Horse Anti-Bovine Lymphocyte Serum ITS Effects In calves
H e t e r o l o g ~ u santilyriphacyte serum (i',LF)
refers t o
antiserum t h n t w a s m i s e d i n one species zg-:inst
lymphoid
c e l l I n t i g e n s of o t h e r s p e c i e s . Slthough the p r e p ? r n t i o n
of ALS was a l r e a d y r e p o r t e d by Metchnikoff i n 1899 t h e use
of t h i s e n t i s e r ? f o r modulation of the innune response had
t o w 2 i t u n t i l t h e r o l e of lynpknid t i s s u e i n t h e i m ~ u n e
r e z c t i o n w 2 s recognized.
I n t h e f a t e f i f t i e s t h e experiments
of I n d e r b i t e e n (1956) were t h e f i r s t t o demonstrate t h a t
heterologous a n t i s e r u r ~directeif Ei,@inst
lynphoict c e l l s c ~ u l d
F n h i b i t delayed h y p e r s e n s i t i v i t y r e a c t i o n , S e v e r a l y e a r s
l a t e r Woodruff snd Anderson (1963) showed c l e a r l y t h a t ALS
used e i t h e r alone o r as a n a d j u n c t t ? t h o r o c i c duct drainage
could prolong the s u r v i v a l of s k i n a l l o g r a f t i n r a t s .
Since 1967 ALS h a s been t h e s u b j e c t of i n c r e a s i n g
n m b e r s o f p u b l i c ~ t i o n sand reviews.
f i e l d of irmnunosuppression.
I t s main use is i n t h e
I t hEts t h e ~ b i l i t yt o msrkedly
diminish c e l l u l a r im-iune responses p a r t i c u l a r l y s k i n a l l o g r a f t
and h e t e r o p a f t r e j e c t i o n i n experimental animals. It a l s o
czn n b o l i s h t h e inmunity evoked by a previous e l l o g r a f t .
The g r a f t v e r s u s h o s t r e ~ c t i o ni n i r r a d i a t e d animals such
a s mice o r monkeys caused by t h e i n j e c t i o n o f a l l o g e n i c s p l e e n
o r bone narrow c e l l s can be n e u t r a l i z e d by :?LS i n j e c t i o n .
ALS w a s one of t h e first t o o l s used t o s t u d y thymus dependent
!I?
c e l l f u n c t i o n s and antibody mediated bone nnrrow dependent
3 cell functions in the immune response. Ls a n experimental
tool it is still used to inliibit preferentially cellular
immune response, while lesving humoral antibody mechanism
generally intact. With regard to clinical immunosuppr~saion
ALS represents the first agent whose actions were limited
to cells of the lymphoid syxtcm and which would specifically
interact with lymphocytes in both resting and proliferative
stages. Thc immunosuppressive effects of ALS can be increased
by prior thymectomy, whole body irradiation, administration
of corticosteroid and lymphocyte depletion by thoracic duct
fistulaALE has been raised in horses, rabbits, pigs and rumi-
nants against lymphocytes of the dos, mouse, rat, monkey and
human. The sera usually possess optimal immunosuppressive
activity when injected into the lymphocyte donor species.
Anti-rat and anti-mouse antisera have also Seen rcised in
chickens a n d ducks, but their spectra of activity are somewhat narrower than mammalian antisera, because of their poor
interaction with mammalian complement. Xany different immunization techniques have been tried, yielding antisera of
different potency.
Non-lymphoid tissues have been used to produce ALS with
effective immunosuppressive action including mouse cell
cultures (L cells),
epidermal cells o r plasma cells. Lymphoid
cell a n d thymocyte cell fractions have also been used to produce potent, more highly specific and less toxic sera.
The 7 5 I&
f r a c t i o n s o f ALS c o n t a i n v i r t u a l l y a l l immuno
suppressive potency,
-
b u t t h a i n t a c t a n t i b o 5 g m o l ~ c u l ci s r e -
q u i r e d f o r o p t i m a l immunosuppressive a c t i v i t y .
Thz p u r e 1 9 s
f r a c t i o n o f ALS s e p . ? r a t e d by i m f i u n o e l e c t r o p h o r ~ s i sh a s l i t t l e
o r no immunosuppressiva a c t i v i t y , The ~ ( a b ) 2p o r t i o n of
the
a n t i b o d y molecule, o b t a i n e d by d i g e s t i o n w i t h p e p s i n from h o r s e - anti-rut
l y n p h o c y t I&
f z i l c d t o p r o l o n y t h e s u r v i v , z l of s k i n
a l l o g r a f t i n r a t s a s r e p o r t e d by ?Foodruff
~4
?&-(1967)-
S i n c e t h e r e p o r t o f S t s r z l g t a&, (1367) ALS has been
usad c l i n i c a l l y i n humrns. A l t h o u g h A L 7 r e m a i n s t h e most p o w e r f u l s i n g l e agent f o r prolongation of s k i n g r a f t s i n mice, i t s
e f f e c t s i n hwu:?ns hnva b e e n f a r l e s s d r a m a t i c .
I t h a s bccome
c l e a r t h a t ALS alone c a n n o t be used t o o b t a i n n c o n t i n u o u s
i m m u n o s u p p r e s s i o n , but r a t h e r must be a p p l i e d i n c o m b i n a t i o n
w i t h c o n v e n t i o n a l immunosuppressive a : y c n t s used i n c l i n i c a l
transplantations.
The m a j o r c l i n i c a l u s c o f ALS a t p r e s e n t i s
a s nn . a d j u n c t t o u s u < ? l islmunosupprcssive 6 r u . z ~s u c h a s a z a t h i o p r i n e a n d p r c d n i s o n e i n t h o management o f p a t i o n t s w i t h k i d n c y
o r l i v e r a l l o s r , a f t , where t h o ALS a c t s on t h e c e l l u l a r i r m u n i t y
a n d a z a t h i o p r i n c on t h e humoral ivmuuli t y
I n c a r d i sc transplan -
t a t i o n ALS i s now r o u t i n e l y added t o t h e u s u a l c o r l b i n a t i o n o f
a z a t h i o p r i n e , p r e d n i s o n c and r s d i n t i o n .
An i m p o r t a n t and r a
t i o n a l i n d i c c t i o n of ALS and a z a t h i u p r i n c i s p r o d u c i n g ternpor a r y immunosuppression t o f a c i l i t ? t e t h e t a k e o f e l l o g r a f t
s k i n on p a t i e n t s w i t h m a s s i v e s e c o n d %nd t h i r d d e g r e e b u r n s
u n t i l h e a l i n g by s e c o n d i n t e n t i o n c a n t?lie p l a c e .
A n o t h e r i n p o r t a n t use o f a n t i l y l l p h o c y t e g l o b u l i n ( A L G )
i s i n p a t i e n t s w i t h fymphoS1NsZ;ic anemia t : ~allo-f: t h e t a k e
of HLA n o n i d e n t i c a l bone marrow. ALS h a s a l s o been a d d e d t o
the
xist
of n g e n t s which may be of v a l u e i n t h e ! 3 u t o i m u n e
d i s o r d e r s o r o t h c r d i s e n c e s w i t h irniune fc2ntures.
I n the past
4LG h a s a l s o been used i n t h e t r e a t m e n t o r l y m p h o p r o l i f e r a t i v e
d i s o r d e r s l i k e l y m p h a t i c 1eukr:min.
I n V e t e r i n a r y Medicinr. t h e usi! o f ALS i s m o s t l y f o r
experimental purposos,
Ths e f f e c t s of A L S i n m i c e , r a t s , dogs
and primatez. hava been s t u d i e d i n t e n s i v e l y * I t i n known t h a t
t h e chimpznze,r; s h ~ r e sorgan a n t i g e n s w i t h humans.
?$any human
l u u c o c y t e a n t i g e n s a r e p r a s e n t on t h e l c u c o c y t e s of chimpan-,
zecs.
S e v e r a l p r i m a t e s p e c i e s ( c h i n p n n z c e ) and r h c s u s monkegs
have been used f o r t h z q u a l i t y a n d s s f a t y c o n t r o l of a n t i human lymphocyte scrum, A n t i - d o s lymphocyte s e r u m h a s been used i n
transplantation studies.
Howcvor,
t h e c l i n i c a l a p p l i c a t i o n of
animal A L B i s very l i n i t a d .
So f a r t h e e f f s c t s o f a n t i Lymphocyt serum i n c a t t l e h a s
n o t been p u b l i s h e d .
O r i g i n a l l y t h e purpose o f t h e production of h o r s e ~ n t i - b o v i n e lymphocyt serum (ABLc;)
w a s f o r t h e t r a n s m i s s i o n o f ju-
v e n i l e bovine l e u k o s e , which i s a d i s e a s e e s p e c i a l l y a f f e c t i n 2
calves
.
The c h a r z c t e r s o f t h e h o r s e A R T S ,
however, ia n o t y e t
known- T h e r e f o r e i t s e f f e c t s on some of t h e humor.51 and c e f l u
l a r immune f u n c t i o n s i n c a l v e s should be determined.
2.1.
P r o d u c t i o n . o f ~ n t i l y r ? p h o c y t os c r u r ? "
FTethods o f r a i s i n g A L S havc b e c n d e s c r i b e d by m a n y
i n v e s t i q n t o r s u s i n ? lymphoid c e l l s suspended i n a nedima
o r e m u l s i f i e d i n complc t e F r o u n d . * s a d j u v a n t
. M':ny
immu-
n i z a t i o n t o c h n i q u e o h a v e b e e n used y i e l d i n g ALS o f h i g h
i m r ~ u n o s u p p r c s s i v ep o t cncy.
T ~ most
L
cornrfion 2 r e t h e tvuo
o r t h r c o nubcutaneous o r i n t r a v e n o u s i n j e c t i o n s q i v e n
3-t
t w o weeks ~ p n r ft o l l o w e d by n b o o s t s r i m m u n i z z t i o n
f o u r weeks t h e r e e f t e r . M n n y d i f f e r e n t h o s t s p e c i e s h a v e
b c e n used t o r a i s c o f f c c t i v e ALS, The most used s p z c i e s
a r e t h e r a b b i t and t h e h o r s s .
P i g s and rurciinrnts h a v e
a l s o b e e n used b u t t h e y proiluca = n t i c c ? r e xof l o w e r q u - ? l i t g .
n
~n%isem
h a v e boon r a i s c C a g a i n s t lymphoid t i s s u e s o f
human, monkey, d o g , r a t , b o v i n e a n d n o u s e The p r o d u c t i o n o f a n t i - m t B L R i n r z b b i t s wes
r e p o r t e d by Woodruff nnc? Anderson ( 1 9 6 3 ) .
i n t r a p e r i t o n c a l l y 200.0
They injected
x l o 6 r a t t h o r a c i c d u c t lympho-
c y t e s t h r e e t i m e s a t weekly i n t e n r " 1 s .
T h c r l b b i t s \-?ere
b l e d t e n d:?ys a f t e r t h e l a a t i n j e c t i o n and b o o s t e r i n t r a p e r i t o n c z d i n j z c t i o n o f 1-2
x lo6 l y m p h o c y t e s were given
t e n days b e f o r e each subsequent bleeding.
The p r o d u c t i o n o f anti-mo u s e A L S w a s communicated
by Levey an2 Pa?cdawar ( 1 3 6 6 a ) , S r z y
naco
et ale
(1966a).
~5 51. (1966) and Ho-
Lavey and Medawar ( 1 9 6 6 ~ )a d m i n i s t e r e d
two i n t r a v e n o u s i n j e c t i o n s of
4-10
r a b b i t s a t f o u r t c a n d ~ y sc g s r t .
x
lo8
lymphoid c e l l s i n
The r a b b i t n TA?eret h e n b l e d
7 d a y s a f t e r t h e s e c o n d i n j a c t i o n and t h e s e r ? were c o l l c c t c d
and h c n t i n ~ c t i v a t e d( 5 6 O ~f o r 30 m i n u t e s ) , S e i t z f i l t e r e d
a n d s t o r e d a t -20°C
~ - 5%&.
u n t i l u s e d . Gray e_f: 21, ( 1 9 6 6 ) and blonaco
( 1 9 6 6 ) r e p o r t e d t h e i r methoci o f r a i s i r i f : A I - T i n r ~ b b i t -
a s f o l l o w s , Each r a b b i t r e c e i v e d a t o t s 1 o f 100.0
x 10' lymph
node c e l l s e m u l s i f i e d i n complete P r e u n d S s a d j u v a n t .
The e ~ . l u l -
s i o n was e q u a l l y d i v i d e d and i n j e c t e d i n t o e a c h orr" t h e f o u r
footpads.
100.0
The b o o s t e r i n j u c t i o n a o f s p l i n t ? c e l l s u s p e n s i o n of
x lo6 c e l l s w i t h o u t
a d j u v n n t weri: g i v e n i n t r n v c n o u s l y
on t h r e e s u c c e s s i v e d a y s f o u r weeks l a t e r .
bled f o u r days a f t e r t h e l a s t i n j e c t i o n .
Thc r a h b i t a wGrc
The blood was l e f t
t o c l o t a t room t e m p e r a t u r e a n d s e r m was c ~ l l e c t a dt h e n e x t
day, h e a t i n z c t i v a t e d and s t o r e d n t - 2 0 ' ~ .
The mode o f p r o d u c t i o n o f ALS in h o r s o s was r e p o r t e d by
a nwnbcr o f w o r k e r s .
IwasaZri o
...t
. - -.
sl.
..
(1967) i ~ x l u n i z e d t e n
h o r s e s w i t h d o g ' s s p l a n i c and lymph node c e l l s a n d 2 h o r s e s
w i t h human lymphoid t i s s u e s .
The h o r s e s were g i v e n 4 t o 6 in-
i n o c u l r t i o n s s t weekly i n t e r v a l n w i t h 0.2
lynphoid c e l l s i n s a l i n e .
t o 194 b i l l i o n
They found t h a t t h e s e r a when i n - -
j e c t e d i n t o does caused l p p h o p c n i a .
They o b s e r v e d t h a t d o s e s
of up t o 1 9 4 b i l l i o n l y n p h o i d c e l l s would r a p i d l y i n c r e z s e
the cytotoxic t i t e r .
Balncr
ct c&. (1968)
2 n d She51 e s
s-
(1971) i n j c c t e d h o r s e s by t h s s u b c u t s r i e o u s r o u t e w i t h 1.0 x f 0 l U
lymphoid c a l l s on 4 t o 6 o c c a s i o n s a t 2 t o 4 weeks i n t e m l o
f o r t h e p r o d u c t i o n o f anti-hu.nan A L S .
one week a f t e r t h e l a s t i n j z c t r o n .
produced i n t h i s manner prolang,d
Thc. i ~ o r s e swere b l e d
They r e p o r t e d t h n t s e r a
s k i n . g r a f t s u r v i v a l i n zlon-
k e y s . Lucko @ a2. (1968) imlunizud h o r s e s by, 3 s u b c u t z n e o u s
i n j e c t i o n s o f 1.0
x 10'
p i g lymph node c e l l s i n m e d i a 1 9 9
at 7 d r y s . Blood was c o f l e c $ c d one wcek a f t o r t h e l a s t i n j e c t
ion.
Twenty f o u r h o u r s t h e r e a f t e r t h e h o r s e s werc g i v e n a n o t h e r
i n j e c t i o n of 0.44
x 10' l y n p h noiic c e l l s and b l e d 7 dcys l a t e r .
The blood wes r l l o w e d t o s t a n d f o r 2 h o u r s zt room t e n p e r Q t u r a
and t h e n k e p t o v e r n i g h t n t
~ O C .
The s a r m w a s s e p a r r t e d , a&
sorbed w i t h one t h i r d of i t s v o l w l e w i t h p i g e r y t h r o c y t e s end
s t o r i l i z c d by S c i t z f i l t r a t i o n and s t o r e d a t - 2 0 O ~ u n t i l used.
They observed t h a t s e r a produced i n t h i s n s n n c r were e f f e c t i v e
and p r o l o n g e d s k i n g r a f t s u r v i v a l in p i g s .
Clroll e_t EC.
immunized 2 h o r s e s by two s u b c u t a n e o u s i n j e c t i o n s of 2.0
b o v i n e m i l k l e u c o c y t e s i n c o r ~ 7 l e t eP r e u n d ' s
(1976)
x 1 06
adjuv?nt folloned
by intravenous injections o f leucocytcs s u s p e n s i o c i n s a l i n e
e v e r y wsek t h e r e a f t e r u n t i l 5 1 wcek.
week.
Blood was c o f l o c t e d e v e r y
The h o r s e a n t i b o v i n e l e u c o c y t e serum when i n j c c t e Z i n t o
c a t t l a caused n e u t r o p e n i a ~ i t h i n1 h o u r ,
Bi-s
et 41.
of anti-mouse
(1971) r e p o r t e d t h e t e c h n i q u e of p r o d u c t i o n
, anti-rat,
i n e i t h e r pigs,
anti-dog
an2 a n t i - c h i c k e n
ALS r z i s e d
c a l v e s , one g o e t o r s h c e p , Each of t h e a n i m a l s
r e c e i v e d v a r y i n g q u z n t i t i e s o f donor thymic c e l l s i n s a l i n e
s u s p e n s i o n o r e m u l s i f i e d i n complete F r e u n d ' s
a d j u v s n t . Nost
a n i m a l s were given two i n j e c t i o n s ~t t w o week i n t o r v a l s .
They
observed t h a t t h e nethod developed f o r t h e groduction of imnunosuppressive scrurl i n t h e p i g n f l i n s t m o u s e lymphocytes proved
s z t i s f a c t o r y f o r t h e p r e p l r a t i o n of a n t i - r s t ,
a n t i - c h i c k e n dLS.
2nti--dog 2nd
B o v i n < ~sheep
,
and gont ALS nnd* a g a i n s t
mouse thyinocytcs a l s o had s t r o n g irmunosuppressivo n c t i v i t y .
I n t h e bovine, production o f im-tunosuppressive ~ c t i v i t yseemed
age dependent.
They noted t h a t poor immunosuppressive a c t i v i t y
was found i n c n l v c s which were l e s s than 2 months o l d .
Th.3 p r o d u c t i o n of anti-nouac,
anti-guinea
a1.
-.-
anti-hunan,
anti-dog
2nd
pi.? ALS i n g o a t s has been r c p o r t e d by Roll-d
(1971). These nnimnls reccivud f o u r i n t r a v e n o u s i n j e c t i o n s
of 5.0
x lo7 cells p e r kg body weight
.qt
weelrly interv.?.ls and
were k i l l e d and bled out one week z f t e r the l e s t 5nj::ction.
They found a c o r r e l n t i o n bet~voen t h e
2.q
vLKr2 membrcne i m u n o -
f l u o r e s c e n c e t i t e r of t h e s e r a s m i n s t nouse thymocytes t o the
in
--
vivo
----- i m u n o s u ~ p r e s s i v ea c t i v i t y
0s
judge6 by profon,+?tion
of t a i l s k i n h o r ~ o g r a f ts u r v i v a l ,
S h e i l g& 9 2 . (1971) communicated t h e p r o d u c t i o n of 8 n t i -
human lymphocyte s c r m i n 3 g o e t s . Eech goc:t rec-eivad 5.0
x 109
thynocytes i n m u l t i p l e subcutaneous i n j e c t i o n s on 3 occasions.
The f i r s t imunizntion
was foflovred a f t o r 2 weeks by 2 i n j e c t - -
i o n s a t weekly i n t e r v a l s .
O n e week n f t e r the t h i r d i n j e c t i o n
t h e g o a t s were bled o u t and 1.5
l i t e r o f serum w::s
obtained
f r o m aach, The antilymphocyte g l o b u l i n prepared from t h i s ALS
was i r ~ ~ u n o s u p p r e s s i v i -n no.nkeys w i t h s k i n xi?nogc.ft.
E f f o c t i v c anti-mouso 6LS i n c h i c k e n s
-3-1 ducks
was pro-
-
( l c 6 R ) . Thcy found t h a t i n v
.-. i t g
. o
- the
d u c d bg j o ~ s t c~ t 21.
,
s e r a wera s t r o n g l y c y t o t o x i c t o roouse l y n ~ h o c y t e si _ n . t h a p r e
s e n c e o f duck o r r a b b i t c o s p l c n e n t b u t i n j c c t i o n i n t o 'nice
b r o u s h t nbout o n l y z i n o r t r z n s i c n t l p s h o p e n i r : .
Many h y p o t h e s e s have been ~ r o p o s e don t h e node of a c t i o n
o f ALS. Nost o f t h c s z hypothesi-s have b e s n t r i e d o u t a n 3
d i s c a r d e d and o r ~ yt h e h y p t h c s i s proposcd by Lance (1968)
vias a c c e p t n b l c . Lance :€nun? t h R t tho T l o b u l i n f r a c t i o n of BLS
w 3 s a c t i v e i n i;munosugpressiun.
H e r e p o r t e d t h a t t h e mti-
lynphocjtte . z l o b u l i n a c h i e v e 3 i t s irmuno-suppressive a c t i o n by
c a u s i n g s o l c c t i r r i ~d e p l e t i o n of c a l l s ~f t h o r e c i r c u l a t i n g
pool
of l y n p h o c y t e s . T h i s
ic
refers t o
antiserum t h n t w a s m i s e d i n one species zg-:inst
lymphoid
c e l l I n t i g e n s of o t h e r s p e c i e s . Slthough the p r e p ? r n t i o n
of ALS was a l r e a d y r e p o r t e d by Metchnikoff i n 1899 t h e use
of t h i s e n t i s e r ? f o r modulation of the innune response had
t o w 2 i t u n t i l t h e r o l e of lynpknid t i s s u e i n t h e i m ~ u n e
r e z c t i o n w 2 s recognized.
I n t h e f a t e f i f t i e s t h e experiments
of I n d e r b i t e e n (1956) were t h e f i r s t t o demonstrate t h a t
heterologous a n t i s e r u r ~directeif Ei,@inst
lynphoict c e l l s c ~ u l d
F n h i b i t delayed h y p e r s e n s i t i v i t y r e a c t i o n , S e v e r a l y e a r s
l a t e r Woodruff snd Anderson (1963) showed c l e a r l y t h a t ALS
used e i t h e r alone o r as a n a d j u n c t t ? t h o r o c i c duct drainage
could prolong the s u r v i v a l of s k i n a l l o g r a f t i n r a t s .
Since 1967 ALS h a s been t h e s u b j e c t of i n c r e a s i n g
n m b e r s o f p u b l i c ~ t i o n sand reviews.
f i e l d of irmnunosuppression.
I t s main use is i n t h e
I t hEts t h e ~ b i l i t yt o msrkedly
diminish c e l l u l a r im-iune responses p a r t i c u l a r l y s k i n a l l o g r a f t
and h e t e r o p a f t r e j e c t i o n i n experimental animals. It a l s o
czn n b o l i s h t h e inmunity evoked by a previous e l l o g r a f t .
The g r a f t v e r s u s h o s t r e ~ c t i o ni n i r r a d i a t e d animals such
a s mice o r monkeys caused by t h e i n j e c t i o n o f a l l o g e n i c s p l e e n
o r bone narrow c e l l s can be n e u t r a l i z e d by :?LS i n j e c t i o n .
ALS w a s one of t h e first t o o l s used t o s t u d y thymus dependent
!I?
c e l l f u n c t i o n s and antibody mediated bone nnrrow dependent
3 cell functions in the immune response. Ls a n experimental
tool it is still used to inliibit preferentially cellular
immune response, while lesving humoral antibody mechanism
generally intact. With regard to clinical immunosuppr~saion
ALS represents the first agent whose actions were limited
to cells of the lymphoid syxtcm and which would specifically
interact with lymphocytes in both resting and proliferative
stages. Thc immunosuppressive effects of ALS can be increased
by prior thymectomy, whole body irradiation, administration
of corticosteroid and lymphocyte depletion by thoracic duct
fistulaALE has been raised in horses, rabbits, pigs and rumi-
nants against lymphocytes of the dos, mouse, rat, monkey and
human. The sera usually possess optimal immunosuppressive
activity when injected into the lymphocyte donor species.
Anti-rat and anti-mouse antisera have also Seen rcised in
chickens a n d ducks, but their spectra of activity are somewhat narrower than mammalian antisera, because of their poor
interaction with mammalian complement. Xany different immunization techniques have been tried, yielding antisera of
different potency.
Non-lymphoid tissues have been used to produce ALS with
effective immunosuppressive action including mouse cell
cultures (L cells),
epidermal cells o r plasma cells. Lymphoid
cell a n d thymocyte cell fractions have also been used to produce potent, more highly specific and less toxic sera.
The 7 5 I&
f r a c t i o n s o f ALS c o n t a i n v i r t u a l l y a l l immuno
suppressive potency,
-
b u t t h a i n t a c t a n t i b o 5 g m o l ~ c u l ci s r e -
q u i r e d f o r o p t i m a l immunosuppressive a c t i v i t y .
Thz p u r e 1 9 s
f r a c t i o n o f ALS s e p . ? r a t e d by i m f i u n o e l e c t r o p h o r ~ s i sh a s l i t t l e
o r no immunosuppressiva a c t i v i t y , The ~ ( a b ) 2p o r t i o n of
the
a n t i b o d y molecule, o b t a i n e d by d i g e s t i o n w i t h p e p s i n from h o r s e - anti-rut
l y n p h o c y t I&
f z i l c d t o p r o l o n y t h e s u r v i v , z l of s k i n
a l l o g r a f t i n r a t s a s r e p o r t e d by ?Foodruff
~4
?&-(1967)-
S i n c e t h e r e p o r t o f S t s r z l g t a&, (1367) ALS has been
usad c l i n i c a l l y i n humrns. A l t h o u g h A L 7 r e m a i n s t h e most p o w e r f u l s i n g l e agent f o r prolongation of s k i n g r a f t s i n mice, i t s
e f f e c t s i n hwu:?ns hnva b e e n f a r l e s s d r a m a t i c .
I t h a s bccome
c l e a r t h a t ALS alone c a n n o t be used t o o b t a i n n c o n t i n u o u s
i m m u n o s u p p r e s s i o n , but r a t h e r must be a p p l i e d i n c o m b i n a t i o n
w i t h c o n v e n t i o n a l immunosuppressive a : y c n t s used i n c l i n i c a l
transplantations.
The m a j o r c l i n i c a l u s c o f ALS a t p r e s e n t i s
a s nn . a d j u n c t t o u s u < ? l islmunosupprcssive 6 r u . z ~s u c h a s a z a t h i o p r i n e a n d p r c d n i s o n e i n t h o management o f p a t i o n t s w i t h k i d n c y
o r l i v e r a l l o s r , a f t , where t h o ALS a c t s on t h e c e l l u l a r i r m u n i t y
a n d a z a t h i o p r i n c on t h e humoral ivmuuli t y
I n c a r d i sc transplan -
t a t i o n ALS i s now r o u t i n e l y added t o t h e u s u a l c o r l b i n a t i o n o f
a z a t h i o p r i n e , p r e d n i s o n c and r s d i n t i o n .
An i m p o r t a n t and r a
t i o n a l i n d i c c t i o n of ALS and a z a t h i u p r i n c i s p r o d u c i n g ternpor a r y immunosuppression t o f a c i l i t ? t e t h e t a k e o f e l l o g r a f t
s k i n on p a t i e n t s w i t h m a s s i v e s e c o n d %nd t h i r d d e g r e e b u r n s
u n t i l h e a l i n g by s e c o n d i n t e n t i o n c a n t?lie p l a c e .
A n o t h e r i n p o r t a n t use o f a n t i l y l l p h o c y t e g l o b u l i n ( A L G )
i s i n p a t i e n t s w i t h fymphoS1NsZ;ic anemia t : ~allo-f: t h e t a k e
of HLA n o n i d e n t i c a l bone marrow. ALS h a s a l s o been a d d e d t o
the
xist
of n g e n t s which may be of v a l u e i n t h e ! 3 u t o i m u n e
d i s o r d e r s o r o t h c r d i s e n c e s w i t h irniune fc2ntures.
I n the past
4LG h a s a l s o been used i n t h e t r e a t m e n t o r l y m p h o p r o l i f e r a t i v e
d i s o r d e r s l i k e l y m p h a t i c 1eukr:min.
I n V e t e r i n a r y Medicinr. t h e usi! o f ALS i s m o s t l y f o r
experimental purposos,
Ths e f f e c t s of A L S i n m i c e , r a t s , dogs
and primatez. hava been s t u d i e d i n t e n s i v e l y * I t i n known t h a t
t h e chimpznze,r; s h ~ r e sorgan a n t i g e n s w i t h humans.
?$any human
l u u c o c y t e a n t i g e n s a r e p r a s e n t on t h e l c u c o c y t e s of chimpan-,
zecs.
S e v e r a l p r i m a t e s p e c i e s ( c h i n p n n z c e ) and r h c s u s monkegs
have been used f o r t h z q u a l i t y a n d s s f a t y c o n t r o l of a n t i human lymphocyte scrum, A n t i - d o s lymphocyte s e r u m h a s been used i n
transplantation studies.
Howcvor,
t h e c l i n i c a l a p p l i c a t i o n of
animal A L B i s very l i n i t a d .
So f a r t h e e f f s c t s o f a n t i Lymphocyt serum i n c a t t l e h a s
n o t been p u b l i s h e d .
O r i g i n a l l y t h e purpose o f t h e production of h o r s e ~ n t i - b o v i n e lymphocyt serum (ABLc;)
w a s f o r t h e t r a n s m i s s i o n o f ju-
v e n i l e bovine l e u k o s e , which i s a d i s e a s e e s p e c i a l l y a f f e c t i n 2
calves
.
The c h a r z c t e r s o f t h e h o r s e A R T S ,
however, ia n o t y e t
known- T h e r e f o r e i t s e f f e c t s on some of t h e humor.51 and c e f l u
l a r immune f u n c t i o n s i n c a l v e s should be determined.
2.1.
P r o d u c t i o n . o f ~ n t i l y r ? p h o c y t os c r u r ? "
FTethods o f r a i s i n g A L S havc b e c n d e s c r i b e d by m a n y
i n v e s t i q n t o r s u s i n ? lymphoid c e l l s suspended i n a nedima
o r e m u l s i f i e d i n complc t e F r o u n d . * s a d j u v a n t
. M':ny
immu-
n i z a t i o n t o c h n i q u e o h a v e b e e n used y i e l d i n g ALS o f h i g h
i m r ~ u n o s u p p r c s s i v ep o t cncy.
T ~ most
L
cornrfion 2 r e t h e tvuo
o r t h r c o nubcutaneous o r i n t r a v e n o u s i n j e c t i o n s q i v e n
3-t
t w o weeks ~ p n r ft o l l o w e d by n b o o s t s r i m m u n i z z t i o n
f o u r weeks t h e r e e f t e r . M n n y d i f f e r e n t h o s t s p e c i e s h a v e
b c e n used t o r a i s c o f f c c t i v e ALS, The most used s p z c i e s
a r e t h e r a b b i t and t h e h o r s s .
P i g s and rurciinrnts h a v e
a l s o b e e n used b u t t h e y proiluca = n t i c c ? r e xof l o w e r q u - ? l i t g .
n
~n%isem
h a v e boon r a i s c C a g a i n s t lymphoid t i s s u e s o f
human, monkey, d o g , r a t , b o v i n e a n d n o u s e The p r o d u c t i o n o f a n t i - m t B L R i n r z b b i t s wes
r e p o r t e d by Woodruff nnc? Anderson ( 1 9 6 3 ) .
i n t r a p e r i t o n c a l l y 200.0
They injected
x l o 6 r a t t h o r a c i c d u c t lympho-
c y t e s t h r e e t i m e s a t weekly i n t e n r " 1 s .
T h c r l b b i t s \-?ere
b l e d t e n d:?ys a f t e r t h e l a a t i n j e c t i o n and b o o s t e r i n t r a p e r i t o n c z d i n j z c t i o n o f 1-2
x lo6 l y m p h o c y t e s were given
t e n days b e f o r e each subsequent bleeding.
The p r o d u c t i o n o f anti-mo u s e A L S w a s communicated
by Levey an2 Pa?cdawar ( 1 3 6 6 a ) , S r z y
naco
et ale
(1966a).
~5 51. (1966) and Ho-
Lavey and Medawar ( 1 9 6 6 ~ )a d m i n i s t e r e d
two i n t r a v e n o u s i n j e c t i o n s of
4-10
r a b b i t s a t f o u r t c a n d ~ y sc g s r t .
x
lo8
lymphoid c e l l s i n
The r a b b i t n TA?eret h e n b l e d
7 d a y s a f t e r t h e s e c o n d i n j a c t i o n and t h e s e r ? were c o l l c c t c d
and h c n t i n ~ c t i v a t e d( 5 6 O ~f o r 30 m i n u t e s ) , S e i t z f i l t e r e d
a n d s t o r e d a t -20°C
~ - 5%&.
u n t i l u s e d . Gray e_f: 21, ( 1 9 6 6 ) and blonaco
( 1 9 6 6 ) r e p o r t e d t h e i r methoci o f r a i s i r i f : A I - T i n r ~ b b i t -
a s f o l l o w s , Each r a b b i t r e c e i v e d a t o t s 1 o f 100.0
x 10' lymph
node c e l l s e m u l s i f i e d i n complete P r e u n d S s a d j u v a n t .
The e ~ . l u l -
s i o n was e q u a l l y d i v i d e d and i n j e c t e d i n t o e a c h orr" t h e f o u r
footpads.
100.0
The b o o s t e r i n j u c t i o n a o f s p l i n t ? c e l l s u s p e n s i o n of
x lo6 c e l l s w i t h o u t
a d j u v n n t weri: g i v e n i n t r n v c n o u s l y
on t h r e e s u c c e s s i v e d a y s f o u r weeks l a t e r .
bled f o u r days a f t e r t h e l a s t i n j e c t i o n .
Thc r a h b i t a wGrc
The blood was l e f t
t o c l o t a t room t e m p e r a t u r e a n d s e r m was c ~ l l e c t a dt h e n e x t
day, h e a t i n z c t i v a t e d and s t o r e d n t - 2 0 ' ~ .
The mode o f p r o d u c t i o n o f ALS in h o r s o s was r e p o r t e d by
a nwnbcr o f w o r k e r s .
IwasaZri o
...t
. - -.
sl.
..
(1967) i ~ x l u n i z e d t e n
h o r s e s w i t h d o g ' s s p l a n i c and lymph node c e l l s a n d 2 h o r s e s
w i t h human lymphoid t i s s u e s .
The h o r s e s were g i v e n 4 t o 6 in-
i n o c u l r t i o n s s t weekly i n t e r v a l n w i t h 0.2
lynphoid c e l l s i n s a l i n e .
t o 194 b i l l i o n
They found t h a t t h e s e r a when i n - -
j e c t e d i n t o does caused l p p h o p c n i a .
They o b s e r v e d t h a t d o s e s
of up t o 1 9 4 b i l l i o n l y n p h o i d c e l l s would r a p i d l y i n c r e z s e
the cytotoxic t i t e r .
Balncr
ct c&. (1968)
2 n d She51 e s
s-
(1971) i n j c c t e d h o r s e s by t h s s u b c u t s r i e o u s r o u t e w i t h 1.0 x f 0 l U
lymphoid c a l l s on 4 t o 6 o c c a s i o n s a t 2 t o 4 weeks i n t e m l o
f o r t h e p r o d u c t i o n o f anti-hu.nan A L S .
one week a f t e r t h e l a s t i n j z c t r o n .
produced i n t h i s manner prolang,d
Thc. i ~ o r s e swere b l e d
They r e p o r t e d t h n t s e r a
s k i n . g r a f t s u r v i v a l i n zlon-
k e y s . Lucko @ a2. (1968) imlunizud h o r s e s by, 3 s u b c u t z n e o u s
i n j e c t i o n s o f 1.0
x 10'
p i g lymph node c e l l s i n m e d i a 1 9 9
at 7 d r y s . Blood was c o f l e c $ c d one wcek a f t o r t h e l a s t i n j e c t
ion.
Twenty f o u r h o u r s t h e r e a f t e r t h e h o r s e s werc g i v e n a n o t h e r
i n j e c t i o n of 0.44
x 10' l y n p h noiic c e l l s and b l e d 7 dcys l a t e r .
The blood wes r l l o w e d t o s t a n d f o r 2 h o u r s zt room t e n p e r Q t u r a
and t h e n k e p t o v e r n i g h t n t
~ O C .
The s a r m w a s s e p a r r t e d , a&
sorbed w i t h one t h i r d of i t s v o l w l e w i t h p i g e r y t h r o c y t e s end
s t o r i l i z c d by S c i t z f i l t r a t i o n and s t o r e d a t - 2 0 O ~ u n t i l used.
They observed t h a t s e r a produced i n t h i s n s n n c r were e f f e c t i v e
and p r o l o n g e d s k i n g r a f t s u r v i v a l in p i g s .
Clroll e_t EC.
immunized 2 h o r s e s by two s u b c u t a n e o u s i n j e c t i o n s of 2.0
b o v i n e m i l k l e u c o c y t e s i n c o r ~ 7 l e t eP r e u n d ' s
(1976)
x 1 06
adjuv?nt folloned
by intravenous injections o f leucocytcs s u s p e n s i o c i n s a l i n e
e v e r y wsek t h e r e a f t e r u n t i l 5 1 wcek.
week.
Blood was c o f l o c t e d e v e r y
The h o r s e a n t i b o v i n e l e u c o c y t e serum when i n j c c t e Z i n t o
c a t t l a caused n e u t r o p e n i a ~ i t h i n1 h o u r ,
Bi-s
et 41.
of anti-mouse
(1971) r e p o r t e d t h e t e c h n i q u e of p r o d u c t i o n
, anti-rat,
i n e i t h e r pigs,
anti-dog
an2 a n t i - c h i c k e n
ALS r z i s e d
c a l v e s , one g o e t o r s h c e p , Each of t h e a n i m a l s
r e c e i v e d v a r y i n g q u z n t i t i e s o f donor thymic c e l l s i n s a l i n e
s u s p e n s i o n o r e m u l s i f i e d i n complete F r e u n d ' s
a d j u v s n t . Nost
a n i m a l s were given two i n j e c t i o n s ~t t w o week i n t o r v a l s .
They
observed t h a t t h e nethod developed f o r t h e groduction of imnunosuppressive scrurl i n t h e p i g n f l i n s t m o u s e lymphocytes proved
s z t i s f a c t o r y f o r t h e p r e p l r a t i o n of a n t i - r s t ,
a n t i - c h i c k e n dLS.
2nti--dog 2nd
B o v i n < ~sheep
,
and gont ALS nnd* a g a i n s t
mouse thyinocytcs a l s o had s t r o n g irmunosuppressivo n c t i v i t y .
I n t h e bovine, production o f im-tunosuppressive ~ c t i v i t yseemed
age dependent.
They noted t h a t poor immunosuppressive a c t i v i t y
was found i n c n l v c s which were l e s s than 2 months o l d .
Th.3 p r o d u c t i o n of anti-nouac,
anti-guinea
a1.
-.-
anti-hunan,
anti-dog
2nd
pi.? ALS i n g o a t s has been r c p o r t e d by Roll-d
(1971). These nnimnls reccivud f o u r i n t r a v e n o u s i n j e c t i o n s
of 5.0
x lo7 cells p e r kg body weight
.qt
weelrly interv.?.ls and
were k i l l e d and bled out one week z f t e r the l e s t 5nj::ction.
They found a c o r r e l n t i o n bet~voen t h e
2.q
vLKr2 membrcne i m u n o -
f l u o r e s c e n c e t i t e r of t h e s e r a s m i n s t nouse thymocytes t o the
in
--
vivo
----- i m u n o s u ~ p r e s s i v ea c t i v i t y
0s
judge6 by profon,+?tion
of t a i l s k i n h o r ~ o g r a f ts u r v i v a l ,
S h e i l g& 9 2 . (1971) communicated t h e p r o d u c t i o n of 8 n t i -
human lymphocyte s c r m i n 3 g o e t s . Eech goc:t rec-eivad 5.0
x 109
thynocytes i n m u l t i p l e subcutaneous i n j e c t i o n s on 3 occasions.
The f i r s t imunizntion
was foflovred a f t o r 2 weeks by 2 i n j e c t - -
i o n s a t weekly i n t e r v a l s .
O n e week n f t e r the t h i r d i n j e c t i o n
t h e g o a t s were bled o u t and 1.5
l i t e r o f serum w::s
obtained
f r o m aach, The antilymphocyte g l o b u l i n prepared from t h i s ALS
was i r ~ ~ u n o s u p p r e s s i v i -n no.nkeys w i t h s k i n xi?nogc.ft.
E f f o c t i v c anti-mouso 6LS i n c h i c k e n s
-3-1 ducks
was pro-
-
( l c 6 R ) . Thcy found t h a t i n v
.-. i t g
. o
- the
d u c d bg j o ~ s t c~ t 21.
,
s e r a wera s t r o n g l y c y t o t o x i c t o roouse l y n ~ h o c y t e si _ n . t h a p r e
s e n c e o f duck o r r a b b i t c o s p l c n e n t b u t i n j c c t i o n i n t o 'nice
b r o u s h t nbout o n l y z i n o r t r z n s i c n t l p s h o p e n i r : .
Many h y p o t h e s e s have been ~ r o p o s e don t h e node of a c t i o n
o f ALS. Nost o f t h c s z hypothesi-s have b e s n t r i e d o u t a n 3
d i s c a r d e d and o r ~ yt h e h y p t h c s i s proposcd by Lance (1968)
vias a c c e p t n b l c . Lance :€nun? t h R t tho T l o b u l i n f r a c t i o n of BLS
w 3 s a c t i v e i n i;munosugpressiun.
H e r e p o r t e d t h a t t h e mti-
lynphocjtte . z l o b u l i n a c h i e v e 3 i t s irmuno-suppressive a c t i o n by
c a u s i n g s o l c c t i r r i ~d e p l e t i o n of c a l l s ~f t h o r e c i r c u l a t i n g
pool
of l y n p h o c y t e s . T h i s
ic