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Homotransplantation by parabiosis
Homotransplantation by Parabiosis J U S T I N V. S C H W I N D , M . D . ,
Santa Barbara, Cali[ornia
S u c c e s s f u l h o m o t r a n s p l a n t a t i o n of t i s s u e in t h e h i g h e r a n i m a l s d e p e n d s n o t o n l y u p o n the e s t a b l i s h m e n t of an a d e q u a t e blood s u p p l y to t h e t r a n s p l a n t e d t i s s u e b u t a l s o upon r e n d e r i n g the host and g r a f t i m m u n o l o g i c a l l y a c c e p t a b l e to e a c h o t h e r . In r e c e n t Soviet a t t e m p t s at t r a n s p l a n t a t i o n of p a r t s as l a r g e ms a n e n t i r e e x t r e m i t y o r h e a d , a v e r y p r e c i s e s u r g i c a l union was a c h i e v e d with the a i d of a n e w s u t u r i n g d e v i c e f o r b l o o d v e s s e l s . S o m e of t h e s e leg and h e a d h o m o grafts persisted for as long as four or five w e e k s , with function of the e y e s , tongue, and o t h e r p a r t s of the t r a n s p l a n t e d h e a d . All a n i m a l s above the i n v e r t e b r a t e s have a n a t u r a l r e s i s t a n c e to f o r e i g n t i s s u e s , but t h i s u n i v e r s a l t r a n s p l a n t a t i o n i m m u n i t y is not p r e s e n t in the e a r l y s t a g e s of d e v e l o p m e n t . Thus when foreign tissues are introduced before immunologic maturity has appeared, the host does not r e c o g n i z e such t i s s u e s as f o r e i g n , a n d w i l l n o t r e j e c t g r a f t s of t i s s u e from this donor at a laterdate. Inthe present series of experiments we are taking advantage of t h i s n a t u r a l l a c k o f a n t i b o d y r e a c t i o n a t a n e a r l y a g e to p r o d u c e a n i m a l s i m m u n o l o g i c a l l y t o l e r a n t to t h e t i s s u e s o f o n e o r m o r e d o n o r s . In t h e r a t i m m u n o l o g i c m a t u r i t y d o e s not a p p e a r until a f t e r two w e e k s . When a n i m a l s y o u n g e r t h a n two w e e k s a r e j o i n e d i n p a r a biosis their blood streams fuse after a few days, as has been demonstrated many times with dye a n d l a b e l e d i r o n s t u d i e s . T h i s a d m i x t u r e of b l o o d a l l o w s a t r a d i n g o f t r a n s p l a n t a t i o n a n t i g e n s b e t w e e n p r o s p e c t i v e h o s t and d o n o r , so t h a t a n y l a t e r t i s s u e c r o s s g r a f t s act like a u t o g r a f t s , i m m u n e f r o m the usual homograft rejection.
p a r t i a l l y a m p u t a t e d l e g , a s s e e n i n F i g u r e 1. The host's own leg was completely amputated a n d d i s c a r d e d , in s o m e e a s e s a t m i d - t h i g h l e v e l , and in o t h e r s at m i d - t i b i a l e v e l . A p a r t i a l a m p u t a t i o n at a s i m i l a r l e v e l was p e r f o r m e d o n t h e l e g to b e t r a n s p l a n t e d , so t h a t w h e n t h e r e c i p i e n t b o n e w a s j o i n e d to the donor bone a relatively normal length of e x t r e m i t y w a s r e s t o r e d . T h e r e m a i n i n g p e d i c l e a t t a c h m e n t to t h e d o n o r c o n t a i n e d the u n d i v i d e d f e m o r a l a r t e r y and vein, and t h e s e w e r e l i g a t e d and d i v i d e d at the t i m e of the final c o m p l e t i o n of a m p u t a t i o n f r o m the d o n o r . T h e c u t e n d s of t h e t i b i a ( o r f e m u r ) were pulled together over an intramedullary pin made from a small hypodermic needle. T h e s c i a t i c n e r v e o f the d o n o r w a s u n i t e d with the p r o x i m a l cut end of the s c i a t i c n e r v e in the leg being t r a n s f e r r e d . These c u t n e r v e e n d s w e r e l a l d e n d to e n d i n a s m a l l s p l i t m a d e in a n e a r b y m u s c l e m a s s , a n d a f i n e s u t u r e p l a c e d to h o l d t h e m u s c l e together over the joined nerves. S u t u r e m a t e r i a l w a s m a d e f r o m N o . 60 cotton thread untwisted into its sixcomposite strands. Seven millimeter eye needles were m a d e s t i l l s m a l l e r by g r i n d i n g down on a sharpening stone. With this tiny needle and a v e r y f i n e s u t u r e m a t e r i a l it w a s p o s s i b l e to p r o d u c e a m o r e p r e c i s e s u r g i c a l a p p o s i t i o n
E,
tt~ S.G. 9 SPLEEN
MATERIALS AND METHODS In t h e w o r k r e p o r t e d h e r e t e n d a y o l d animals, usually littermates, or nonlittermates of s i m i l a r age, w e r e j o i n e d by a
2235 Castillo St., Santa B a r b a r a , C a l i f o r n i a . Submitted for publication N o v e m b e r 29, 1961. 332
F i g . 1. IMethod of attaching partial17 amputated leg to the stump of the r e c i p i e n t . After p a r a b i o s i s the animal on the right becomes the p o s s e s s o r of the right hind leg of the donor on the left. ]SR
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of the nerves and muscles. T h e skin w a s joined over the leg attached to the parabiotic partners. During this period of parabiosis the host and donor w e r e bound together with two layers of adhesive tape, which w a s adjusted as the animals g r e w larger. Figure 2 is a radiograph of two such animals bound together with a metal pin connecting donor tibia with recipient tibia. After about ten days the transplanted leg was well attached to the recipient and no sign of the skin incision remained. At this stage a small incision was made in the skin of the leg on the donor side and a ligature was placed across a part of the remaining attachment to the donor. And in two or three more stages, with the aid of procaine, the leg was completely amputated from the donor. If good vascular union had been established w i t h the" h o s t , the leg remained pink and began to grow at the same rate as the host's own legs. In approximately one month sensation to painful stimulus appeared in the new leg, and if a particularly precise nerve union was established some motion appeared after one month in the muscles of the new extremity. In ordinary parabiotic unions successful transfer of a leg has been accomplished in 30 t o 4 0 p e r c e n t o f t h e a t t e m p t s . In one
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series II out of 31 attempts resulted in a p e r m a n e n t l y accepted leg. But if a small piece of spleen tissue w a s r e m o v e d f r o m each animal, m i n c e d and placed subcutaneously into the other animal, the percentage of successful parabiotic unions and successful l e g transfer w a s increased t o about 75 t o 80 p e r c e n t . I n o n e s m a l l series of five parabiotic unions, four resulted in a successful transfer of a leg from donor to host. Medawar, Martinez, Good and others have shown that spleen cell suspensions have the ability to induce tolerance to tissues of the s p l e e n c e l l d o n o r . 2.3. 4 W h e n t h e y o u n g r a t s are about two days old, an incision is made over the spleen area and about one half of the spleen is ligated with the fine cotton suture and removed. It is minced and immediately placed in a subcutaneous pocket of the other animal, and the incisions are closed with the same type of cotton suture. If a host animal is to be conditioned to two or more prospective donors, this is accomplished by a piece of spleen being implanted into the host from each of the possible donors and the host spleen is divided a m o n g the donors. In a search for a m o r e readily procurable tissue than spleen, it was found that cross implantation of a
F i g . 2. R a d i o g r a p h of two a n i m a l s j o i n e d in p a r a b i o s i s w i t h a m e t a l p i n c o n n e c t i n g d o n o r t i b i a to t h e t i b i a of the r e c i p i e n t . T h e r e m a i n i n g p e d i c l e to the d o n o r on t h e l e f t c a n be s e e n . W i t h i n t h i s p e d i c l e a r e the u n c u t f e m o r a l v e s s e l s f r o m t h e d o n o r w h i c h n o u r i s h the leg u n t i l it e s t a b l i s h e s a s e l f s u s t a i n i n g b l o o d s u p p l y f r o m the r e c i p i e n t on t h e r i g h t .
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p i e c e of s a l i v a r y g l a n d b e t w e e n d o n o r a n d recipient rats was effective in inducing immunologic tolerance for subsequent direct t r a n s p l a n t s of s k i n f r o m e a c h to the o t h e r . S k i n g r a f t t e s t i n g of the o r i g i n a l b r e e d i n g s t o c k w a s d o n e to a s s u r e a n o n - i n b r e d genetic b a c k g r o u n d . White f e m a l e s w e r e m a t e d w i t h c o l o r e d m a l e s of a d i f f e r e n t s t r a i n , w h i c h r e s u l t e d i n o f f s p r i n g of m a n y color combinations, and this facilitated skin graft testing observations. Dark haired skin was p l a c e d on white b a c k g r o u n d s and vice v e r s a . I n a d d i t i o n the s k i n g r a f t s w e r e r o t a t e d 180 d e g r e e s to m a k e t h e h a i r s t a n d out f r o m the h o s t ' s h a i r . T h e s k i n g r a f t s w e r e s e c u r e d to the h o s t w i t h f i v e o r s i x s t i t c h e s , a n d a l a y e r of s p o n g e r u b b e r w a s t a p e d o v e r the e n t i r e g r a f t .
RESULTS A t the d a t e of w r i t i n g it i s t h i r t e e n months since three rats had amputated legs r e p l a c e d by legs f r o m o t h e r a n i m a l s and these rats are still walking about on their replaced legs. Four other animals with replaced right rear legs are still using these legs ten months after transplantation. F u n c t i o n a l c a p a c i t y of t h e s e r e p l a c e d l e g s is n o t p e r f e c t , s i n c e a f t e r t r a n s p l a n t a t i o n the h o s t h a s two l e f t h i n d l e g s . B e c a u s e of the d i f f i c u l t y of p o s i t i o n i n g t h e a n i m a l s d u r i n g p a r a b i o s i s , it w a s e a s i e r to r e p l a c e a right hind leg with a left hind leg f r o m the donor. In twelve other cases extra legs were p l a c e d u p o n t h e b a c k s of a n i m a l s ( F i g . 3). These dorsally placed legs were innervated b y a b r a n c h of t h e h o s t ' s s c i a t i c n e r v e r e d i r e c t e d u p the b a c k to t h e n e w l e g a s i n F i g u r e 4. W h e n the f i f t h l e g i s s t i m u l a t e d by pinching, the host leg, which donated p a r t of i t s n e r v e , i s m o v e d , a n d w h e n the h o s t ' s own leg is s t i m u l a t e d , m o t i o n c a n be d e m o n s t r a t e d in the extra leg on the back by lifting a small weight. After a leg has been transferred by p a r a b i o t i c u n i o n , and the u n i o n is d i s c o n tinued, the s e p a r a t e d p a r t n e r s r e m a i n t o l e r a n t of s u b s e q u e n t d i r e c t t r a n s p l a n t s of t i s s u e from each other, as noted in a previous p a p e r . F o r i n s t a n c e o n e h o s t b e a r i n g a leg from another animal accepted a skin graft f r o m t h e d o n o r t h r e e m o n t h s a f t e r t h e leg graft and still another skin graft placed six m o n t h s a f t e r the o r i g i n a l graft. C o n t r o l g r a f t s f r o m r a t s o t h e r than the donor w e r e rejected.
Fig. 3. Extra leg placed up on the back of the host, 13 months after transplantation. R a d i o g r a p h s of t r a n s p l a n t e d e x t r e m i t i e s , six months after transplantation, showed b o n e g r o w t h a n d a d e g r e e of c a l c i f i c a t i o n i n d i s t i n g u i s h a b l e f r o m t h a t of l e g b o n e s of the h o s t . F i g u r e 5 s h o w s a f u l l y c l e v e l o p e d a n d c o m p l e t e l y c a l c i f i e d foot a s c o m p a r e d to the r e l a t i v e l y i m m a t u r e c o n d i t i o n a t the t i m e of t r a n s p l a n t a t i o n . F i g u r e 6 s h o w s a t r a n s p l a n t e d l e g u n i t e d to t h e h o s t , w i t h the tibia firmly united over a small intramedullary pin. Radiographically and on gross
Fig. 4. I n n e r v a t i o n of extra leg on the back by a branch of the h o s t ' s own sciatic nerve.
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Fig. 5. F u l l y developed f o o t on t h e r i g h t a s c o m p a r e d t o the p o o r l y c a l c i f i e d i m m a t u r e stage at the t i m e of t r a n s p l a n t a t i o n . Fig. 6. H o m o t r a n s p l a n t e d l o w e r leg and foot with the g r a f t e d tibia u n i t e d to the h o s t tibia o v e r a m e t a l pin, and showing f i r m c a l c i f i c a t i o n of the t r a n s p l a n t e d bone. dissection there is a sturdy union of the proximal portion of the host's tibia with the distal portion of donor tibia. The same method of tissue transfer was used to transfer other body parts. Figure 7 shows a rat. with an extra tail. A b d o m i n a l organs which can be pulled out and attached under the skin of an adjoined animal can be transplanted f r o m one animal to another. In three cases a testicle w a s exteriorized, leaving its pedicle intact, and allowed to attach subcutaneously in another animal while the two animals w e r e maintained in parabiosis. Portions of the spleen w e r e also transferred to another in two cases. Histologic sections of these testicles and spleens after two m o n t h s in the n e w hosts s h o w e d n o r m a l morphology. Histologic studies of the m a r r o w , muscle, bone and skin of transplanted legs d e m o n s t r a t e d no fundamental change in their microscopic architecture.
persists. After immunologic maturity is accomplished the host continues to recognize this donor's tissues as self, and the implantation of tissues from this host at a later date is not met with the usual homograft rejection. However, tissues from other donors meet with destructive antibody reaction and eventual rejection.
DISCUSSION Homotransplantation of tissues other than cornea or cartilage evokes an immunologic reaction on the part of the host, resulting in destruction of the graft, unless the host and donor are identical twins, or fraternal twins which had a fusion of their placentas during e m b r y o n i c development. H o w e v e r , if a Prospective host is exposed to living cells f r o m a possible donor at a stage of development before i m m u n o l o g i c maturity is reached, a tolerance for the donor's tissues
F i g . 7. E x t r a upon this h o s t .
tall ten months after p l a c e m e n t
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Combining two or more animals in parabiosis at a stage of development before antibody synthesis is possible renders these parabiotic partners immunologicallytolerant of e a c h o t h e r ' s t i s s u e s , so t h a t a f t e r s e p a r a t i o n o f t h e p a r a b i o s i s e a c h i n d i v i d u a l f a i l s to recognize as foreign the tissues of its former parabiotic partner. This parabiotic unionalso serves the purpose of establishing an adequate b l o o d s u p p l y to a g r a f t e d e x t r e m i t y . For instance a leg being transferred has a double blood supply for a time, partly from the unamputated remaining a t t a c h m e n t to t h e d o n o r , a n d p a r t l y f r o m i t s n e w a t t a c h m e n t to the recipient. At first this vascular attachm e n t to t h e r e c i p i e n t i s m e r e l y t h r o u g h capillary outgrowth fro:~ the donor. Four months after transplantation grafted legs have been removed for study and blood vessels l a r g e e n o u g h to b e s e e n w i t h t h e n a k e d e y e have been observed. Apparently some of the o r i g i n a l c a p i l l a r y o u t g r o w t h s m e r g e d to f o r m a g r o s s l y d e t e c t a b l e s e t o f b l o o d v e s s e l s , to meet the physiologic demands of the enlarging g r a f t e d l e g , SUMMARY AND
CONCLUSIONS
1. T i s s u e h o m o g r a f t r e j e c t i o n c a n b e i n hibited in rats by placing the donor and recipient in parabio s is at an immunologically immature age.
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2. Parabiosis accomplishes the double objective of establishing an adequate blood supply to the graft while it is also inducing tolerance in the host to the graft. 3. This inhibition of antibody synthesis is p e r m a n e n t and complete enough to induce the host to accept a graft as large as an entire extremity and retain it permanently, 4. Transplanted legs m a t u r e at a rate c o m p a r a b l e to that of the host:s o w n legs, and these n e w legs develop sensation and a limited d e g r e e of usefulness.
REFERENCES i. Demikhov, V. P.: The Experimental Transplantation of Organs. Ztschr. Aerztl Fortbild., No. 6, 1959. 2. Billingham, R. E., Brent, L., and IViedawar, P. B.: Actively acquired tolerance offoreigncells. Nature, 172: 603-608, 1953. 3. Mariani, T., Martinez, C., Smith, J. M., and Good, R. A.: Induction of immunological tolerance to male skin isografts in female mice subsequent to the neonatal period. Proc. Soc. Exper. Biol. & Med., 101: 596-599, 1959. 4. N~artinez, C.: Essential duration ofparabiosis and development of tolerance to skin homografts in mice. Proc. Soc. Exper. Biol. Ivled., 104: 256-269, 1960. 5. Schwind, J. V.: Successful homotransplantation of organs and extremities in rats. Westegn Med., 2: 540-544, 1961.
Santa Barbara, Cali[ornia
S u c c e s s f u l h o m o t r a n s p l a n t a t i o n of t i s s u e in t h e h i g h e r a n i m a l s d e p e n d s n o t o n l y u p o n the e s t a b l i s h m e n t of an a d e q u a t e blood s u p p l y to t h e t r a n s p l a n t e d t i s s u e b u t a l s o upon r e n d e r i n g the host and g r a f t i m m u n o l o g i c a l l y a c c e p t a b l e to e a c h o t h e r . In r e c e n t Soviet a t t e m p t s at t r a n s p l a n t a t i o n of p a r t s as l a r g e ms a n e n t i r e e x t r e m i t y o r h e a d , a v e r y p r e c i s e s u r g i c a l union was a c h i e v e d with the a i d of a n e w s u t u r i n g d e v i c e f o r b l o o d v e s s e l s . S o m e of t h e s e leg and h e a d h o m o grafts persisted for as long as four or five w e e k s , with function of the e y e s , tongue, and o t h e r p a r t s of the t r a n s p l a n t e d h e a d . All a n i m a l s above the i n v e r t e b r a t e s have a n a t u r a l r e s i s t a n c e to f o r e i g n t i s s u e s , but t h i s u n i v e r s a l t r a n s p l a n t a t i o n i m m u n i t y is not p r e s e n t in the e a r l y s t a g e s of d e v e l o p m e n t . Thus when foreign tissues are introduced before immunologic maturity has appeared, the host does not r e c o g n i z e such t i s s u e s as f o r e i g n , a n d w i l l n o t r e j e c t g r a f t s of t i s s u e from this donor at a laterdate. Inthe present series of experiments we are taking advantage of t h i s n a t u r a l l a c k o f a n t i b o d y r e a c t i o n a t a n e a r l y a g e to p r o d u c e a n i m a l s i m m u n o l o g i c a l l y t o l e r a n t to t h e t i s s u e s o f o n e o r m o r e d o n o r s . In t h e r a t i m m u n o l o g i c m a t u r i t y d o e s not a p p e a r until a f t e r two w e e k s . When a n i m a l s y o u n g e r t h a n two w e e k s a r e j o i n e d i n p a r a biosis their blood streams fuse after a few days, as has been demonstrated many times with dye a n d l a b e l e d i r o n s t u d i e s . T h i s a d m i x t u r e of b l o o d a l l o w s a t r a d i n g o f t r a n s p l a n t a t i o n a n t i g e n s b e t w e e n p r o s p e c t i v e h o s t and d o n o r , so t h a t a n y l a t e r t i s s u e c r o s s g r a f t s act like a u t o g r a f t s , i m m u n e f r o m the usual homograft rejection.
p a r t i a l l y a m p u t a t e d l e g , a s s e e n i n F i g u r e 1. The host's own leg was completely amputated a n d d i s c a r d e d , in s o m e e a s e s a t m i d - t h i g h l e v e l , and in o t h e r s at m i d - t i b i a l e v e l . A p a r t i a l a m p u t a t i o n at a s i m i l a r l e v e l was p e r f o r m e d o n t h e l e g to b e t r a n s p l a n t e d , so t h a t w h e n t h e r e c i p i e n t b o n e w a s j o i n e d to the donor bone a relatively normal length of e x t r e m i t y w a s r e s t o r e d . T h e r e m a i n i n g p e d i c l e a t t a c h m e n t to t h e d o n o r c o n t a i n e d the u n d i v i d e d f e m o r a l a r t e r y and vein, and t h e s e w e r e l i g a t e d and d i v i d e d at the t i m e of the final c o m p l e t i o n of a m p u t a t i o n f r o m the d o n o r . T h e c u t e n d s of t h e t i b i a ( o r f e m u r ) were pulled together over an intramedullary pin made from a small hypodermic needle. T h e s c i a t i c n e r v e o f the d o n o r w a s u n i t e d with the p r o x i m a l cut end of the s c i a t i c n e r v e in the leg being t r a n s f e r r e d . These c u t n e r v e e n d s w e r e l a l d e n d to e n d i n a s m a l l s p l i t m a d e in a n e a r b y m u s c l e m a s s , a n d a f i n e s u t u r e p l a c e d to h o l d t h e m u s c l e together over the joined nerves. S u t u r e m a t e r i a l w a s m a d e f r o m N o . 60 cotton thread untwisted into its sixcomposite strands. Seven millimeter eye needles were m a d e s t i l l s m a l l e r by g r i n d i n g down on a sharpening stone. With this tiny needle and a v e r y f i n e s u t u r e m a t e r i a l it w a s p o s s i b l e to p r o d u c e a m o r e p r e c i s e s u r g i c a l a p p o s i t i o n
E,
tt~ S.G. 9 SPLEEN
MATERIALS AND METHODS In t h e w o r k r e p o r t e d h e r e t e n d a y o l d animals, usually littermates, or nonlittermates of s i m i l a r age, w e r e j o i n e d by a
2235 Castillo St., Santa B a r b a r a , C a l i f o r n i a . Submitted for publication N o v e m b e r 29, 1961. 332
F i g . 1. IMethod of attaching partial17 amputated leg to the stump of the r e c i p i e n t . After p a r a b i o s i s the animal on the right becomes the p o s s e s s o r of the right hind leg of the donor on the left. ]SR
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of the nerves and muscles. T h e skin w a s joined over the leg attached to the parabiotic partners. During this period of parabiosis the host and donor w e r e bound together with two layers of adhesive tape, which w a s adjusted as the animals g r e w larger. Figure 2 is a radiograph of two such animals bound together with a metal pin connecting donor tibia with recipient tibia. After about ten days the transplanted leg was well attached to the recipient and no sign of the skin incision remained. At this stage a small incision was made in the skin of the leg on the donor side and a ligature was placed across a part of the remaining attachment to the donor. And in two or three more stages, with the aid of procaine, the leg was completely amputated from the donor. If good vascular union had been established w i t h the" h o s t , the leg remained pink and began to grow at the same rate as the host's own legs. In approximately one month sensation to painful stimulus appeared in the new leg, and if a particularly precise nerve union was established some motion appeared after one month in the muscles of the new extremity. In ordinary parabiotic unions successful transfer of a leg has been accomplished in 30 t o 4 0 p e r c e n t o f t h e a t t e m p t s . In one
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series II out of 31 attempts resulted in a p e r m a n e n t l y accepted leg. But if a small piece of spleen tissue w a s r e m o v e d f r o m each animal, m i n c e d and placed subcutaneously into the other animal, the percentage of successful parabiotic unions and successful l e g transfer w a s increased t o about 75 t o 80 p e r c e n t . I n o n e s m a l l series of five parabiotic unions, four resulted in a successful transfer of a leg from donor to host. Medawar, Martinez, Good and others have shown that spleen cell suspensions have the ability to induce tolerance to tissues of the s p l e e n c e l l d o n o r . 2.3. 4 W h e n t h e y o u n g r a t s are about two days old, an incision is made over the spleen area and about one half of the spleen is ligated with the fine cotton suture and removed. It is minced and immediately placed in a subcutaneous pocket of the other animal, and the incisions are closed with the same type of cotton suture. If a host animal is to be conditioned to two or more prospective donors, this is accomplished by a piece of spleen being implanted into the host from each of the possible donors and the host spleen is divided a m o n g the donors. In a search for a m o r e readily procurable tissue than spleen, it was found that cross implantation of a
F i g . 2. R a d i o g r a p h of two a n i m a l s j o i n e d in p a r a b i o s i s w i t h a m e t a l p i n c o n n e c t i n g d o n o r t i b i a to t h e t i b i a of the r e c i p i e n t . T h e r e m a i n i n g p e d i c l e to the d o n o r on t h e l e f t c a n be s e e n . W i t h i n t h i s p e d i c l e a r e the u n c u t f e m o r a l v e s s e l s f r o m t h e d o n o r w h i c h n o u r i s h the leg u n t i l it e s t a b l i s h e s a s e l f s u s t a i n i n g b l o o d s u p p l y f r o m the r e c i p i e n t on t h e r i g h t .
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p i e c e of s a l i v a r y g l a n d b e t w e e n d o n o r a n d recipient rats was effective in inducing immunologic tolerance for subsequent direct t r a n s p l a n t s of s k i n f r o m e a c h to the o t h e r . S k i n g r a f t t e s t i n g of the o r i g i n a l b r e e d i n g s t o c k w a s d o n e to a s s u r e a n o n - i n b r e d genetic b a c k g r o u n d . White f e m a l e s w e r e m a t e d w i t h c o l o r e d m a l e s of a d i f f e r e n t s t r a i n , w h i c h r e s u l t e d i n o f f s p r i n g of m a n y color combinations, and this facilitated skin graft testing observations. Dark haired skin was p l a c e d on white b a c k g r o u n d s and vice v e r s a . I n a d d i t i o n the s k i n g r a f t s w e r e r o t a t e d 180 d e g r e e s to m a k e t h e h a i r s t a n d out f r o m the h o s t ' s h a i r . T h e s k i n g r a f t s w e r e s e c u r e d to the h o s t w i t h f i v e o r s i x s t i t c h e s , a n d a l a y e r of s p o n g e r u b b e r w a s t a p e d o v e r the e n t i r e g r a f t .
RESULTS A t the d a t e of w r i t i n g it i s t h i r t e e n months since three rats had amputated legs r e p l a c e d by legs f r o m o t h e r a n i m a l s and these rats are still walking about on their replaced legs. Four other animals with replaced right rear legs are still using these legs ten months after transplantation. F u n c t i o n a l c a p a c i t y of t h e s e r e p l a c e d l e g s is n o t p e r f e c t , s i n c e a f t e r t r a n s p l a n t a t i o n the h o s t h a s two l e f t h i n d l e g s . B e c a u s e of the d i f f i c u l t y of p o s i t i o n i n g t h e a n i m a l s d u r i n g p a r a b i o s i s , it w a s e a s i e r to r e p l a c e a right hind leg with a left hind leg f r o m the donor. In twelve other cases extra legs were p l a c e d u p o n t h e b a c k s of a n i m a l s ( F i g . 3). These dorsally placed legs were innervated b y a b r a n c h of t h e h o s t ' s s c i a t i c n e r v e r e d i r e c t e d u p the b a c k to t h e n e w l e g a s i n F i g u r e 4. W h e n the f i f t h l e g i s s t i m u l a t e d by pinching, the host leg, which donated p a r t of i t s n e r v e , i s m o v e d , a n d w h e n the h o s t ' s own leg is s t i m u l a t e d , m o t i o n c a n be d e m o n s t r a t e d in the extra leg on the back by lifting a small weight. After a leg has been transferred by p a r a b i o t i c u n i o n , and the u n i o n is d i s c o n tinued, the s e p a r a t e d p a r t n e r s r e m a i n t o l e r a n t of s u b s e q u e n t d i r e c t t r a n s p l a n t s of t i s s u e from each other, as noted in a previous p a p e r . F o r i n s t a n c e o n e h o s t b e a r i n g a leg from another animal accepted a skin graft f r o m t h e d o n o r t h r e e m o n t h s a f t e r t h e leg graft and still another skin graft placed six m o n t h s a f t e r the o r i g i n a l graft. C o n t r o l g r a f t s f r o m r a t s o t h e r than the donor w e r e rejected.
Fig. 3. Extra leg placed up on the back of the host, 13 months after transplantation. R a d i o g r a p h s of t r a n s p l a n t e d e x t r e m i t i e s , six months after transplantation, showed b o n e g r o w t h a n d a d e g r e e of c a l c i f i c a t i o n i n d i s t i n g u i s h a b l e f r o m t h a t of l e g b o n e s of the h o s t . F i g u r e 5 s h o w s a f u l l y c l e v e l o p e d a n d c o m p l e t e l y c a l c i f i e d foot a s c o m p a r e d to the r e l a t i v e l y i m m a t u r e c o n d i t i o n a t the t i m e of t r a n s p l a n t a t i o n . F i g u r e 6 s h o w s a t r a n s p l a n t e d l e g u n i t e d to t h e h o s t , w i t h the tibia firmly united over a small intramedullary pin. Radiographically and on gross
Fig. 4. I n n e r v a t i o n of extra leg on the back by a branch of the h o s t ' s own sciatic nerve.
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Fig. 5. F u l l y developed f o o t on t h e r i g h t a s c o m p a r e d t o the p o o r l y c a l c i f i e d i m m a t u r e stage at the t i m e of t r a n s p l a n t a t i o n . Fig. 6. H o m o t r a n s p l a n t e d l o w e r leg and foot with the g r a f t e d tibia u n i t e d to the h o s t tibia o v e r a m e t a l pin, and showing f i r m c a l c i f i c a t i o n of the t r a n s p l a n t e d bone. dissection there is a sturdy union of the proximal portion of the host's tibia with the distal portion of donor tibia. The same method of tissue transfer was used to transfer other body parts. Figure 7 shows a rat. with an extra tail. A b d o m i n a l organs which can be pulled out and attached under the skin of an adjoined animal can be transplanted f r o m one animal to another. In three cases a testicle w a s exteriorized, leaving its pedicle intact, and allowed to attach subcutaneously in another animal while the two animals w e r e maintained in parabiosis. Portions of the spleen w e r e also transferred to another in two cases. Histologic sections of these testicles and spleens after two m o n t h s in the n e w hosts s h o w e d n o r m a l morphology. Histologic studies of the m a r r o w , muscle, bone and skin of transplanted legs d e m o n s t r a t e d no fundamental change in their microscopic architecture.
persists. After immunologic maturity is accomplished the host continues to recognize this donor's tissues as self, and the implantation of tissues from this host at a later date is not met with the usual homograft rejection. However, tissues from other donors meet with destructive antibody reaction and eventual rejection.
DISCUSSION Homotransplantation of tissues other than cornea or cartilage evokes an immunologic reaction on the part of the host, resulting in destruction of the graft, unless the host and donor are identical twins, or fraternal twins which had a fusion of their placentas during e m b r y o n i c development. H o w e v e r , if a Prospective host is exposed to living cells f r o m a possible donor at a stage of development before i m m u n o l o g i c maturity is reached, a tolerance for the donor's tissues
F i g . 7. E x t r a upon this h o s t .
tall ten months after p l a c e m e n t
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Combining two or more animals in parabiosis at a stage of development before antibody synthesis is possible renders these parabiotic partners immunologicallytolerant of e a c h o t h e r ' s t i s s u e s , so t h a t a f t e r s e p a r a t i o n o f t h e p a r a b i o s i s e a c h i n d i v i d u a l f a i l s to recognize as foreign the tissues of its former parabiotic partner. This parabiotic unionalso serves the purpose of establishing an adequate b l o o d s u p p l y to a g r a f t e d e x t r e m i t y . For instance a leg being transferred has a double blood supply for a time, partly from the unamputated remaining a t t a c h m e n t to t h e d o n o r , a n d p a r t l y f r o m i t s n e w a t t a c h m e n t to the recipient. At first this vascular attachm e n t to t h e r e c i p i e n t i s m e r e l y t h r o u g h capillary outgrowth fro:~ the donor. Four months after transplantation grafted legs have been removed for study and blood vessels l a r g e e n o u g h to b e s e e n w i t h t h e n a k e d e y e have been observed. Apparently some of the o r i g i n a l c a p i l l a r y o u t g r o w t h s m e r g e d to f o r m a g r o s s l y d e t e c t a b l e s e t o f b l o o d v e s s e l s , to meet the physiologic demands of the enlarging g r a f t e d l e g , SUMMARY AND
CONCLUSIONS
1. T i s s u e h o m o g r a f t r e j e c t i o n c a n b e i n hibited in rats by placing the donor and recipient in parabio s is at an immunologically immature age.
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2. Parabiosis accomplishes the double objective of establishing an adequate blood supply to the graft while it is also inducing tolerance in the host to the graft. 3. This inhibition of antibody synthesis is p e r m a n e n t and complete enough to induce the host to accept a graft as large as an entire extremity and retain it permanently, 4. Transplanted legs m a t u r e at a rate c o m p a r a b l e to that of the host:s o w n legs, and these n e w legs develop sensation and a limited d e g r e e of usefulness.
REFERENCES i. Demikhov, V. P.: The Experimental Transplantation of Organs. Ztschr. Aerztl Fortbild., No. 6, 1959. 2. Billingham, R. E., Brent, L., and IViedawar, P. B.: Actively acquired tolerance offoreigncells. Nature, 172: 603-608, 1953. 3. Mariani, T., Martinez, C., Smith, J. M., and Good, R. A.: Induction of immunological tolerance to male skin isografts in female mice subsequent to the neonatal period. Proc. Soc. Exper. Biol. & Med., 101: 596-599, 1959. 4. N~artinez, C.: Essential duration ofparabiosis and development of tolerance to skin homografts in mice. Proc. Soc. Exper. Biol. Ivled., 104: 256-269, 1960. 5. Schwind, J. V.: Successful homotransplantation of organs and extremities in rats. Westegn Med., 2: 540-544, 1961.