transplanta- prior to transplantation. The onset of retion when cyclophosphamide and methyl- jection is delayed and graft survival is prednisolone were used together for donor significantly improved by this form of donor pretreatment with cryoprecipitated plasma pretreatment. and pulsatile perfusion for 11 hr. Although the exact mechanism for the acOur present study indicates that pro- tion of ALG in donor pretreatment is not longed graft survival of canine nonperfused known, one of the targets for ALG is bekidney allografts can be obtained using lieved to be the “passenger leukocytes” cyclophosphamide and methylprednisolone in the allograft. Stuart and co-workers [9] donor pretreatment. However, when kidney identified these cells as a “drug-sensitive, allografts are perfused in addition to donor highly immunogenic donor-cell population pretreatment for 24 hr rather than 11 hr as that was of nonparenchymal origin.” Their utilized by Zincke et al. [ 111,graft rejection work confirmed previous experimental data and survival are statistically comparable to by Snell [8] suggesting that donor leufresh non-pretreated nonperfused controls. kocytes present in the allograft might conProlonged perfusion after cyclophosphamide tribute an immunogenic stimulus for the and methylprednisolone donor pretreat- host’s response to the allograft. The lymment, therefore, appears to remove the phocytotoxic effects of anti-lymphocyte beneficial effects of either treatment alone. sera have been demonstrated by other inSince hypothermic pulsatile perfusion for vestigators [7]. Evidence has also been pre24 hr did not damage the allografts, we sented by Guttman et a/. [5] suggesting that would have to assume that long-term recir- leukocytes in the transplanted kidney may culation of CY and MP is harmful to the be important immunogenic agents in the kidneys or that damage induced by donor etiology of rejection. Experiments done by pretreatment is augmented by 24 hr of hypo- his group demonstrated that kidneys from donors pretreated with anti-thymocyte serum thermic pulsatile perfusion. Preliminary data indicating this effect led were not rejected as rapidly as controls. us to utilize a new modality of graft Thus, donor pretreatment with ALG might pretreatment in conjunction with pulsatile affect the primary cellular response to the kidney allograft . perfusion of the renal grafts. Anti-lymphoAnti-lymphoblast globulin appears to offer blast globulin (ALG) was chosen for several reasons. ALG can be administered to the a practical means of donor pretreatment donor in a shorter amount of time prior to which could be tested clinically; whereas, nephrectomy (1 hr). It has been proven to be cyclophosphamide should be administered
TOLEDO-PEREYRA
ET AL.: ALG DONOR PRETREATMENT
approximately more than 8 to 24 hr prior to nephrectomy, followed by methylprednisolone 2 hr before excision; ALG can be given to the donor 1 hr before the kidneys are removed. In many instances, this would be of significant value since it is often difficult to maintain a potential donor in a stable condition long enough to adhere to the longterm donor pretreatment protocol necessary for utilization of cyclophosphamide and methylprednisolone. In summary, our study indicates that ALG is a feasible alternative to cyclophosphamide and methylprednisolone as a means of donor pretreatment and can be successfully used to prolong canine renal allograft survival after pulsatile perfusion for up to 24 hr. Further experimental work in assessing the various doses and type of ALG, the time of administration, and the combination with other cytochemicals is necessary in order to make final recommendations as to the best use of this potentially beneficial modality of treatment. REFERENCES 1. Callender, C. O., Simmons, R. L., Toledo-Pereyra, L. H. ef al. Prolongation of kidney allografts perfused by antilymphocyte globulin in vitro. Transplantation 16: 377, 1973. 2. Chassot, P., Beaudoin, J., and Guttman, R. D.
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Prolongation of kidney allograft survival in dogs with donor pretreatment. Surg. Forum 26: 314, 1973. 3. Freeman, J. S., Chamberlain, E., Reemtsma, K. et al. Prolongation of rat heart allograII by donor pretreatment with immunosuppressive agents. TrAnSplAnt Proc. k 580, 1971. 4. Freeman, J. S., Chamberlain, E., Reemtsma, K. et al. Rat heart allograft survival with donor pretreatment. Circulation 43(Suppl. 1): 120, 1971. 5. Guttman, R. D., Carpenter, C. B., Lindquist, R. R. et al. Renal transplantation in the inbred rat. III. A study of heterologous anti-thymocyte sera. .I. Exp. Med. 126: 1099, 1967. 6. Guttman, R. D., and Lindquist, R. R. Renal transplantation in the inbred rat. XI. Reduction of allograft immunogenicity by cytotoxic drug pretreatment of donors. Transplantation 8: 490, 1969. 7. Monaco, A. P., Wood, M. L., Gray, J. G. et al. Studies on heterologous anti-lymphocyte serum in mice. II. Effect on the immune response. J. Immunol. 96: 229, 1%6. 8. Snell, G. D. The homograft reaction. Annu. Rev. Microbial. 11: 439, 1957. 9. Stuart, F. P., Larrick, T., Holter, A. et al. Delayed rejection of renal allografts in the rat and dog by reduction of passenger leucocytes. Surgery 70: 128, 1971. 10. Woods, J. E., Cooperman, A. M., and Holley, K. E. Attempted antigenic alteration of canine kidneys by 24 or 48 hours perfusion with immunosuppressive agents. TrAnSplAntAtiOn 13: 336,1972. 11. Zincke, H., Woods, J. E., Khan, A. V. et al. Immunological donor pretreatment in combination with pulsatile preservation in cadaver renal transplantation. Transplantation 26: 207, 1978.