ALS AND BONE MARROW
INFUSION IN IMMUNOSUPPRESSION
CARIDIS
THE COMBINED USE OF ANTILYMPHOCYTIC , . SERUM AND BONE MARROW INFUSION IN IMMUNOLOGICAL SUPPRESSION D. T. CARIDIS University Department of Surgery, St Vincent's Hospital, Melbourne
The combined use of antilymphocytic serum and live donor-specific bone marrow cells has produced significant immunosuppressionln dogs as judged by renal allograft survival. This combined action is specific in that it cannot be reproduced whether given alone or when live donor-specific bone marrow cells are replaced by dead donor-specific bone marrow cells or other antigens. The study has paved the way for human trials.
IN SPITE of many developments, the late, unremitting, slow failure of renal allografts has remained essentially the same. This is largely due to the fact that suppression of the recipient's immune reactivity is ineffective against chronic rejection. The advantages, therefore, of rendering the recipient immunologically tolerant are immense. In this respect Monaco and Wood (1970) showed that low doses (10-50 x lo6)of non-hybrid, homozygous allogeneic bone marrow cells induced specific augmented survival of skin allografts in nonthymectomized, ALS-treated adult mice. They postulated that this combined therapy induced a state of immunological tolerance. These experiments suggested a potential application of the principle to human renal transplantation. However, this could not be attempted until it was demonstrated that extension to large animals produced similarly encouraging results, and that the principle was specific. METHODS Randomly selected, unrelated, unmatched mongrel dogs were used throughoutthestudy. Recipient donor combination was always of the opposite sex. The recipients were always males, and females were used for renal allograft and live donor-specific bone marrow cell (LDSBMC) procurement. Nineteen groups of experimental animals were studied, each consisting of 5 to 10 dogs. Rabbit anti-dog lymphocyte serum (RADLS), made from canine mesenteric Reprints University of Melbourne Department of Surgery, St Vincent's Hospital, Fitzroy. Victoria 3000
AUST. N.Z. J. SURG.VOL. 49 - No. 6, DECEMBER, 1979
lymph nodes, was given subcutaneously from Day -7 to Day +7, relative to renal transplant on Day 0. The rabbit antidog lymphocytic serum (RADLS) was prepared in adult white New Zealand rabbits. Canine mesenteric lymph nodes were cleaned of perinodal fat and adventitia, minced with scissors, and then passed through stainless steel wire meshes (60 gauge). The suspension was washed three times in saline, following which the lymphocytes were resuspended to a concentration of 50 x lo6 cell/ml. One ml of the suspension was emulsified with an equal volume of complete Freund's adjuvant and injected into the rabbits' footpads. Four weeks later the rabbits were injected subcutaneously in multiple sites with 50 x lo6 saline-suspended cells (no adjuvant) on three successive days. Seven days after the last booster injection the rabbits were exsanguinated, and the blood was kept at 4" C for 24 hours. The serum was then separated and centrifugalized. Subsequently it was inactivated by heating at 50" C for 30 minutes and absorbed twice with thricewashed erythrocytes in order to remove the haemolysin. After addition of 1:lOO.OOO methiolate the serum was pooled and stored at -60°C in 10 ml ampoules. For the procurement of bone marrow cells, dog ribs were resected subperiosteally, cut into 2 cm fragments, and crushed with a heavy clamp. The marrow cells that protruded from thecut ends were flushed into a cold Petri dish by jets of Hank's Balanced Salt Solution (HBSS) containing 10 U heparin/ml at 4OC. The suspension was centrifugalized 709
CARIDIS
ALS AND BONE MARROW INFdslON IN IMMUNOSUPPRESSION TABLE1 Renal Allograft Survival in Controls, and Recipients Pretreated wifh Rabbit Antidog Lymphocyte SerurnfRADLS) and Later Infused with Live Donor-Speciftc Bone Marrow Cells (LDSBMC) Group 'Control A
B C D E
F
Number
7 7 6 7 6 5 10
RADLS LDSBMC 1 mlikg 1 ml/ko 2-4 x 2 mlik; 2 mlikg 2-4 x 2 mbkg 6-8 x 2 rnlikg 2-4 x
Survival (Days)
10Vko
10Vkg lOVkg(Day +lo) lOVkg(Day +10 +20)
10 10 1 1 12 14 14 14 15 16 16 16 17 14 17 19 28 31 33 '14 '17 23 23 24 25 '20 28 '38 41 56 '78 26 28 37 43 44 114 134 142 163 174 204 222 246 391 420
17 24 31
'No clinical or histological evidence of rejection at time of death
(200 G for 10 minutes) and washed twice with HBSS. Each preparation produced ayield of 1.6 x109+0.2x l o 9 nucleated cells per rib, of which more than 95% were viable as tested by the trypan blue exclusion method. Immediately after this viability test, the predetermined dose of LDSBMC was infused intravenously over a period of 10 to 15 minutes. Renal allografts were placed in the iliac fossa with an end-to-side anastomosis of the renal artery and vein into the external iliac artery and vein respectively. The ureter was implanted into the bladder. After completion of the renal transplant all recipient dogs underwent bilateral nephrectomy. Chromosome analysis was performed on cells from peripheral blood and splenic lymphocytes. For this 10 ml of blood or splenic suspension, to which 0.1 ml of preservative-free heparin and 0.1 ml of PHA were added, was allowed to separate at room temperature for two hours. Then 2 ml of plasma were added to 6 m l of culture medium (TC199:CSL). T o stimulate mitosis, 0.1 ml of 1 5 0 phytohaemagglutinin (Difco) with 0.2 ml of dog blood was used. This culture was then incubated at 37" C for 72 hours, at which time colchinine (0.05g/ml) was added, followed by an additional three hours' incubation. The cells were then washed with pre-warmed HBSS and suspended for seven minutes at 37" C in a hypotonic (0.75 M KCL) solution. Following centrifugalization the cells were fixed in a solution containing onethird glacial acetic acid and two-thirds methanol for 30 minutes. Slides were prepared and stained first with 1:lO Giemsa and then Grenisa buffer. In the dog, in contrast to the76achrocentricautosomes, the X chromosome is a prominent metacentric chromosome. The Y chromosome is also metacentric, but considerably smaller. Thus the distinction between male and female cells can be made easily. The rabbit antidog lymphocytic serum and donorspecific bone marrow cells were given on a per kilogram body weight basis, hereafter referred to as/kg. 710
-
The efficacy of RADLS was tested by total lymphocyte and white cell counts. In order to detect any GVH (graft versus host) linked endotoxaemia, endotoxin studies were performed at random using the lymulus lysate test (Reinhold and Fine, 1971). All dogs underwent autopsy at death. The pathological findings were recorded and the renal allografts were removed for subsequent histological studies. EXPERIMENTS AND RESULTS
Details of the composition of groups, treatment given, and times of survival of animals are given in Tables 1, 2, 3 and 4. In the control group (7 dogs - no treatment), rejection occurred within 10 to 17 days. After initial good function, kidney function deteriorated three to five days prior to death. Following this control experiment, the next group of dogs (Group A) were injected subcutaneously with low doses of RADLS ( 1 ml/kg) daily, from Day -7 to Day+7 relative to renal allograft transplantation on Day 0. These dogs exhibited only a modest renal TABLE2 Statistical Analysis of the Results, with Cornparison of Survival Times of the Groups Shown
Group
Mean Survival (Days) + S E M
Control
12 6 t 0 97
A
169t12
Control
12 6 i 0 97
P Value
INFUSION IN IMMUNOSUPPRESSION
CARIDIS
THE COMBINED USE OF ANTILYMPHOCYTIC , . SERUM AND BONE MARROW INFUSION IN IMMUNOLOGICAL SUPPRESSION D. T. CARIDIS University Department of Surgery, St Vincent's Hospital, Melbourne
The combined use of antilymphocytic serum and live donor-specific bone marrow cells has produced significant immunosuppressionln dogs as judged by renal allograft survival. This combined action is specific in that it cannot be reproduced whether given alone or when live donor-specific bone marrow cells are replaced by dead donor-specific bone marrow cells or other antigens. The study has paved the way for human trials.
IN SPITE of many developments, the late, unremitting, slow failure of renal allografts has remained essentially the same. This is largely due to the fact that suppression of the recipient's immune reactivity is ineffective against chronic rejection. The advantages, therefore, of rendering the recipient immunologically tolerant are immense. In this respect Monaco and Wood (1970) showed that low doses (10-50 x lo6)of non-hybrid, homozygous allogeneic bone marrow cells induced specific augmented survival of skin allografts in nonthymectomized, ALS-treated adult mice. They postulated that this combined therapy induced a state of immunological tolerance. These experiments suggested a potential application of the principle to human renal transplantation. However, this could not be attempted until it was demonstrated that extension to large animals produced similarly encouraging results, and that the principle was specific. METHODS Randomly selected, unrelated, unmatched mongrel dogs were used throughoutthestudy. Recipient donor combination was always of the opposite sex. The recipients were always males, and females were used for renal allograft and live donor-specific bone marrow cell (LDSBMC) procurement. Nineteen groups of experimental animals were studied, each consisting of 5 to 10 dogs. Rabbit anti-dog lymphocyte serum (RADLS), made from canine mesenteric Reprints University of Melbourne Department of Surgery, St Vincent's Hospital, Fitzroy. Victoria 3000
AUST. N.Z. J. SURG.VOL. 49 - No. 6, DECEMBER, 1979
lymph nodes, was given subcutaneously from Day -7 to Day +7, relative to renal transplant on Day 0. The rabbit antidog lymphocytic serum (RADLS) was prepared in adult white New Zealand rabbits. Canine mesenteric lymph nodes were cleaned of perinodal fat and adventitia, minced with scissors, and then passed through stainless steel wire meshes (60 gauge). The suspension was washed three times in saline, following which the lymphocytes were resuspended to a concentration of 50 x lo6 cell/ml. One ml of the suspension was emulsified with an equal volume of complete Freund's adjuvant and injected into the rabbits' footpads. Four weeks later the rabbits were injected subcutaneously in multiple sites with 50 x lo6 saline-suspended cells (no adjuvant) on three successive days. Seven days after the last booster injection the rabbits were exsanguinated, and the blood was kept at 4" C for 24 hours. The serum was then separated and centrifugalized. Subsequently it was inactivated by heating at 50" C for 30 minutes and absorbed twice with thricewashed erythrocytes in order to remove the haemolysin. After addition of 1:lOO.OOO methiolate the serum was pooled and stored at -60°C in 10 ml ampoules. For the procurement of bone marrow cells, dog ribs were resected subperiosteally, cut into 2 cm fragments, and crushed with a heavy clamp. The marrow cells that protruded from thecut ends were flushed into a cold Petri dish by jets of Hank's Balanced Salt Solution (HBSS) containing 10 U heparin/ml at 4OC. The suspension was centrifugalized 709
CARIDIS
ALS AND BONE MARROW INFdslON IN IMMUNOSUPPRESSION TABLE1 Renal Allograft Survival in Controls, and Recipients Pretreated wifh Rabbit Antidog Lymphocyte SerurnfRADLS) and Later Infused with Live Donor-Speciftc Bone Marrow Cells (LDSBMC) Group 'Control A
B C D E
F
Number
7 7 6 7 6 5 10
RADLS LDSBMC 1 mlikg 1 ml/ko 2-4 x 2 mlik; 2 mlikg 2-4 x 2 mbkg 6-8 x 2 rnlikg 2-4 x
Survival (Days)
10Vko
10Vkg lOVkg(Day +lo) lOVkg(Day +10 +20)
10 10 1 1 12 14 14 14 15 16 16 16 17 14 17 19 28 31 33 '14 '17 23 23 24 25 '20 28 '38 41 56 '78 26 28 37 43 44 114 134 142 163 174 204 222 246 391 420
17 24 31
'No clinical or histological evidence of rejection at time of death
(200 G for 10 minutes) and washed twice with HBSS. Each preparation produced ayield of 1.6 x109+0.2x l o 9 nucleated cells per rib, of which more than 95% were viable as tested by the trypan blue exclusion method. Immediately after this viability test, the predetermined dose of LDSBMC was infused intravenously over a period of 10 to 15 minutes. Renal allografts were placed in the iliac fossa with an end-to-side anastomosis of the renal artery and vein into the external iliac artery and vein respectively. The ureter was implanted into the bladder. After completion of the renal transplant all recipient dogs underwent bilateral nephrectomy. Chromosome analysis was performed on cells from peripheral blood and splenic lymphocytes. For this 10 ml of blood or splenic suspension, to which 0.1 ml of preservative-free heparin and 0.1 ml of PHA were added, was allowed to separate at room temperature for two hours. Then 2 ml of plasma were added to 6 m l of culture medium (TC199:CSL). T o stimulate mitosis, 0.1 ml of 1 5 0 phytohaemagglutinin (Difco) with 0.2 ml of dog blood was used. This culture was then incubated at 37" C for 72 hours, at which time colchinine (0.05g/ml) was added, followed by an additional three hours' incubation. The cells were then washed with pre-warmed HBSS and suspended for seven minutes at 37" C in a hypotonic (0.75 M KCL) solution. Following centrifugalization the cells were fixed in a solution containing onethird glacial acetic acid and two-thirds methanol for 30 minutes. Slides were prepared and stained first with 1:lO Giemsa and then Grenisa buffer. In the dog, in contrast to the76achrocentricautosomes, the X chromosome is a prominent metacentric chromosome. The Y chromosome is also metacentric, but considerably smaller. Thus the distinction between male and female cells can be made easily. The rabbit antidog lymphocytic serum and donorspecific bone marrow cells were given on a per kilogram body weight basis, hereafter referred to as/kg. 710
-
The efficacy of RADLS was tested by total lymphocyte and white cell counts. In order to detect any GVH (graft versus host) linked endotoxaemia, endotoxin studies were performed at random using the lymulus lysate test (Reinhold and Fine, 1971). All dogs underwent autopsy at death. The pathological findings were recorded and the renal allografts were removed for subsequent histological studies. EXPERIMENTS AND RESULTS
Details of the composition of groups, treatment given, and times of survival of animals are given in Tables 1, 2, 3 and 4. In the control group (7 dogs - no treatment), rejection occurred within 10 to 17 days. After initial good function, kidney function deteriorated three to five days prior to death. Following this control experiment, the next group of dogs (Group A) were injected subcutaneously with low doses of RADLS ( 1 ml/kg) daily, from Day -7 to Day+7 relative to renal allograft transplantation on Day 0. These dogs exhibited only a modest renal TABLE2 Statistical Analysis of the Results, with Cornparison of Survival Times of the Groups Shown
Group
Mean Survival (Days) + S E M
Control
12 6 t 0 97
A
169t12
Control
12 6 i 0 97
P Value
