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The problem of the noninbred animal in tissue transplantation
The Problem of the Noninbred Animal in Tissue Transplantation D. G. McQUARRIE, M.D., ROBERT S. BROWN, B. A., AND RICHARD L. VARCO, M. D., Ph.D.,
University o[ Minnesota Hospitals
Billingham and Silvers s have pointed out the desirability and e s s e n t i a l criteria for inbred experimental animals for transplantation s t u d i e s . In some types of experimental s t udi e s the id eal is not r e a l i s t i c a l l y obtainable, and it is n e c e s sary to use experimental a n i m a l s that are not inbred for histocompatibility factors and g e n e t i c identity. Such use of noninbred animals may be n e c e s s a r y in order to expand observations to other s p e c i e s , to get larger t i s s u e samples, or to d o procedures and make observations not possi'bte in the more ubiquitous inbred mice. Although there are inbred s t r a i n s of rats, rabbits, c h i c k e n s and hamsters, the problem of obtaining adequate n u m b e r s , in a variety of strains and at a r e a s o n a b l e cost, frequently forces the a c c e p t a n c e of that l e s s d e s i r a b l e alternative: use of animals of unknown g e n e t i c composition. There are no i s o I o g o u s inbred strains of dogs* known to the authors. There are a large number of s i gni f i c a nt contributions in the field of t i s s u e transplantation i n which noninbred rats a n d rabbits,9,13,1e.22 dogs Is,z1 and the g e n e t i c a l l y h e t e r o g e n e o u s h u m a n ~ ° , l s have been the experimental s p e c i e s . An occasional study is seen in which the experimental design has erroneously implied that animals derived from d i f f e r e n t s o u r c e s have g e n e t i c heterogenity and that there is some positive information on the genetic c o m p o s i t i o n o f the animals b e c a u s e they h a v e been s e l e c t e d from mixed populations or separate c o l o n i e s .
From the Department o f Surgery, University of Minnesota Hospitals, Minneapolis, Minnesota. This study was aided by grants from ~the U. S. Publlc HeaIth Service, The Graduate School of the University of Minnesota, and Richard L. V a r c o Surgical Research Fund. Submitted for publication April 19, 1962. *Several attempt s ate under way to establish inbred beagle Colonies.
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The following s t u d i e s were done for three reasons. First, the authors frequently find it n e c e s s a r y to u s e large noninbred animals from commercial s o u r c e s . Second, there appeared to be some marked effect of " s t r a i n ' ' t of commercial origin on graft survival in rats. Third, we wished to find some of the v a r i a b l e s that may exist when noninbred animals are u s e d in order to improve experimefftat design. We b e l i e v e the data of these simple s t u d i e s may have i n t e r e s t b e c a u s e they i l l u s t r a t e the c o n s i d e r a b l e range of some s y s t e m a t i c v a r i a b l e s which may creep into experiments and confuse interpretation when noninbred animals are used.
METII 0 I}S For more nearly related " s t r a i n s , " s e p a r a t e albino rats of Sprague-Dawley origin were obtained in 1958-I959 from the Holtzman Company and from the Rolfsmeyer Company. T h e s e colonies had been c l o s e d s i n c e 1952 and 1954, r e s p e c t i v e l y . For comparison of " s t r a i n s " with more divergent g e n e t i c background, L c n g - E v a n s rats from Rockland Farms were u sed . All animals for reciprocal grafting were males weighing 150 to I75 gin. R e c i p r o c a l grafts were performed simultaneously between each of the ' * s t r a i n " pairs, giving the six groups of animals l i s t e d in Table I. To study the effects of " s t r a i n " on the induction of t o l e r a n c e , newborn rats in groups with the same six " s t r a i n " combinations were i n j e c t e d intraperitoneally with spleen brei (approximately 10 million ceils) during the first 24 hours after birth. G r a f t i n g was done at two months. Spleen donors were adult animals, later to be u s e d as skin donors. Animals were considered tolerant if the grafted skin appeared ,The use of the work **straln" does not imply any degree of inbreeding b u t s e r v e s to distinguish different commercial sources of noninbred rats.
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normal in s i z e and texture, had regrowth of hair, and survived 90 days. Grafting p r o c e d u r e s were done similarly in all groups. After shaving the ventral s u r f a c e of the animals with e l e c t r i c clippers, and c l e a n s i n g with 70 per c e n t alcohol, s u p r a p a n n i c u l a r g r a f t s , 9 sq. cm. in area, were taken from the ventral thorax and abdomen. In at1 i n s t a n c e s each animal r e c e i v e d an autograft from the thorax to the abdomen and a homograft from the p a i r e d donor to the prepared s i t e on the chest. Grafts were sutured in p l a c e after r e v e r s i n g the hair direction of the graft. A thin coating of bacitracin ointment was applied to the wound edges. A clean gauze square was placed o v e r the grafts and s e c u r e d with three or four turns of waterproof a d h e s i v e tape. This d r e s s i n g was changed every two days. In addition, each graft was i n s p e c t e d daily by dividing the tape on the dorsal s u r f a c e and then r e f a s t e n i n g the protective bandage. All t r a n s p l a n t s were c h e c k e d g r o s s l y for texture and a p p e a r a n c e and intactn e s s of epithelium. Abrupt c e s s a t i o n of the capillary circulation and the s u b s e q u e n t r a p i d g r a f t rejection produced a r e a s o n a b l y c l e a r end point for determining the time of r e j e c t i o n . However, in t h o s e i n s t a n c e s where r e j e c t i o n c o n s i s t e d of a r e t a r d e d r e a c t i o n , a slow r e p e t i t i v e desquamation of i s o l a t e d a r e a s of the epithelium took p l a c e . T h i s retarded rej e c t i o n made it difficult to e s t a b l i s h a d i s t i n c t end point as p r e c i s e a s the c e s s a t i o n of capillary c i r c u l a t i o n . We have n o t e d . t h a t when a c l e a r l y d e f i n a b l e follicular pattern remains in the graft, a r e a s with the t h i c k n e s s and chara c t e r i s t i c s of normal donor e p i t h e l i u m are h i s t o l o g i c a l l y d i s t i n g u i s h a b l e . In q u e s t i o n a b l e i n t e r p r e t a t i o n s , b i o p s i e s were examined for the p r e s e n c e of i n t a c t epithelium. T h i s epidermis with l o s s of rete pegs w h i c h would be sugg e s t i v e of overgrowth of host epithelium was regarded as r e j e c t e d . E l a s t i c s t a i n s were not done. D i s a p p e a r a n c e of the follicular pattern or h l s t o l o g i c c r i t e r i a were taken as the end point in t h o s e i n s t a n c e s in which r e j e c t i o n was prolonged. In all i n s t a n c e s ending in ultimate graft r e j e c t i o n , t h i s end point c a m e after shrinkage o f the graft and was promptly followed by s c a r formation.
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than in the other four groups; and second, the d i f f e r e n c e in mean r e j e c t i o n time between the two groups (15.6 d a y s ) is significant (P<0.01) when compared by a T t e s t . The difference in r e j e c t i o n pattern between groups 1 and 2 si~ould be mentioned. Eight of the 19 grafts in combination 2 ( T a b l e 1) underwent abrupt r e j e c t i o n with rapid c e s s a t i o n of circulation. The remaining grafts s l o w l y shrank, desquamated, and were partially overgrown by h o s t epithelium. In contrast, only three out of 19 homografts in group 1 ~anderwent rapid rejection. In two i n s t a n c e s in group 1, the skin from Holtzman rats (on Rollsmeyer rats) survived permanently with abundant hair regrowth. In computing mean time of r e j e c tion, t h e s e two permanent grafts were included in the computation as 57 day survivals. T h r e e additional grafts survived 55 to 57 d a y s and were then r e j e c t e d . The two longest durations of graft survival in group 2 were 31 and 42 days. When hooded Long-Evan s rats were cross grafted with the Holtzman rats (groups 3 and 4, Section A, T a b l e 1), survival times were shortened. T h e d i f f e r e n c e in mean survival time between groups 3 and 4 (3.6 days), though somewhat smaller, was s t a t i s t i c a l l y s i g n i f i c a n t at the 95 per c e n t probability level. With one exception, r e j e c t i o n was a b r u p t and c l e a r - c u t . In the reciprocal grafts in groups 5 and 6 (Section A, T a b l e 1) ( L o n g - E v a n s and Rolfsmeyer rats), r e j e c t i o n time was sharp in all i n s t a n c e s and there was no d i f f e r e n c e in mean time of graft rejection between groups. Control autografts survived and had regrowth of hair in all but one i n s t a n c e . In the induction of t o l e r a n c e , all attempts in group 1 were s u c c e s s f u l while a l e s s e r proportion ( 7 / 1 1 ) of homografts in group 2 survived (Section B, T a b l e 1). No " r u n t i n g " was n o t e d in i n j e c t e d animals in this group, and the drop from " n u m b e r i n j e c t e d " to the number grafted r e p r e s e n t s neonatal d e a t h s or l o s s during a n e s t h e s i a and grafting. In the combinations where g e n e t i c d i v e r s i t y would be e x p e c t e d to be g r e a t e r (groups 3, 4, 5, and 6, Section B, T a b l e 1), only 2 out o f 13 animals in group 6 showed t o l e r a n c e to homologous grafts. There was an apparent prolongation of mean time of graft survival in g r o u p s 3 and 4 which could p o s s i b l y be interpreted as an exp r e s s i o n of some degree of t o l e r a n c e .
i{ ESU L T S The r e s u l t s are summarized on Table 1. In examining the d i f f e r e n t r e j e c t i o n times in the first tw o groups o f the r e c i p r o c a l grafts (Section A), t h e r e are two apparent facts. F i r s t , t h e m e a n graft survival was longer in the~e combinations
DISCUSSION Although t h e s e o b s e r v a t i o n s in commercial rats seem pedestrian, they d o serve to unders c o r e a number of e x p e r i m e n t a l factors. The
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MC Q U A R R I E , BROWN and V A R C O
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Table I B - Induction of Tolerance between Commercial Rat Strains
A - Reciprocal Grafts between Commercial Rat Strains
Strain Combination
Range Mean Grab in Rejection Difference No. Days i Time* of Means
P
Mean Rejection Time of Those Grafts Not No. No. No. Surviving Beyond Injected Tolerant Grahed 90 Days 36
16/16
19 9-42 ~ 23.4 -+5.5
40
7/I I
30
3Holtzman to Long-Evans
12 7-23
11.3~-1.3
60
0/17
21
4Long-Evans to Holtzman
12 5-13
7.7-+1.3
54
0/20
20.9
12-21 15.5"-3.0
41
0/12
19.5
45
2/'13
19
1 Holtzman to Rol fsmeyer
19 8-57 t
2 Rolfsmeyer to Holtzman
39 -*6.3
15.6
3.6
<0.01
1
5Long-Evans 8~ to Rolfsmeyer
1.8 6Rolfsmeyerto Long-Evans
10 t7"21 t
13.7-*3.8
I
>0.01
Plus or minus 2 standard errors of the mean. Three grafts survived 55 to 57 days. An additional two grafts appear normal with abundant hair regrowth. They were included in the calculations as having survived 57 days. These grafts were followed for five months. Only one graft survived 42 days. Two animals died shortly after grafting. d a t a s h o w the e x t r e m e s of s o m e of the e x p e r i mental r e s u l t s which may o c c u r in g r a f t s u r v i v a l time with the o n l y e x p e r i m e n t a l v a r i a b l e b e i n g s o u r c e of origin of n o n i n b r e d r a t s . Many of the following c o n s i d e r a t i o n s a r e w e l l u n d e r s t o o d by i n v e s t i g a t o r s primarily e n g a g e d in d e f i n i n g the g e n e t i c s of t r a n s p l a n t a t i o n immunity. To the l e s s e x p e r i e n c e d , t h e s e d a t a i n d i c a t e the interp r e t i v e " c o s t " of u s i n g n o n i n b r e d a n i m a l s . If such v a r i a t i o n s in g r a f t s u r v i v a l may b e p r o d u c e d by the s e l e c t i o n of the e x p e r i m e n t a l r a t s , o n e must reasonably presume that such variations may o c c u r in any o t h e r s p e c i e s which are not inbred for d e f i n i t i o n of h i s t o c o m p a t i b i l i t y f a c tors. A p r e v i o u s p a p e r 10 s h o w e d s a m e of the p i t f a l l s of i n t r a s t r a i n g r a f t s in c o m m e r c i a l r a t s . H e n c e , it is not r e a l l y " s a f e " to indulge in broad a s s u m p t i o n s as to g e n e t i c s i m i l a r i t y or d i s s i m i l a r i t y in u s i n g random b r e d or p e n - b r e d p o p u l a t i o n s . Since g e n e t i c c o m p o s i t i o n for a number of h i s t o c o m p a t i b i l i t y f a c t o r s i s e s s e n t i a l l y a d i s c o n t i n u o u s v a r i a b l e , it may a f f e c t
the d i s t r i b u t i o n of the m e a s u r e d r e s u l t of g r a f t s u r v i v a l time in s u c h a way a s to g i v e m a r k e d l y s k e w e d d i s t r i b u t i o n s or e v e n d i m o d a l d i s t r i b u t i o n s . P r o b l e m s c a n a r i s e when the u s u a l s t a t i s t i c a l m e t h o d s w h i c h a s s u m e an a p p r o x i m a t e l y normal d i s t r i b u t i o n are a p p l i e d w i t h o u t a p p r o p r i a t e a n a l y s i s or c o r r e c t i o n . As e x a m p l e s of the m a n y p o s s i b l e i n t e r p r e t a t i o n s of t h e s e d a t a , o n e m i g h t a s s u m e t h a t the d i f f e r e n c e in m e a n r e j e c t i o n time of g r a f t s b e t w e e n g r o u p s 1 and 2 a n d b e t w e e n 3 and 4 in T a b l e 1 , P a r t A, c o u l d be due to any or all of the f o l l o w i n g f a c t o r s : F i r s t , it i s p o s s i b l e t h a t the o b s e r v e d diff e r e n c e s may r e f l e c t a d i f f e r i n g a b i l i t y of a " c o m m e r c i a l s t r a i n " t o r e s p o n d ta the a n t i g e n i c s t i m u l u s of a t e s t g r a f t . T h e f a c t t h a t a l l s t r a i n s s h o w e d a v i g o r o u s r e s p o n s e to t h e a n t i g e n s of at l e a s t o n e s t r a i n s p e a k s a g a i n s t t h i s b e i n g t h e major factor. S e c o n d , the two s u r v i v i n g g r a f t s , in g r o u p 1, s u g g e s t the i m p o r t a n c e of d i f f e r e n c e s with re-
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g a r d to homo- or h e t e r o z y g o s i t y at a p a r t i c u l a r h i s t o c o m p a t l b i l l t y l o c u s . If she donor a n d r e c i p i e n t animal d i f f e r at o n l y o n e h i s t o c o m p a t i b i l i t y f a c t o r , t h e n , w h e t h e r t h e a n i m a l s are h o m o z y g o u s or h e t e r o z y g o u s for that f a c t o r b e c o m e s of imp o r t a n c e . If o n e a n i m a l of a p a i r is h o m o z y g o u s for a h i s t o c o m p a t i b i l i t y l o c u s (HH), and the o t h e r a n i m a l i s h e t e r o z y g o u s for the s a m e l o c u s (HX), it would be e x p e c t e d t h a t g r a f t s from HH to fIX would p e r s i s t i n d e f i n i t e l y , while the r e c i p r o c a l g r a f t s from HX to Hit would be r e j e c t e d . T h i s p o s s i b i l i t y would h a v e to be c o n s i d e r e d whenever permanent survivals occur between animals of unknown g e n e t i c i d e n t i t y s u c h a s s e e n in g r o u p 1. H o w e v e r , it i s s u r p r i s i n g to s e e a s p o n t a n e o u s a c c e p t a n c e r a t e of a p p r o x i m a t e l y 10 per cent in c o m m e r c i a l r a t s from d i f f e r e n t c l o s e d colon],' s o u r c e s . Third, it is p o s s i b l e that s o m e r a t s m a y exh i b i t s o m e d e g r e e of n o n r e a c t i v i t y to h o m o g r a / t s s i m i l a r to t h a t which h a s been found in c r o s s g r a f t s b e t w e e n h a m s t e r s from w i d e l y u n r e l a t e d n o n i n b r e d s t r a i n s , l . 6 T h e s u g g e s t i o n of " n o n r e a c t i v i t y " i s p a r t i c u l a r l y a t t r a c t i v e in the f i r s t two s t r a i n c o m b i n a t i o n s of this s t u d y . Is i s p o s s i b l e in r a t s , as w e l l a s in h a m s t e r s , t h a t t h i s represents a degree of "relatedness for-histocompatibility factors." As B i l l i n g h a m and H i l d e m a n 7 c o n c l u d e d , ' " T h e r e are o n l y a few *strong' but at l e a s t s e v e r a l ' w e a k ' h i s t o c o m p a t i b i l i t y a n t i g e n s ( g e n e s ) s e g r e g a t i n g in the Syrian h a m s t e r s t o c k s . " F o u r t h , t h e r e m a y be a d e f i n a b l e d i f f e r e n c e in the s t i m u l u s p r e s e n t e d to the h o s t by a l l e l i c and n o n a I l e l i c h i s t o c o m p a t i b i l i t y a n t i g e n s . T h e v a r i a t i o n in the p o t e n c y of a l l e l i c a n t i g e n s h a s b e e n n o t e d b e f o r e in blood group antigeras. T h e A and B s u b s t a n c e s are s t r o n g l y a n t i g e n i c , w h i l e O s u b s t a n c e is w e a k l y a n t i g e n i c . ~4 T h e d i f f e r e n c e in the s t i m u l u s of the t-11 l o c u s a s c o m p a r e d to the s t r o n g e r a n t i g e n s a s s o c i a t e d with the H 2 l o c u s i s well s t u d i e d ir~ inbred m i c e . 4,1v,~9, a° B i l l i n g h a m et al. 8 h a v e demons t r a t e d in m i c e the p h e n o m e n o n of v a r i a t i o n in the s t r e n g t h of h i s t o c o m p a t i b i l i t y a n t i g e n s in a c o n c l u s i v e w a y . T h e r e s u l t s in g r o u p s 1 and 2, 3 and 4 ( T a b l e 1) would s u g g e s t t h a t v a r i a t i o n in s t r e n g t h of h i s t o c o m p a t i b i l i t y a n t i g e n i s of c o n s i d e r a b l e s i g n i f i c a n c e in n o n i n b r e d s p e c i e s o t h e r than m i c e . It would n e v e r be p o s s i b l e to d e m o n s t r a t e the p r e s e n c e or a b s e n c e of t h i s f a c t o r in n o n i n b r e d a n i m a l s without m a i n t a i n i n g r e s e r v a t i o n s . In d e s i g n i n g e x p e r i m e n t s in w h i c h a n i m a l s o f unknown g e n e t i c c o m p o s i t i o n are u s e d , t h i s v a r i a b l e s h o u l d be a s s u m e d to be p r e s e n t . When g e n e t i c a l l y h e t e r o g e n o u s a n i m a l s are e m p l o y e d , an o c c a s i o n a l ' m a r k e d p r o l o n g a t i o n of g r a f t s u r v i v a l m u s t be e x p e c t e d . An i s o l a t e d
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p e r m a n e n t s u r v i v a l must be v i e w e d c r i t i c a l l y . Fifth, smaller differences such as occurred in the H o l t z m a n to L o n g - E v a n s g r a f t s ( g r o u p s 3 a n d 4, S e c t i o n A) c o u l d r e s u l t from c h a n c e s e l e c t i o n of a n i m a l s at d i f f e r e n t s t a g e s of a hair c y c l e . E a r l y studi e s by B a l l a n t y n e and C o n v e r s e 2 h a v e s h o w n a s much a s s i x day:g' d i f f e r e n c e in m e a n r e j e c t i o n t i m e s of s u p r a p a n n i c u l a r g r a f t s in n o n i n b r e d r a t s . T h e i r s u b s e q u e n t s t u d i e s 3 t e n d to m i n i m i z e the i m p o r t a n c e of t h i s f a c t o r and e m p h a s i z e the v a s c u l a r i t y of the graft bed f o l l o w i n g p l u c k i n g of hair. In t h e s e c o m b i n a t i o n s , it is d o u b t f u l t h a t the 15.6 day mean d i f f e r e n c e c o u l d be an e x p r e s s i o n of d i f f e r i n g skin c y c l e s . Skin c y c l e d i f f e r e n c e s do not explain the two g r a f t s in the H o l t z m a n to R o l f s m e y e r g r o u p (group 1) which p e r s i s t e d and had v i g o r o u s h a i r growth long a f t e r the r e c i p r o c a l p a i r e d c r o s s g r a f t from t h e s e a n i m a l s to the d o n o r s had been r e j e c t e d . In s u m m a r y , the s e c o n d and fourth p o s s i b i l i t i e s s e e m m o s t l i k e l y . It can b e s e e n that inbred s t r a i n s of r a t s with known g e n e t i c c o m p o s i t i o n would h a v e to be u s e d to m a k e a c o n c l u s i v e d i s tinction between these five factors. If the v a r i a b l e of i n d u c t i o n of t o l e r a n c e is a d d e d to the e x p e r i m e n t a l d e s i g n , as is done in the s e c o n d part of the s t u d i e s , one s e e s d a t a s u g g e s t i v e l y p a r a l l e l to the o b s e r v a t i o n s of M a r t i n e z e t al. l l in m i c e . T h e s e w o r k e r s demo n s t r a t e d t h a t t h e r e i s a m a r k e d d i f f e r e n c e in the p e r c e n t a g e of s u c c e s s e s in the i n d u c t i o n o f t o l e r a n c e in m i c e d e p e n d i n g on the p a r t i c u l a r c o m b i n a t i o n of s t r a i n s i n v o l v e d . At f i r s t g l a n c e , the d a t a in S e c t i o n B ( T a b l e I ) w o u l d s u g g e s t t h a t the a n t i g e n i c s t r e n g t h of the i n j e c t e d hom o l o g o u s t i s s u e is of i m p o r t a n c e . I n d u c t f b n of t o l e r a n c e i s m o s t s u c c e s s f u l in the " s t r a i n " c o m b i n a t i o n where normal h o m o g r a f t s u r v i v a l w a s l o n g e s t . Again, a d e f i n i t i v e s t a t e m e n t c a n not b e m a d e from our d a t a in n o n i n b r e d r a t s . The five possibilitiesmentioned a b o v e may all c o n t r i b u t e to the p r o d u c t i o n of t o l e r a n c e to skin h o m o g r a f t s . I n t e r p r e t a t i o n i s further comp o u n d e d by v a r y i n g d e g r e e s of immunologic maturity at the time of s p l e e n c e l l i n j e c t i o n ; d i f f e r i n g a n t i g e n i c i t y of i n j e c t e d d o n o r c e l l s both b e t w e e n " s t r a i n s " and within " s t r a i n s " ; and v a r y i n g l e t h a l i t y (due to runt d i s e a s e ) of inj e c t e d c e l l s . C o n s i d e r a t i o n of t h e s e m a n y f a c t o r s may be p a r t i c u l a r l y u s e f u l in i n t e r p r e t i n g much of the c o n f l i c t i n g d a t a in e n d o c r i n e t r a n s p l a n t a t i o n , w h e r e n o n i n b r e d a n i m a l s h a v e been e x t e n sively used. Nevertheless, noninbred animals can h a v e s o m e u s e f u l n e s s , when t h e i r u s e s e e m s n e c e s s a r y , for p u r p o s e s of l a r g e r s p e c i m e n s i z e or t e c h n i q u e r e q u i r i o g l a r g e r a n i m a l s , or for o b s e r v a t i o n s in a n o n i n b r e d s p e c i e s . T h e ex-
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p e r i m e n t will of n e c e s s i t y h a v e to be o n e which d o e s not a t t e m p t to d i s t i n g u i s h d i s c r e t e g e n e t i c or h i s t o c o m p a t i b i l i t y f a c t o r s . One m u s t p r e s u m e the a b s e n c e of g e n e t i c k n o w l e d g e of the a n i m a l s . E x p e r i m e n t s m u s t be d e s i g n e d a c c o r d i n g l y . B a s i c p r e c a u t i o n s s h o u l d include: (a) S i m u l t a n e o u s s e l e c t i o n on a s t r i c t l y random b a s i s (by a t a b l e of random n u m b e r s ) of the e x p e r i m e n t a l and control g r o u p s . (b) Any m e a s u r e d v a r i a b l e s h o u l d h a v e a r e a s o n a b l y random d i s t r i b u t i o n through the e x p e r i m e n t a l and control g r o u p s . N o t e the m a r k e d l y s k e w e d d i s t r i b u t i o n e v i d e n t in group 1, Section A, to h a v e a mean of 39 d a y s with a maximum o f 57 d a y s . (c) One s h o u l d f a c e the n e c e s s i t y t h a t with a h e t e r o g e n e o u s p o p u l a t i o n of v a r y i n g c h a r a c t e r i s t i c s , e n l a r g e m e n t of the e x p e r i m e n t a l error i s the r e s u l t . T h i s is e v i d e n t here in the wide s t a n d a r d error of the m e a n . T h i s s h o u l d be c o m p a r e d to t h e s m a l l s t a n d a r d error in s i m i l a r s i z e d g r o u p s in s t u d i e s in i n b r e d m i c e . I n c r e a s e in v a r i a n c e within e x p e r i m e n t a l groups d e m a n d s e n l a r g e m e n t of g r o u p s i z e and d i m i n i s h e s t h e c h a n c e s of d e t e c t i n g s m a l l e r eff e c t s . O c c a s i o n a l l y , the u s e o f n o n i n b r e d a n i m a l s for " e c o n o m y " p u r p o s e s i s no e c o n o m y at all. (d) It s h o u l d n e v e r be p r e s u m e d t h a t s i m i l a r studies done at different times using commercial a n i m a l s are in any way a d d i t i v e . T h e a u t h o r s h a v e had a s u p p l i e r c h a n g e without n o t i c e from one c o l o n y to a new, g e n e t i c a l l y d i f f e r e n t , partially inbred c o l o n y . In s o m e t r a n s p l a n t a t i o n p r o b l e m s , in which u s e of a n i m a l s of unknown g e n e t i c i d e n t i t y i s either n e c e s s a r y or u n a v o i d a b l e , it i s w e l l to recognize that significant strain differences and s y s t e m a t i c v a r i a b l e s may be p r e s e n t . G e o g r a p h i c s e p a r a t i o n and c l o s e d c o l o n y r a n d o m mating do not a l w a y s g u a r a n t e e g e n e t i c d i v e r s i t y of two s t r a i n s of r a t s . T h i s would p r e s u m a b l y be e q u a l l y true for o t h e r s p e c i e s . E x p e r i m e n t a l design s h o u l d be c r i t i c a l and e x p e r i m e n t a l g r o u p s i z e s h o u l d be l a r g e enough to a s s e s s the true e f f e c t of " s t r a i n " and unknown g e n e t i c f a c t o r s when the u s e of n o n i s o t o g o u s a n i m a l s of a n y s p e c i e s is r e q u i r e d . An o b v i o u s o p t i m u m s o l u tion would b e to h a v e inbred s t r a i n s of the m a j o r l a b o r a t o r y a n i m a l s p e c i e s r e a d i l y a v a i l a b l e from c o m m e r c i a l or o t h e r s o u r c e s .
SUMMARY 1. O b s e r v a t i o n s b e t w e e n s t r a i n s of commercially bred noniSologou~ rats show marked variation in.homograft survival between six comb i n a t i o n s of t h r e e s t r a i n s . D i f f e r e n c e in m e a n s u r v i v a l of g r o u p s of r e c i p r o c a l interstrain g r a f t s w a s a s g r e a t a s 15 d a y s .
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2. T h e p r o p o r t i o n of s u c c e s s f u l i n d u c t i o n of t o l e r a n c e to h o m o g r a f t s b e t w e e n s t r a i n s v a r i e d , d e p e n d i n g on s t r a i n c o m b i n a t i o n . 3. T h e d a t a d e m o n s t r a t e the d e g r e e of s o m e v a r i a t i o n s of e x p e r i m e n t a l error which can a r i s e in u s i n g n o n i n b r e d c o m m e r c i a l a n i m a l s in t i s s u e transplantation studies. 4. Some of the p r o b l e m s in i n t e r p r e t a t i o n and d e s i g n of h o m o g r a f t e x p e r i m e n t s are d i s c u s s e d .
ll E F E II F]~ C E S 1. Adams, R.A., Patt, D.I., and Lutz, B.R.: Longterm p e r s i s t e n c e of skin homografts in untreated hamsters. Transpl. Bull., 3:41, 1956. 2. Batlantyne, D.L., and Converse, J.M.: Effect of hair cycles on rat skin homografts. Ann. N.Y. Acad. Sci., 64:958, 1957. 3. Battantyne, D.L., and Converse, J.M.: Further observations of halr-skin cycles and the survival of skin homografts in rats. Transpl. Bull., 6:93, 1959. 4. Berrian, J.tt., and Jacobs, R.L.: Diversity of transplantation antigens in the mouse. Biological Problems of Grafting. University of Liege (Belgium), 1959, p. 131. 5. Billingham, R.E., and Silvers, W.K.: Inbred animals and tissue transplantation immunity. Transpl. Bull., 6:399, 1959. 6. Billingham, R.E., and ltildeman, W.tt.: Studies of transplantation immunity in hamsters. Ann. N.Y. Acad. Sci., 73:676, t958. 7. Billingham, R.E., and tIildeman, m.ll.: Studies on the i:amunological responses of hamsters to skin homografts. Proc. Roy. Soc. London, s. B, 149:216, 1958. 8. Billingham, R.E., Brent, L., Medawar, P.B., and Sparrow, E.M.: Quantitative studies on tissue transplantation immunity. L. The survival times of skin homografts exchanged between members of different inbred strains of mice. Proc. Royal Soc., s. B, 143:43, 1954. 9. Dempster, W.J., Lennox, B., and Boag, J.W.: Prolongation of survival of skin homotransplants in the rabbit by irradiation of the host. Brit. J. Exp. Path., 31-670, 1950. 10. Good, R.A., Varco, R.L., Aust, J.B., and Zak, S.J.: Transplantation studies in patients with agammaglobulinemia. Ann. N.Y. Acad. Sci., 64:882, 1957. I I . Martinez, C., Smith, J.M., Aust, J.B., and Good, R.A.: Acquired tolerance to skin hOmografts in mice of different strains. Proc. Soc. Exper. Biol. & Med., 97:736, 1958. 12. McQuarrie, D.G., Kim, J.H., and Varco, R.L.: The long-term surviwlt of intrastrain homografts in comm~:rcia|ly bred rats. Transpl. Bull., 6:97, 1959. 13. Medawar, P.B., and Woodruff, M.F.A.: The induction of tolerance by skin homografts on newborn rats. Immunology, 1:27, 1958. 14. Morgan, W.T.J., and Waddell, M.B.R.: Specific blood group O substance. Brit. J. Exp. Path., 26:387, 1945. 15. Peer, L.A.: Behavior of skin grafts exchanged between parents and offspring. Ann. N.Y. Acad. Sci;, 73:584, 1958.
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16. Puza, A., and Gombas, A.: Acquired tolerance of skin homografts in dogs. Transpl. Bull., 5:3(J, 1958. 17. Rubin, B.A.: The determination of the H-2 genotype of CE mice. Transpl. Bull., 7:153, 1960. 18. Scothorne, R.J.: Studies on the response of regional lymph nodes to skin homografts. Ann. N.Y. Acad. Sci., 64:1028, 1957. 19. Snell, G.D.: The immunogenetics of tumor transplantation. Cancer Res., 12:543, 1952.
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20. Snell, G.D., Smith, P., and Gabrielson, F.: Analysis of the histocompatibility-2 locus in the mouse. J. Nat. Cancer Inst., 14:457, 1953. 21. Tomas, L., Murray, J.E., and Couch, N.P.: Consecutive skin homografts in the dog. Transpl. Bull., 4:156, 1957. 22. Woodruff, M.F.A.: Postpartum induction of tolerance to homologous skin in rats. Ann. N.Y. Acad. Sci., 64:792, 1957.
University o[ Minnesota Hospitals
Billingham and Silvers s have pointed out the desirability and e s s e n t i a l criteria for inbred experimental animals for transplantation s t u d i e s . In some types of experimental s t udi e s the id eal is not r e a l i s t i c a l l y obtainable, and it is n e c e s sary to use experimental a n i m a l s that are not inbred for histocompatibility factors and g e n e t i c identity. Such use of noninbred animals may be n e c e s s a r y in order to expand observations to other s p e c i e s , to get larger t i s s u e samples, or to d o procedures and make observations not possi'bte in the more ubiquitous inbred mice. Although there are inbred s t r a i n s of rats, rabbits, c h i c k e n s and hamsters, the problem of obtaining adequate n u m b e r s , in a variety of strains and at a r e a s o n a b l e cost, frequently forces the a c c e p t a n c e of that l e s s d e s i r a b l e alternative: use of animals of unknown g e n e t i c composition. There are no i s o I o g o u s inbred strains of dogs* known to the authors. There are a large number of s i gni f i c a nt contributions in the field of t i s s u e transplantation i n which noninbred rats a n d rabbits,9,13,1e.22 dogs Is,z1 and the g e n e t i c a l l y h e t e r o g e n e o u s h u m a n ~ ° , l s have been the experimental s p e c i e s . An occasional study is seen in which the experimental design has erroneously implied that animals derived from d i f f e r e n t s o u r c e s have g e n e t i c heterogenity and that there is some positive information on the genetic c o m p o s i t i o n o f the animals b e c a u s e they h a v e been s e l e c t e d from mixed populations or separate c o l o n i e s .
From the Department o f Surgery, University of Minnesota Hospitals, Minneapolis, Minnesota. This study was aided by grants from ~the U. S. Publlc HeaIth Service, The Graduate School of the University of Minnesota, and Richard L. V a r c o Surgical Research Fund. Submitted for publication April 19, 1962. *Several attempt s ate under way to establish inbred beagle Colonies.
48
The following s t u d i e s were done for three reasons. First, the authors frequently find it n e c e s s a r y to u s e large noninbred animals from commercial s o u r c e s . Second, there appeared to be some marked effect of " s t r a i n ' ' t of commercial origin on graft survival in rats. Third, we wished to find some of the v a r i a b l e s that may exist when noninbred animals are u s e d in order to improve experimefftat design. We b e l i e v e the data of these simple s t u d i e s may have i n t e r e s t b e c a u s e they i l l u s t r a t e the c o n s i d e r a b l e range of some s y s t e m a t i c v a r i a b l e s which may creep into experiments and confuse interpretation when noninbred animals are used.
METII 0 I}S For more nearly related " s t r a i n s , " s e p a r a t e albino rats of Sprague-Dawley origin were obtained in 1958-I959 from the Holtzman Company and from the Rolfsmeyer Company. T h e s e colonies had been c l o s e d s i n c e 1952 and 1954, r e s p e c t i v e l y . For comparison of " s t r a i n s " with more divergent g e n e t i c background, L c n g - E v a n s rats from Rockland Farms were u sed . All animals for reciprocal grafting were males weighing 150 to I75 gin. R e c i p r o c a l grafts were performed simultaneously between each of the ' * s t r a i n " pairs, giving the six groups of animals l i s t e d in Table I. To study the effects of " s t r a i n " on the induction of t o l e r a n c e , newborn rats in groups with the same six " s t r a i n " combinations were i n j e c t e d intraperitoneally with spleen brei (approximately 10 million ceils) during the first 24 hours after birth. G r a f t i n g was done at two months. Spleen donors were adult animals, later to be u s e d as skin donors. Animals were considered tolerant if the grafted skin appeared ,The use of the work **straln" does not imply any degree of inbreeding b u t s e r v e s to distinguish different commercial sources of noninbred rats.
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normal in s i z e and texture, had regrowth of hair, and survived 90 days. Grafting p r o c e d u r e s were done similarly in all groups. After shaving the ventral s u r f a c e of the animals with e l e c t r i c clippers, and c l e a n s i n g with 70 per c e n t alcohol, s u p r a p a n n i c u l a r g r a f t s , 9 sq. cm. in area, were taken from the ventral thorax and abdomen. In at1 i n s t a n c e s each animal r e c e i v e d an autograft from the thorax to the abdomen and a homograft from the p a i r e d donor to the prepared s i t e on the chest. Grafts were sutured in p l a c e after r e v e r s i n g the hair direction of the graft. A thin coating of bacitracin ointment was applied to the wound edges. A clean gauze square was placed o v e r the grafts and s e c u r e d with three or four turns of waterproof a d h e s i v e tape. This d r e s s i n g was changed every two days. In addition, each graft was i n s p e c t e d daily by dividing the tape on the dorsal s u r f a c e and then r e f a s t e n i n g the protective bandage. All t r a n s p l a n t s were c h e c k e d g r o s s l y for texture and a p p e a r a n c e and intactn e s s of epithelium. Abrupt c e s s a t i o n of the capillary circulation and the s u b s e q u e n t r a p i d g r a f t rejection produced a r e a s o n a b l y c l e a r end point for determining the time of r e j e c t i o n . However, in t h o s e i n s t a n c e s where r e j e c t i o n c o n s i s t e d of a r e t a r d e d r e a c t i o n , a slow r e p e t i t i v e desquamation of i s o l a t e d a r e a s of the epithelium took p l a c e . T h i s retarded rej e c t i o n made it difficult to e s t a b l i s h a d i s t i n c t end point as p r e c i s e a s the c e s s a t i o n of capillary c i r c u l a t i o n . We have n o t e d . t h a t when a c l e a r l y d e f i n a b l e follicular pattern remains in the graft, a r e a s with the t h i c k n e s s and chara c t e r i s t i c s of normal donor e p i t h e l i u m are h i s t o l o g i c a l l y d i s t i n g u i s h a b l e . In q u e s t i o n a b l e i n t e r p r e t a t i o n s , b i o p s i e s were examined for the p r e s e n c e of i n t a c t epithelium. T h i s epidermis with l o s s of rete pegs w h i c h would be sugg e s t i v e of overgrowth of host epithelium was regarded as r e j e c t e d . E l a s t i c s t a i n s were not done. D i s a p p e a r a n c e of the follicular pattern or h l s t o l o g i c c r i t e r i a were taken as the end point in t h o s e i n s t a n c e s in which r e j e c t i o n was prolonged. In all i n s t a n c e s ending in ultimate graft r e j e c t i o n , t h i s end point c a m e after shrinkage o f the graft and was promptly followed by s c a r formation.
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than in the other four groups; and second, the d i f f e r e n c e in mean r e j e c t i o n time between the two groups (15.6 d a y s ) is significant (P<0.01) when compared by a T t e s t . The difference in r e j e c t i o n pattern between groups 1 and 2 si~ould be mentioned. Eight of the 19 grafts in combination 2 ( T a b l e 1) underwent abrupt r e j e c t i o n with rapid c e s s a t i o n of circulation. The remaining grafts s l o w l y shrank, desquamated, and were partially overgrown by h o s t epithelium. In contrast, only three out of 19 homografts in group 1 ~anderwent rapid rejection. In two i n s t a n c e s in group 1, the skin from Holtzman rats (on Rollsmeyer rats) survived permanently with abundant hair regrowth. In computing mean time of r e j e c tion, t h e s e two permanent grafts were included in the computation as 57 day survivals. T h r e e additional grafts survived 55 to 57 d a y s and were then r e j e c t e d . The two longest durations of graft survival in group 2 were 31 and 42 days. When hooded Long-Evan s rats were cross grafted with the Holtzman rats (groups 3 and 4, Section A, T a b l e 1), survival times were shortened. T h e d i f f e r e n c e in mean survival time between groups 3 and 4 (3.6 days), though somewhat smaller, was s t a t i s t i c a l l y s i g n i f i c a n t at the 95 per c e n t probability level. With one exception, r e j e c t i o n was a b r u p t and c l e a r - c u t . In the reciprocal grafts in groups 5 and 6 (Section A, T a b l e 1) ( L o n g - E v a n s and Rolfsmeyer rats), r e j e c t i o n time was sharp in all i n s t a n c e s and there was no d i f f e r e n c e in mean time of graft rejection between groups. Control autografts survived and had regrowth of hair in all but one i n s t a n c e . In the induction of t o l e r a n c e , all attempts in group 1 were s u c c e s s f u l while a l e s s e r proportion ( 7 / 1 1 ) of homografts in group 2 survived (Section B, T a b l e 1). No " r u n t i n g " was n o t e d in i n j e c t e d animals in this group, and the drop from " n u m b e r i n j e c t e d " to the number grafted r e p r e s e n t s neonatal d e a t h s or l o s s during a n e s t h e s i a and grafting. In the combinations where g e n e t i c d i v e r s i t y would be e x p e c t e d to be g r e a t e r (groups 3, 4, 5, and 6, Section B, T a b l e 1), only 2 out o f 13 animals in group 6 showed t o l e r a n c e to homologous grafts. There was an apparent prolongation of mean time of graft survival in g r o u p s 3 and 4 which could p o s s i b l y be interpreted as an exp r e s s i o n of some degree of t o l e r a n c e .
i{ ESU L T S The r e s u l t s are summarized on Table 1. In examining the d i f f e r e n t r e j e c t i o n times in the first tw o groups o f the r e c i p r o c a l grafts (Section A), t h e r e are two apparent facts. F i r s t , t h e m e a n graft survival was longer in the~e combinations
DISCUSSION Although t h e s e o b s e r v a t i o n s in commercial rats seem pedestrian, they d o serve to unders c o r e a number of e x p e r i m e n t a l factors. The
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Table I B - Induction of Tolerance between Commercial Rat Strains
A - Reciprocal Grafts between Commercial Rat Strains
Strain Combination
Range Mean Grab in Rejection Difference No. Days i Time* of Means
P
Mean Rejection Time of Those Grafts Not No. No. No. Surviving Beyond Injected Tolerant Grahed 90 Days 36
16/16
19 9-42 ~ 23.4 -+5.5
40
7/I I
30
3Holtzman to Long-Evans
12 7-23
11.3~-1.3
60
0/17
21
4Long-Evans to Holtzman
12 5-13
7.7-+1.3
54
0/20
20.9
12-21 15.5"-3.0
41
0/12
19.5
45
2/'13
19
1 Holtzman to Rol fsmeyer
19 8-57 t
2 Rolfsmeyer to Holtzman
39 -*6.3
15.6
3.6
<0.01
1
5Long-Evans 8~ to Rolfsmeyer
1.8 6Rolfsmeyerto Long-Evans
10 t7"21 t
13.7-*3.8
I
>0.01
Plus or minus 2 standard errors of the mean. Three grafts survived 55 to 57 days. An additional two grafts appear normal with abundant hair regrowth. They were included in the calculations as having survived 57 days. These grafts were followed for five months. Only one graft survived 42 days. Two animals died shortly after grafting. d a t a s h o w the e x t r e m e s of s o m e of the e x p e r i mental r e s u l t s which may o c c u r in g r a f t s u r v i v a l time with the o n l y e x p e r i m e n t a l v a r i a b l e b e i n g s o u r c e of origin of n o n i n b r e d r a t s . Many of the following c o n s i d e r a t i o n s a r e w e l l u n d e r s t o o d by i n v e s t i g a t o r s primarily e n g a g e d in d e f i n i n g the g e n e t i c s of t r a n s p l a n t a t i o n immunity. To the l e s s e x p e r i e n c e d , t h e s e d a t a i n d i c a t e the interp r e t i v e " c o s t " of u s i n g n o n i n b r e d a n i m a l s . If such v a r i a t i o n s in g r a f t s u r v i v a l may b e p r o d u c e d by the s e l e c t i o n of the e x p e r i m e n t a l r a t s , o n e must reasonably presume that such variations may o c c u r in any o t h e r s p e c i e s which are not inbred for d e f i n i t i o n of h i s t o c o m p a t i b i l i t y f a c tors. A p r e v i o u s p a p e r 10 s h o w e d s a m e of the p i t f a l l s of i n t r a s t r a i n g r a f t s in c o m m e r c i a l r a t s . H e n c e , it is not r e a l l y " s a f e " to indulge in broad a s s u m p t i o n s as to g e n e t i c s i m i l a r i t y or d i s s i m i l a r i t y in u s i n g random b r e d or p e n - b r e d p o p u l a t i o n s . Since g e n e t i c c o m p o s i t i o n for a number of h i s t o c o m p a t i b i l i t y f a c t o r s i s e s s e n t i a l l y a d i s c o n t i n u o u s v a r i a b l e , it may a f f e c t
the d i s t r i b u t i o n of the m e a s u r e d r e s u l t of g r a f t s u r v i v a l time in s u c h a way a s to g i v e m a r k e d l y s k e w e d d i s t r i b u t i o n s or e v e n d i m o d a l d i s t r i b u t i o n s . P r o b l e m s c a n a r i s e when the u s u a l s t a t i s t i c a l m e t h o d s w h i c h a s s u m e an a p p r o x i m a t e l y normal d i s t r i b u t i o n are a p p l i e d w i t h o u t a p p r o p r i a t e a n a l y s i s or c o r r e c t i o n . As e x a m p l e s of the m a n y p o s s i b l e i n t e r p r e t a t i o n s of t h e s e d a t a , o n e m i g h t a s s u m e t h a t the d i f f e r e n c e in m e a n r e j e c t i o n time of g r a f t s b e t w e e n g r o u p s 1 and 2 a n d b e t w e e n 3 and 4 in T a b l e 1 , P a r t A, c o u l d be due to any or all of the f o l l o w i n g f a c t o r s : F i r s t , it i s p o s s i b l e t h a t the o b s e r v e d diff e r e n c e s may r e f l e c t a d i f f e r i n g a b i l i t y of a " c o m m e r c i a l s t r a i n " t o r e s p o n d ta the a n t i g e n i c s t i m u l u s of a t e s t g r a f t . T h e f a c t t h a t a l l s t r a i n s s h o w e d a v i g o r o u s r e s p o n s e to t h e a n t i g e n s of at l e a s t o n e s t r a i n s p e a k s a g a i n s t t h i s b e i n g t h e major factor. S e c o n d , the two s u r v i v i n g g r a f t s , in g r o u p 1, s u g g e s t the i m p o r t a n c e of d i f f e r e n c e s with re-
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1Ioi. !11, No. I - J a n u a r y , 1963
g a r d to homo- or h e t e r o z y g o s i t y at a p a r t i c u l a r h i s t o c o m p a t l b i l l t y l o c u s . If she donor a n d r e c i p i e n t animal d i f f e r at o n l y o n e h i s t o c o m p a t i b i l i t y f a c t o r , t h e n , w h e t h e r t h e a n i m a l s are h o m o z y g o u s or h e t e r o z y g o u s for that f a c t o r b e c o m e s of imp o r t a n c e . If o n e a n i m a l of a p a i r is h o m o z y g o u s for a h i s t o c o m p a t i b i l i t y l o c u s (HH), and the o t h e r a n i m a l i s h e t e r o z y g o u s for the s a m e l o c u s (HX), it would be e x p e c t e d t h a t g r a f t s from HH to fIX would p e r s i s t i n d e f i n i t e l y , while the r e c i p r o c a l g r a f t s from HX to Hit would be r e j e c t e d . T h i s p o s s i b i l i t y would h a v e to be c o n s i d e r e d whenever permanent survivals occur between animals of unknown g e n e t i c i d e n t i t y s u c h a s s e e n in g r o u p 1. H o w e v e r , it i s s u r p r i s i n g to s e e a s p o n t a n e o u s a c c e p t a n c e r a t e of a p p r o x i m a t e l y 10 per cent in c o m m e r c i a l r a t s from d i f f e r e n t c l o s e d colon],' s o u r c e s . Third, it is p o s s i b l e that s o m e r a t s m a y exh i b i t s o m e d e g r e e of n o n r e a c t i v i t y to h o m o g r a / t s s i m i l a r to t h a t which h a s been found in c r o s s g r a f t s b e t w e e n h a m s t e r s from w i d e l y u n r e l a t e d n o n i n b r e d s t r a i n s , l . 6 T h e s u g g e s t i o n of " n o n r e a c t i v i t y " i s p a r t i c u l a r l y a t t r a c t i v e in the f i r s t two s t r a i n c o m b i n a t i o n s of this s t u d y . Is i s p o s s i b l e in r a t s , as w e l l a s in h a m s t e r s , t h a t t h i s represents a degree of "relatedness for-histocompatibility factors." As B i l l i n g h a m and H i l d e m a n 7 c o n c l u d e d , ' " T h e r e are o n l y a few *strong' but at l e a s t s e v e r a l ' w e a k ' h i s t o c o m p a t i b i l i t y a n t i g e n s ( g e n e s ) s e g r e g a t i n g in the Syrian h a m s t e r s t o c k s . " F o u r t h , t h e r e m a y be a d e f i n a b l e d i f f e r e n c e in the s t i m u l u s p r e s e n t e d to the h o s t by a l l e l i c and n o n a I l e l i c h i s t o c o m p a t i b i l i t y a n t i g e n s . T h e v a r i a t i o n in the p o t e n c y of a l l e l i c a n t i g e n s h a s b e e n n o t e d b e f o r e in blood group antigeras. T h e A and B s u b s t a n c e s are s t r o n g l y a n t i g e n i c , w h i l e O s u b s t a n c e is w e a k l y a n t i g e n i c . ~4 T h e d i f f e r e n c e in the s t i m u l u s of the t-11 l o c u s a s c o m p a r e d to the s t r o n g e r a n t i g e n s a s s o c i a t e d with the H 2 l o c u s i s well s t u d i e d ir~ inbred m i c e . 4,1v,~9, a° B i l l i n g h a m et al. 8 h a v e demons t r a t e d in m i c e the p h e n o m e n o n of v a r i a t i o n in the s t r e n g t h of h i s t o c o m p a t i b i l i t y a n t i g e n s in a c o n c l u s i v e w a y . T h e r e s u l t s in g r o u p s 1 and 2, 3 and 4 ( T a b l e 1) would s u g g e s t t h a t v a r i a t i o n in s t r e n g t h of h i s t o c o m p a t i b i l i t y a n t i g e n i s of c o n s i d e r a b l e s i g n i f i c a n c e in n o n i n b r e d s p e c i e s o t h e r than m i c e . It would n e v e r be p o s s i b l e to d e m o n s t r a t e the p r e s e n c e or a b s e n c e of t h i s f a c t o r in n o n i n b r e d a n i m a l s without m a i n t a i n i n g r e s e r v a t i o n s . In d e s i g n i n g e x p e r i m e n t s in w h i c h a n i m a l s o f unknown g e n e t i c c o m p o s i t i o n are u s e d , t h i s v a r i a b l e s h o u l d be a s s u m e d to be p r e s e n t . When g e n e t i c a l l y h e t e r o g e n o u s a n i m a l s are e m p l o y e d , an o c c a s i o n a l ' m a r k e d p r o l o n g a t i o n of g r a f t s u r v i v a l m u s t be e x p e c t e d . An i s o l a t e d
TISSUE T R A N S P L A N T A T I O N
51
p e r m a n e n t s u r v i v a l must be v i e w e d c r i t i c a l l y . Fifth, smaller differences such as occurred in the H o l t z m a n to L o n g - E v a n s g r a f t s ( g r o u p s 3 a n d 4, S e c t i o n A) c o u l d r e s u l t from c h a n c e s e l e c t i o n of a n i m a l s at d i f f e r e n t s t a g e s of a hair c y c l e . E a r l y studi e s by B a l l a n t y n e and C o n v e r s e 2 h a v e s h o w n a s much a s s i x day:g' d i f f e r e n c e in m e a n r e j e c t i o n t i m e s of s u p r a p a n n i c u l a r g r a f t s in n o n i n b r e d r a t s . T h e i r s u b s e q u e n t s t u d i e s 3 t e n d to m i n i m i z e the i m p o r t a n c e of t h i s f a c t o r and e m p h a s i z e the v a s c u l a r i t y of the graft bed f o l l o w i n g p l u c k i n g of hair. In t h e s e c o m b i n a t i o n s , it is d o u b t f u l t h a t the 15.6 day mean d i f f e r e n c e c o u l d be an e x p r e s s i o n of d i f f e r i n g skin c y c l e s . Skin c y c l e d i f f e r e n c e s do not explain the two g r a f t s in the H o l t z m a n to R o l f s m e y e r g r o u p (group 1) which p e r s i s t e d and had v i g o r o u s h a i r growth long a f t e r the r e c i p r o c a l p a i r e d c r o s s g r a f t from t h e s e a n i m a l s to the d o n o r s had been r e j e c t e d . In s u m m a r y , the s e c o n d and fourth p o s s i b i l i t i e s s e e m m o s t l i k e l y . It can b e s e e n that inbred s t r a i n s of r a t s with known g e n e t i c c o m p o s i t i o n would h a v e to be u s e d to m a k e a c o n c l u s i v e d i s tinction between these five factors. If the v a r i a b l e of i n d u c t i o n of t o l e r a n c e is a d d e d to the e x p e r i m e n t a l d e s i g n , as is done in the s e c o n d part of the s t u d i e s , one s e e s d a t a s u g g e s t i v e l y p a r a l l e l to the o b s e r v a t i o n s of M a r t i n e z e t al. l l in m i c e . T h e s e w o r k e r s demo n s t r a t e d t h a t t h e r e i s a m a r k e d d i f f e r e n c e in the p e r c e n t a g e of s u c c e s s e s in the i n d u c t i o n o f t o l e r a n c e in m i c e d e p e n d i n g on the p a r t i c u l a r c o m b i n a t i o n of s t r a i n s i n v o l v e d . At f i r s t g l a n c e , the d a t a in S e c t i o n B ( T a b l e I ) w o u l d s u g g e s t t h a t the a n t i g e n i c s t r e n g t h of the i n j e c t e d hom o l o g o u s t i s s u e is of i m p o r t a n c e . I n d u c t f b n of t o l e r a n c e i s m o s t s u c c e s s f u l in the " s t r a i n " c o m b i n a t i o n where normal h o m o g r a f t s u r v i v a l w a s l o n g e s t . Again, a d e f i n i t i v e s t a t e m e n t c a n not b e m a d e from our d a t a in n o n i n b r e d r a t s . The five possibilitiesmentioned a b o v e may all c o n t r i b u t e to the p r o d u c t i o n of t o l e r a n c e to skin h o m o g r a f t s . I n t e r p r e t a t i o n i s further comp o u n d e d by v a r y i n g d e g r e e s of immunologic maturity at the time of s p l e e n c e l l i n j e c t i o n ; d i f f e r i n g a n t i g e n i c i t y of i n j e c t e d d o n o r c e l l s both b e t w e e n " s t r a i n s " and within " s t r a i n s " ; and v a r y i n g l e t h a l i t y (due to runt d i s e a s e ) of inj e c t e d c e l l s . C o n s i d e r a t i o n of t h e s e m a n y f a c t o r s may be p a r t i c u l a r l y u s e f u l in i n t e r p r e t i n g much of the c o n f l i c t i n g d a t a in e n d o c r i n e t r a n s p l a n t a t i o n , w h e r e n o n i n b r e d a n i m a l s h a v e been e x t e n sively used. Nevertheless, noninbred animals can h a v e s o m e u s e f u l n e s s , when t h e i r u s e s e e m s n e c e s s a r y , for p u r p o s e s of l a r g e r s p e c i m e n s i z e or t e c h n i q u e r e q u i r i o g l a r g e r a n i m a l s , or for o b s e r v a t i o n s in a n o n i n b r e d s p e c i e s . T h e ex-
52
MC Q U A R R I E , BRO~'N and V A R C O
p e r i m e n t will of n e c e s s i t y h a v e to be o n e which d o e s not a t t e m p t to d i s t i n g u i s h d i s c r e t e g e n e t i c or h i s t o c o m p a t i b i l i t y f a c t o r s . One m u s t p r e s u m e the a b s e n c e of g e n e t i c k n o w l e d g e of the a n i m a l s . E x p e r i m e n t s m u s t be d e s i g n e d a c c o r d i n g l y . B a s i c p r e c a u t i o n s s h o u l d include: (a) S i m u l t a n e o u s s e l e c t i o n on a s t r i c t l y random b a s i s (by a t a b l e of random n u m b e r s ) of the e x p e r i m e n t a l and control g r o u p s . (b) Any m e a s u r e d v a r i a b l e s h o u l d h a v e a r e a s o n a b l y random d i s t r i b u t i o n through the e x p e r i m e n t a l and control g r o u p s . N o t e the m a r k e d l y s k e w e d d i s t r i b u t i o n e v i d e n t in group 1, Section A, to h a v e a mean of 39 d a y s with a maximum o f 57 d a y s . (c) One s h o u l d f a c e the n e c e s s i t y t h a t with a h e t e r o g e n e o u s p o p u l a t i o n of v a r y i n g c h a r a c t e r i s t i c s , e n l a r g e m e n t of the e x p e r i m e n t a l error i s the r e s u l t . T h i s is e v i d e n t here in the wide s t a n d a r d error of the m e a n . T h i s s h o u l d be c o m p a r e d to t h e s m a l l s t a n d a r d error in s i m i l a r s i z e d g r o u p s in s t u d i e s in i n b r e d m i c e . I n c r e a s e in v a r i a n c e within e x p e r i m e n t a l groups d e m a n d s e n l a r g e m e n t of g r o u p s i z e and d i m i n i s h e s t h e c h a n c e s of d e t e c t i n g s m a l l e r eff e c t s . O c c a s i o n a l l y , the u s e o f n o n i n b r e d a n i m a l s for " e c o n o m y " p u r p o s e s i s no e c o n o m y at all. (d) It s h o u l d n e v e r be p r e s u m e d t h a t s i m i l a r studies done at different times using commercial a n i m a l s are in any way a d d i t i v e . T h e a u t h o r s h a v e had a s u p p l i e r c h a n g e without n o t i c e from one c o l o n y to a new, g e n e t i c a l l y d i f f e r e n t , partially inbred c o l o n y . In s o m e t r a n s p l a n t a t i o n p r o b l e m s , in which u s e of a n i m a l s of unknown g e n e t i c i d e n t i t y i s either n e c e s s a r y or u n a v o i d a b l e , it i s w e l l to recognize that significant strain differences and s y s t e m a t i c v a r i a b l e s may be p r e s e n t . G e o g r a p h i c s e p a r a t i o n and c l o s e d c o l o n y r a n d o m mating do not a l w a y s g u a r a n t e e g e n e t i c d i v e r s i t y of two s t r a i n s of r a t s . T h i s would p r e s u m a b l y be e q u a l l y true for o t h e r s p e c i e s . E x p e r i m e n t a l design s h o u l d be c r i t i c a l and e x p e r i m e n t a l g r o u p s i z e s h o u l d be l a r g e enough to a s s e s s the true e f f e c t of " s t r a i n " and unknown g e n e t i c f a c t o r s when the u s e of n o n i s o t o g o u s a n i m a l s of a n y s p e c i e s is r e q u i r e d . An o b v i o u s o p t i m u m s o l u tion would b e to h a v e inbred s t r a i n s of the m a j o r l a b o r a t o r y a n i m a l s p e c i e s r e a d i l y a v a i l a b l e from c o m m e r c i a l or o t h e r s o u r c e s .
SUMMARY 1. O b s e r v a t i o n s b e t w e e n s t r a i n s of commercially bred noniSologou~ rats show marked variation in.homograft survival between six comb i n a t i o n s of t h r e e s t r a i n s . D i f f e r e n c e in m e a n s u r v i v a l of g r o u p s of r e c i p r o c a l interstrain g r a f t s w a s a s g r e a t a s 15 d a y s .
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2. T h e p r o p o r t i o n of s u c c e s s f u l i n d u c t i o n of t o l e r a n c e to h o m o g r a f t s b e t w e e n s t r a i n s v a r i e d , d e p e n d i n g on s t r a i n c o m b i n a t i o n . 3. T h e d a t a d e m o n s t r a t e the d e g r e e of s o m e v a r i a t i o n s of e x p e r i m e n t a l error which can a r i s e in u s i n g n o n i n b r e d c o m m e r c i a l a n i m a l s in t i s s u e transplantation studies. 4. Some of the p r o b l e m s in i n t e r p r e t a t i o n and d e s i g n of h o m o g r a f t e x p e r i m e n t s are d i s c u s s e d .
ll E F E II F]~ C E S 1. Adams, R.A., Patt, D.I., and Lutz, B.R.: Longterm p e r s i s t e n c e of skin homografts in untreated hamsters. Transpl. Bull., 3:41, 1956. 2. Batlantyne, D.L., and Converse, J.M.: Effect of hair cycles on rat skin homografts. Ann. N.Y. Acad. Sci., 64:958, 1957. 3. Battantyne, D.L., and Converse, J.M.: Further observations of halr-skin cycles and the survival of skin homografts in rats. Transpl. Bull., 6:93, 1959. 4. Berrian, J.tt., and Jacobs, R.L.: Diversity of transplantation antigens in the mouse. Biological Problems of Grafting. University of Liege (Belgium), 1959, p. 131. 5. Billingham, R.E., and Silvers, W.K.: Inbred animals and tissue transplantation immunity. Transpl. Bull., 6:399, 1959. 6. Billingham, R.E., and ltildeman, W.tt.: Studies of transplantation immunity in hamsters. Ann. N.Y. Acad. Sci., 73:676, t958. 7. Billingham, R.E., and tIildeman, m.ll.: Studies on the i:amunological responses of hamsters to skin homografts. Proc. Roy. Soc. London, s. B, 149:216, 1958. 8. Billingham, R.E., Brent, L., Medawar, P.B., and Sparrow, E.M.: Quantitative studies on tissue transplantation immunity. L. The survival times of skin homografts exchanged between members of different inbred strains of mice. Proc. Royal Soc., s. B, 143:43, 1954. 9. Dempster, W.J., Lennox, B., and Boag, J.W.: Prolongation of survival of skin homotransplants in the rabbit by irradiation of the host. Brit. J. Exp. Path., 31-670, 1950. 10. Good, R.A., Varco, R.L., Aust, J.B., and Zak, S.J.: Transplantation studies in patients with agammaglobulinemia. Ann. N.Y. Acad. Sci., 64:882, 1957. I I . Martinez, C., Smith, J.M., Aust, J.B., and Good, R.A.: Acquired tolerance to skin hOmografts in mice of different strains. Proc. Soc. Exper. Biol. & Med., 97:736, 1958. 12. McQuarrie, D.G., Kim, J.H., and Varco, R.L.: The long-term surviwlt of intrastrain homografts in comm~:rcia|ly bred rats. Transpl. Bull., 6:97, 1959. 13. Medawar, P.B., and Woodruff, M.F.A.: The induction of tolerance by skin homografts on newborn rats. Immunology, 1:27, 1958. 14. Morgan, W.T.J., and Waddell, M.B.R.: Specific blood group O substance. Brit. J. Exp. Path., 26:387, 1945. 15. Peer, L.A.: Behavior of skin grafts exchanged between parents and offspring. Ann. N.Y. Acad. Sci;, 73:584, 1958.
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Vol. 111, No. I - J a n u a r y , 1963
16. Puza, A., and Gombas, A.: Acquired tolerance of skin homografts in dogs. Transpl. Bull., 5:3(J, 1958. 17. Rubin, B.A.: The determination of the H-2 genotype of CE mice. Transpl. Bull., 7:153, 1960. 18. Scothorne, R.J.: Studies on the response of regional lymph nodes to skin homografts. Ann. N.Y. Acad. Sci., 64:1028, 1957. 19. Snell, G.D.: The immunogenetics of tumor transplantation. Cancer Res., 12:543, 1952.
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20. Snell, G.D., Smith, P., and Gabrielson, F.: Analysis of the histocompatibility-2 locus in the mouse. J. Nat. Cancer Inst., 14:457, 1953. 21. Tomas, L., Murray, J.E., and Couch, N.P.: Consecutive skin homografts in the dog. Transpl. Bull., 4:156, 1957. 22. Woodruff, M.F.A.: Postpartum induction of tolerance to homologous skin in rats. Ann. N.Y. Acad. Sci., 64:792, 1957.