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Induction of low zone tolerance to sheep erythrocytes in the lizard, Calotes versicolor
DEVELOPMENTAL AND COMLPARATIVE IM/~/NOLOGY, Vol. 4, pp. 295-308, 0145-305X/80/020295-14502.00/0 Printed in the U.S.A. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
INDUCTION
OF IN
LOW THE
ZORE
TOL~ANCE
LIZARD,
CALOTES
TO
SWak~
1980.
ERYTHROCYTES
VERSICOLOH
So B a s k a r a n d Y R . M u t h u k k - ~ p p a n Department o f Immnnology, S c h o o l o f B i o l o g i c a l S c i e n c e s , Madural KamazaJ U n i v e r s i t y , ~ a d u r a I 625 021, INDIA.
ABSTRACT Adult l i z a r d s p z e t r e a t e d w i t h sublmmuno6en/c d o s e s o f sheep e r y t h r o c y ~ e e (SRBC) and s u b s e q u e n t l y c h a l l e r ~ e d w i t h an o p t t m ~ l dose o f SRBC became t o l e r a n t t o SRBC. I n t r e a t e d l i z a r d s t h e number of plaque-formln~oells (PFC) in the spleen and serum a n t i b o d y l e v e l were r e d u c e d t o 10~ o f t h e r e s p o n s e in n o n - t r e a ted control lizards. This low l e v e l o f r e s p o n s e was due t o t r u e i n d u c t i o n o f t o l e r a n c e and not s i m p l y t o a d e l a y i n t h e o c c u r r e n c e o f peak r e s p o n s e , I n c r e a s e i n t h e c h a l l e n 6 e dose o f SRBC d i d not a l t e r t h e t o l e r a n t s t a t e . A m~nimum p e r i o d o f 3 days was found n e c e s s a r y f o r t h e e s t a b l i s h m e n t o f t o l e r a n c e . The i n d u c e d t o l e r a n c e was antl4~en s p e c i f i c and p e r s i s t e d f o r a t l e a s t 42 d a y s . We d i s c u s s t h e l i k e l i h o o d t h a t t h i s u n r e s p o n s i v e n e s s i s s i m i l a r t o low zone t o l e r a n c e (LET) p r e v i o u s l y r e ported i n o t h e r animal s p e c i e s .
INTRODUCTION Imnmnologieal t o l e r a n c e t o d i f f e r e n t a n t l 6 e n s c a n be I n d u c e d b o t h i n n e o n a t a l and a d u l t ar.lmals by p r i o r t r e a t m e n t w i t h t h e s p e c i f i c a n t i g e n . Serum p r o t e i n s have been used i n a m a j o r i t y o f s t u d i e s d e s i g n e d t o t n v e s ti~te t h e mechanism o f t o l e r a n c e s i n c e t h e s e p r o t e i n s h a t e s e v e r a l p r o p e r t l e s p a r t i c u l a r l y f a v o u r a b l e f o r such s t u d i e s ( 1 ) . S t u d i e s on t h e I n f l u e n c e o f a n t i g e n dose on t h e i n d u c t i o n o f t o l e r a n c e have shown t h a t t o l e r a n c e c a n be induced by a d m i n i s t r a t i o n o f m a s s i v e d o s e s o f a n t i 6 e n ( h J z h
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LOW ZONE TOLERANCE IN LIZARDS
Vol. 4, No. 2
zone t o l e r a n c e , I~T) (2,3,4,5,6), by r e p e a t e d i n j e c t i o n s o f n o n - i m , ~ n o g e n i c p r e p a r a t i o n s o f a n t i g e n o r by a n t i g e n g i v e n u n d e r n o n - i m m u n i z i n g c o n d l t i o n s (low zone t o l e r a n c e , LZT) ( 4 , 7 , 8 , 9 , 1 0 ) . HZT might r e p r e s e n t a highly artificial e x p e r i m e n t a l s y s t e m t h a t would not l e n d i t s e l f r e a d i l y to generalizatiom in terms of ontogenic events leading to the establishment o f t o l e r a n c e ( 7 ) . Hence, s t u d i e s on LZT have g a i n e d i m p o r t a n c e . The p h y l o g e n e t i c a p p r o a c h t o immunity has been s t r e s s e d m a i n l y b e c a u s e o f i t s potentiality i n p r o v i d i n g i n f o r m a t i o n c o n c e r n i n g t h e mechanism o f lmmunogenesis (11). With r e g a r d t o i m m u n o l o g i c a l t o l e r a n c e , r e p o r t s h a v e a p p e a r e d on HZT t o b o v i n e serum a l b u m i n (~SA) i n t h e a d u l t t o a d , Bufo m a r i ~ u s (12) and HZT t o SRBC and human serum a l b u m i n i n n e o n a t a l c h i c k e n s ( 1 9 ) . L i t t l e i s known a b o u t t . - m - n o l o g i c a l t o l e r a n c e i n n o n - z a ~ z a l i a n s p e c i e s . T h e r e f o r e , t h e p r e s e n t s t u d y on t h e i n d u c t i o n of LZT t o SRBC i n l i z a r d s was made u s i n g s u b t , ~ u n o g e n i c d o s e s o f n a t i v e SRBC.
~THODS Animals Adult l i z a r d s o f b o t h s e x e s w e i g h i n g 40 - 65 g were u s e d . a r d s w e r e housed and m a i n t a i n e d as d e s c r i b e d p r e v i o u s l y ( 1 4 ) .
Liz-
A n t i g e n s Blood was drawn from t h e J u g u l a r v e i n o f a s i n g l e s h e e p i n t o A l s e v e r ' s s o l u t i o n (pH 6 . 1 ) . To o b t a i n SRBC, t h e b l o o d was washed t h r e e t i m e s i n p h o s p h a t e - b u f f e r e d s a l i n e (PBS, pH 7 . 2 ) and r e s u s p e n d e d t o t h e required concentrations shortly before use. R a b b i t blood was o b t a i n e d from t h e m a r g i n a l e a r v e i n . Rat and b a t were b l e d by c a r d i a c p u n c t u r e . R a b b i t , r a t and b a t e r y t h r o c y t e e (RabRBC, RRBC, BRBC) were p r e p a r e d i n t h e same ~ y as SRBC. P o l y v i m y l p y r r o l t d o n e (PVP) o f m o l e c u l a r w e i g h t 360,000 (PVP KgO, T o k y o k a s e i , Tokyo) was u s e d a t a c o n c e n t r a t i o n o f 50 mg p e r ad o f PBS. A s s a y s f o r P l a q u e - f o r m i ~ c e l l and Serum Anttbod~ R e s p o n s e s D i r e c t haemol y s i n p r o d u o i n g c e l l s i n t h e o p l e e n a n d h a e m a g E l u t i n i n (HA) and h a e m o l y s l n (HL) a n t i b o d i e s i n t h e serum were d e t e c t e d by t h e methods d e s c r i b e d p r e v i ously (15). Anti-PVP a n t i b o d i e s were d e t e c t e d by p a s s i v e h a e m a g g l u t t n t n a s s a y a s d e s c r i b e d by Anderson and Blomgren ( 1 5 ) and m o d i f i e d f o r C a / o t e e (Unpubl i s h e d ) , Statistics D a t a from e x p e r i m e n t a l and c o n t r o l g r o u p s were compared u s i ~ Student's t-test. Mean d i f f e r e n c e s were c o n s i d e r e d s i g ~ f i c a n t when P ~0.05. Each gToup o o n t a i n s d a m/hiram o f 5 a n i m a l s ~r~ e a c h p o i n t r e p r e s e n t s t h e mean r e s p o n s e o f a g r o u p . V e r t i c a l b a r s i n f i g u r e s d e n o t e s t a n d a r d e r r o r ( S . E . ) o f t h e mean.
RESULTS I n f l u e n o e o f Route and Dose o f t h e AntiKen on PFC Response L i z a r d s were i n j e c t e d w i t h d i f f e r e n t d i l u t i o n s o f SRBC and s p l e n i c PFC were e s t i m a t e d 7 d a y s a f t e r i n t r a m u s c u l a r ( i . m . ) o r i n t r a c a r d i a c ( i . c . ) o r 14 d a y s a f t e r i n t r . a p e r i t o n e a l ( i . p . ) immnunizations. The r e s u l t s a r e shown i n FIG. 1. The maximum r e s p o n s e t o SfiBC a d m i n i s t e r e d i . p . o r i . m . o c c u r r e d a t a dose o f 6 . 0 x 108 SRBC, whereas 108 e l i c i t e d t h e maximum r e s p o n s e a f t e r i . c .
Vol. 4, No. 2
LOW ZONE TOLERANCE IN LIZARDS
297
i~ntzationo SRBC g i v e n by a l l t h e t h r e e r o u t e s p r o d u c e d dose d e p e n d e n t responses. T h i s dose d e p e n d e n c y can be c a t e g o r l s e d as f o l l o w s l noninmmnogenio (103 - 1 0 5 ) , sub-immunogentc (106 - 108, e x c e p t i . e . r o u t e ) , 2°Ptimal09(6°O x 108, e x c e p t i . c , r o u t e ) and s u p r a o p t i m ~ l ( 1 . 2 • 10 ~ and •4 x 1 ). Because o f t h e c o m b i n a t i o n o f e a s e o f i n j e c t i o n and q u i c k r e a c t i o n t i m e , t°m. i m m u n i z a t i o n was p r e f e r r e d i n a l l t h e f o l l o w i z ~ e x periments.
1600
--
1200
....
(J m
A ....
i. c
--@----
i .m
- - m - -
i.p
(00 i, D.
800
•/"
40C
, \
0
I
,o 3
,o ~
,o 5
,o6
,o ~
SRBC
,o ~
6xlO 8
,.2x,o 92.4~1o9
DOSE
FIG. I Influence of route and dose of SRBC on the PFC response.
Lizards received
d i f f e r e n t d o s e s o f SRBC by t . p . , i . m . o r i . c . r o u t e . PFC a s s a y was p e r f o r med 7 (l.m., l.c.) and 14 (l.p.) days after li~urdzatlon.
I n d u c t i o n o f Low Zone T o l e r a n c e
To i n d u c e LZT t o SRBC I n l i z a r d s ,
repeated
In~ectlons of subln~unogeD/c doses of SRBC were given. Lizards received i.m. 107 SRBC dally for a period of 10 days. Control lizards received PBS alone. Both groups were challenged with 6.0 x 108 SR3C 10 days after the last
298
LOW ZONE TOLERANCE IN LIZARDS
Vol. 4, No. 2
TABLE 1 Determination of the tolerizing
schedule
Treatment a Number of injections
SRBC dose
Challenge c
Mean PFC/IO 6 W~C + S.E. 12 + 4
(6) d
SRBC
866 _+ 143
(11)
107 SRBC
SRBC
27 + 11
(9)
14
1o 6 s ~ c
s~Bc
9 _+ 6
(8)
12 10
106 SR3C 106 SRBC
SRBC
(5)
SRBC
18 Z 3 24 _+ 9
7
106 s~BC
SEBC
30 _+ 12
(14)
7b
106 SEBC
SRBC
59 + 19
(7)
10.5 SRBC
SRBC
52 + 6
(7)
1o
P~s
P~s
10
P~S
10
10
(10)
a L i z a r d s r e c e i v e d d a i l y i n j e c t i o n s ( i . m . ) o f t h e i n d i c a t e d dose o f SRBC for the period mentioned. Control ani~ls r e c e i v e d PBS a l o n e . h I n d i c a t e d dose o f SRBC were g i v e n on a l t e r n a t e d a y s . c L i z a r d s were c h a l l e n g e d t . m . w i t h 6 . 0 x 108 SRBC 10 d a y s a f t e r i n j e c t i o n and PFC were a ~ u m e r a t e d 7 d a y s l a t e r . d Number o f - n ~ m a l s t e s t e d i s g i v e n i n p a r e n t h e s e s .
the last
injection. PFC a s s a y was c a r r i e d out 7 d a y s a f t e r t h e c h a l l e n g e i n j e c t i o n . Low dose SRBC t r e a t e d l i z a r d s showed a v e r y low ,,,tuber o f PFC ( a b o u t 1096 o f the control response) after challenge (Table !). Likewise, pretreatment w i t h 7, 10, 12 o r 14 i n j e c t i o n s o f e i t h e r 106 o r 105 SRBC r e n d e r e d l i z a r d s unresponsive to a further i~nogenic challenge. Even t h o u g h t h e r e was no o b v i o u s d i f f e r e n c e i n t h e l e v e l o f t o l e r a n c e i n d u c e d by d i f f e r e n t d o s e s o f SRBC, f o r t h e s a k e o f c o n v e n i e n c e , 10 d a i l y i n j e c t i o n s o f 106 SRBC were g i v e n t o induce t o l e r a n c e i n l i z a r d s . I t was r e q u i r e d t o d e t e r m i n e w h e t h e r t h e t o l e r i z i n g i n j e c t i o n s a l o n e induced any ~mmune response. FIG. 2 shows the result of assays for PFC and serum a n t i b o d y (HA and HL) a t i n t e r v a l s u p t o 21 d a y s a f t e r l a s t t o l e r i z i r ~ injection. I m m e d i a t e l y a f t e r t h e l a s t t o l e r i z i n g i n j e c t i o n , t h e r e were a b o u t 300 PFC/106 WBC, which i s a b o u t 30~ o f t h e r e s p o n s e i n c o n t r o l a n i m a l s t o a n o p t i m a l immunogenic d o s e . PFC rmmber and serum a n t i b o d y r e m a i n e d a t t h e 2 1 s t day l e v e l u n t i l 42 d a y s . From t h e s e d a t a a p e r i o d o f 10 d a y s was considered necessary for establishment of tolerance. Hence, i n t h e f o l l o w i n g e x p e r i m e n t s , t h e c h a l l e n g e i n j e c t i o n was g i v e n 10 d a y s a f t e r t h e l a s t tolerizing Injection.
Vol. 4, No. 2
LOW ZONE TOLERANCE IN LIZARDS
400
1
PFC
1
HA
299
m
HL 300
\
m
O
% -~ 2 0 0 ¢.) M.. II.
:E r~ ~J
G) 4
I00
2
i
2
7 DAYS
I0 AFTER THE
14 SCHEDULE
21
FIG, 2
P~C and serum a n t i b o d y r e s p o n s e s i n t o l e r a n t l i s a r d s b e f o r e c h a l l e ~ 6 e . PFC s~d serum a n t i b o d i e s ( i n r e c i p r o c a l u n i t s ) were measured on d i f £ e r e n t days a f t e r t h e s t a n d a r d t o l e r t s i ~ schedule. HA - h a e a a ~ l u t i n i n ; HL - H a e m o l y s i n .
R . e s p o n n s An T o l e r a n t L i z a r d s : K i n e t i c s o f FYC stud Serum A n t i b g d ~ Rejponse8 An T o l e r a n t Li s s z d' s To d e t ermine w h e t h e r t h e o b s e r T e d l o v l e v e l o f r e s p o n s e An t o l e r a n t l i z a r d s was due t o a t r u e i n d u c t i o n o f t o l e r a n c e and not s i m p l y t o a d e l l 7 i n t h e o c c u r r e n c e o f a peak r e e p o r m e , t h e f o l l o v i r ~ e x p e r i m e n t was c a r r i e d o u t , C o n t r o l and t o l e r a n t l i z a r d s were c h a / l e ~ e d w i t h 6 . 0 x 108 SRBC and t h e JJmUns r e s p o m l e s v e r e a s s e s s e d on d i f f e r e n t da~s a f t e r c h a l l e n g e . C o n t r o l l i z a r d s shoved h i g h ~ u s b e r o f PFC on d a y ? and h i g h l e v e l s o f HA and HL a n t i b o d i e s on day 14 and 210 r e s p e c t i v e l y (FIG. 3 , 4 ) . ~y c o n t r a s t , t o l e r a n t l l s a r d e shoved low : e s p o n e e s (PFC and s e r ~ a a n t i b o d y ) on a l l da~s t e s t e d (FIG. 3 , 4 ) . I n b o t h c o n t r o l and t o l e r a n t l i z a r d s no 2-ME r e sistant antibodies vere detected, From t h e d a t a i t i s e v i d e n t t h a t t h e obserTed t o l e r a n c e v a s not due t o a s h i f t i n t h e time o f o c c u r r e n c e o f peak r e s p o n s e .
300
LOW ZONE TOLERANCE IN LIZARDS
Vol. 4, No.2
2000 --
1000 • 0
• CONTROL O TOLERANT
~0 o 0. I00
I0 3"5
7 DAYS
AFTER
14 CHALLENGE
21
FIG. 3
K i r ~ t t e s o f I~FC r e s p o n s e i n t o l e r a n t l i z a r d s a £ t e r c h a l l e n g e . Tolerant and c o n t r o l l i z a r d s were c h a l l e n g e d i . m . w i t h 6 . 0 x 108 SRBC 10 d a y s after the last tolertztng injection. PFC a s s a y was p e r f o r m e d on d i f f e r e n t days a f t e r c h a l l e n g e .
Vol. 4, No. 2
LOW ZONE TOLERANCE IN LIZARDS
256
-
CONTROL
BB
128 MJ iv I-I."1"
TOLERANT
T
28
T
4 _ ..J
52
52 "~
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0 m
:D ¢¢ M.I u)
256
-
T
II
64
>-
I-Z
301
O
"l-
16
pZ
8 ¸ W
-4
4
0
__i 7
_&
II 21
14
DAYS
7
AFTER
--
14
n
--
0
ZI
CHALLENGE
FIG. 4
Serum a n t i b o d y r e s p o n s e I n t o l e r a n t l i z a r d s a f t e r c h a l l e r ~ e . Tolerant and control lizards were challer~ed i@m. with 6.0 • 108 SRBC 10 days after the last tolerizI~ injection. Serum a n t i b o d i e s were measured on d i f f e r e n t days a f t e r c h a l l e D g e .
I n f l u e n o e o f C h a l l e r ~ e Dose To d e t e r m i n e t h e t n ~ l u e n o e o f t h e dose o f c h a l l e n g e a n t i g e n on i n d u c e d t o l e r a n c e t o SRBC, t o l e r t z e d and c o n t r o l l i z a r d s were c h a l l e ~ e d w i t h d i f f e r e n t c o n o e n t r a t i o n s o f SIIBC. PFC a s s aye were p e r f o r m e d 7 and 14 days a f t e r c h - l l e n g e ( T a b l e 2 ) . Only a few ~FC were found i n t o l e r a n t l i z a r d s on d a y s 7 and 14 a ~ t e r c h a l l e ~ e w i t h 6 . 0 x 108 , 1 . 2 x 109 o r 2 . 4 x 109 SRBC. A f t e r c h a l l e ~ i ~ w i t h a low dose ( t 0 6 o e l l s ) o f SRBC, t h e PFC zoeporme i n t o l e r a n t l i z a r d s d i d not d i f f e r l a t h from t h a t o f c o n t r o l s on d ~ 7 ( P > O . 0 5 ) ! however, a s i g n i f i c a n t d i f f e r e n c e was n o t e d on day 14 ( P < 0 . 0 2 5 ) . This ,met be due t o t h e f a c t t h a t a mmxin~a munber o f PFC was e v i d e n t o n l y on day 14 a f t e r low dose c h a l l e n g e . The d a t a i n d t o a t e t h a t t h e induoed t o l e r a n c e was s t r o n g and c o u l d not be b r o k e n s i m p l y by i n o r e & s i ~ t h e o h a l l e n g e dose o f SR~C.
302
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LOW ZONE TOLERANCE IN LIZARDS
303
Specificity of Tolerance Specificity of tolerance was tested by challenging the SRBC-tolerant lizards either with SRBC or other erythrocytes (RRBC, RabRBC, BRBC). The PFC response to SRBC was very low in tolerant lizards; the responses to RRBC, RabRBC and BRBC were not significantly different from that in the respective controls (Table 3)- PFC response to RRBC and RabRBC were not reduced even when these antigens were given to tolerant lizards along with SRBC (Table 4). Another group of control and SRBC-tolerant lizards received i.m. 20 mg of PVP in 0.4 ml PBS and the anti-PVP antibodies were measured on day 7, 14 and 21. The reciprocal titer values were: 667 + 192, 2080 + 481, 1200 + 440 (control lizards) and 629 ~ 81, 1867 ~ 448, I~80 ~ 197 (t~lerant lizards). The means of the titers of the two groups differed by less than 5~, thereby indicating the ability of SRBC-tolerant lizards to respond to an unrelated antigen, PVP.
TABLE
5
Specificity of tolerance
Mean PFC/IO 6 WBC c ~ S.E. Challenge
b
P value Control a
Tolerant a
26 +
7
SRBC
1088 + 559
RRBC
197 (~)+26
124(+)24~
BRBC
184 + 46
162 + 54
<0.5
152 ~ 55 (6)
<0.5
(7) RabRBC
172 + 41 (V)
a
See explanatory notes in Table 2. b c d
Ten days after the last injection both groups of lizards were challenged i.m. with 6.0 x 108 SRBC, 1.0 x 108 RRBC, BRBC or 2.0 x 108 RabRBC. PFC response against the challenge antigen was assessed 7 days after the challenge. Number of animals tested is given in parentheses.
304
LOW ZONE TOLERANCE IN LIZARDS
TABLE Specificity
Treatment a
Challenge
Vol. 4, No. 2
4
of t o l e r a n c e Mean PFC/IO 6 WBC c ~ S.E.
b
a n t l - S RBC
a n t i-RRBC
a n t i-EabRBC
Control
SRBC + RRBC
668 + 185
192(± 6 59 -)
N.T.
Tolerant
SRBC + RRBC
68(~)+54*
172+)58~
N.T.
Control
Sl~C , Rab]~C
Tolerant
SRBC + RabEBC
a See b Ten ged 2.0
707(~)205
N.T.
178(~)54
45 + 16"
N.T.
151 + 35
(15)
(15)
explanatory notes in Table 2. days after the last injection both groups of lizards were challenl.m. either with 6.0 • 108 SRBC + 1.0 x 108 RRBC or 6.0 • 108 SRBC + x 108 RabRBC.
e PFC response against challenge antigens were assessed separately 7 days after challenge. d Number of Anlmals tested is given in parentheses; N.T. = Not tested. @
Significantly different from the respective control (P<0.01). D u r a t i o n o f T o l e r a n c e To s t u d y t h e d u r a t i o n o f t o l e r a r ~ e , g r o u p s o f l i s a r d s were c h a l l e n g e d w i t h 6 . 0 • 108 SRBC on d i f f e r e n t d a y s a f t e r t h e t o lerlzing regimen. The PFC assaywas performed 7 days later. An appreciable number of PFC (271 ~ 117/106 WBC) was evident when the lizards were challenged immediately after the last tolerlzing injection (day I). The response was, however, meager when challenged 5, 7 or as late as 42 days after the tolerizing injections ( 4 0 ~ 11, 1 9 ~ 5 and 9 ~ 5, respectively). By c o n t r a s t , t h e PFC r e s p o n s e i n n o n - t o l e r a n t c o n t r o l g r o u p s was u n i f o r m l y high (998 ~ 212),
DISCUSSION The d e g r e e o f inmune r e s p o n s e i s p r o f o u n d l y a f f e c t e d by b o t h t h e r o u t e o f a d m i n i s t r a t i o n and t h e d o s e o f a n t i g e n ( 1 7 , 18, 1 9 ) . Results of t h e p r e s e n t e x p e r i m e n t s ( F I G . 1) showed t h a t a n i n c r e a s e i n SRBC d o s e , i r r e s p e c t i v e o f t h e r o u t e o f i m m u n i z a t i o n , was f o l l o w e d by a n i n c r e a s e i n t h e number o f s p l e n i c PFC i n l i z a r d s , as a l r e a d y r e p o r t e d ( 1 9 ) . The s u p e r i o r i t y o f i . m . r o u t e o v e r i . p . i m m u n i z a t i o n i n l i z a r d s c a n be a t t r i b u t e d t o t h e minimum l o s s o f a n t i g e n due t o p h a g o c y t o s i s , ~ the maintenance of a strong antigenic stimulus by a constant, slow release of antigen from t h e i n j e c t i o n s i t e . Intracardiac immunization also elicited a
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quicker response. Howeverp s i n c e t o l e r a n c e i n j e c t i o n s , t h e i . m . r o u t e was p r e f e r r e d .
induction
305
involved repeated
While the administration of an optimal dose of antigen induces effective I ~ n l t y , an antlgen dose hlgher or lower than the im1,no~enlc range would result in a state of unresponsiveness (20). Tolerance to SRBC in l i z a r d s was i n d u c e d by r e p e a t e d i n j e c t i o n s o f subimmunogenic d o s e s o f SEBC. The s t a n d a r d t o l e r i z i r ~ d o s e , 10 b SRBC, i s 600 t i m e s l e s s t h a n t h e o p t i m a / , imm, nogenic dose (6.0 x 108 SRBC). This finding is in agreement with studies In which a similar prolonged injection schedule of low dose of antigen was used. For example: induction of LZT to fla~ellin in neonatal rats
(21), to SRBC i n neonatal mice ( 6 ) , to BSA (3, 4, 5, 20) and pha~e fd part i c l e s ( 9 , 2 2 ) i n a d u l t m i c e . I n a l l t h e s e s t u d i e s , t h e t o l e r i z i n g dose i s a t l e a s t 100 t i m e s s m a l l e r t h a n t h e o p t i m a l d o s e . Huchet and Feldmann i n duced LZT t o d i n i t r o p h e ~ l c o n j u g a t e d human ~ g l o b u l i n (DNP-HGG) w i t h r e p e a t e d i n j e c t i o n s o f 10, 1 o r 0.1 ~ DHP-SHGG. I n d u c t i o n o f c o m p l e t e t o l e r a n c e r e q u i r e d 10 ) ~ E / i n J e c t i o n , whereas i n j e c t i o n o f 1 o r 0.1 )ag r e s u l t e d in partial tolerance (10). By c o n t r a s t , i n t h e p r e s e n t s t u d y , no s u c h d i s c r i m i n a t i o n was e v i d e n t b e t w e e n t h e d i f f e r e n t d o s e s used t o i n d u c e LZT t o SRBC i n l i z a r d s ( T a b l e 1 ) . F a i l u r e t o a l t e r t h e t o l e r a n t s t a t e by i n c r e a s i r ~ t h e c h a l l e r ~ e d o s e o f a n t i g e n ( T a b l e 2) r u l e s out t h e p o s s i b i l i t y that t h e a n t i g e n t h r e s h o l d l e v e l n e c e s s a r y t o I n d u c e a n lr~nune r e s p o n s e c o u l d be gTeater in tolerant lizards. I t was c o n s i d e r e d p o s s i b l e t h a t t h e t o l e r o ~ e n i c e f f e c t s o f a low dose SRBC t r e a t m e n t might d i m i n i s h w i t h t i m e a f t e r c h a l l e n g e i n j e c t i o n and t h a t a normal r e s p o n s e would d e v e l o p a f t e r a l o n g e r i n t e r v a l t h a n u s u ally required. T h i s p o s s i b i l i t y was r u l e d out s i n c e l i z a r d s e x h i b i t e d no e s c a p e from t h e t o l e r a n t s t a t e when t h e PFC a s s a y was p e r f o r m e d 7, 14 and 21 days after challenge (FIG. 2). Thus, the induced unresponsiveness represents a true tolerant state as shown previously in mlee (8). Tolerant lizards showed very few direct PFC and low levels of 2-ME sensitive antibodies (FIG. 2,3,4 and Table 2). In Lice made tolerant to SRBC, b o t h d i r e c t and i n d i r e c t PFCs were r e d u c e d ( 8 ) . C o n v e r s e l y , mice t o l e r a n t t o DNP-I~GG ( 1 0 ) and pha~e f d p a r t i c l e s ( 9 ) showed marked r e d u c t i o n i n IgG a n t i b o d y r e s p o n s e w i t h normal l e v e l s o f IgM a n t i b o d i e s . I t was s u g g e s t e d t h a t t h e I~G r e s p o n s e i s more thymus d e p e n d e n t t h a n t h e I ~ r e s p o n s e and t h a t t o l e r a n c e c o u l d a f f e c t s p e c i f i c a l l y t h e T - c e l l s which h e l p i n t h e development of the I~ response (4,5,9,10,17). I n l i z a r d s , t h e PFC ( d i r e c t PFC) r e s p o n s e t o SEBC i s thymus d e p e n d e n t ( 2 4 , 2 5 ) and no 2-ME r e s i s t a n t a n t i b o d i e s were i d e n t i f i e d e v e n i n s e c o n d a r y tm-,~ne r e s p o n s e t o SRBC (15)o T h e r e f o r e , i n t h e a p p a r e n t a b s e n c e o f an I ~ r e s p o n s e , t o l e r a n t l i z a r d s e x h i b i t a marked s u p p r e s s i o n o f t h e I ~ 4 r e s p o n s e . The PFC response found 2 days after the last tolerizing ir~eotion was not f o l l o w e d by a p p r e c i a b l e l e v e l s o f serum HA o r HL a n t i b o d y ; a l s o , o n l y a v e r y low amount o f serum a n t i b o d ~ was d e t e c t e d i n t o l e r a n t l i z a r d s a t any time a f t e r c h a l l e n g e (FIG. 2 , 3 ) . I n n e w - b o r n mice made t o l e r a n t t o SP~C (LZT and HZT), F r i e d m a n r e p o r t e d t h e p r e s e n c e o f a p p r e c i a b l e amounts o f a n t i b o d y b e f o r e and a f t e r c h a l l e r ~ e , b u t h e r e t h e nnmber o f e p l e n / c PFC was v e r y low ( 6 ) . Our r e c e n t s t u d i e s show t h a t p a s s i v e t r a n s f e r o f serum from t o l e r a n t t o normal l i z a r d s , s h o r t l y b e f o r e o r a l o n g w i t h t h e a d m i n i s t r a t i o n o f SRBC, s u p p r e s s e d t h e PFC r e s p o n s e o f normal l i z a r d s ( u n p u b l i s h e d o b s e r v e -
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tion). Results of the in vitro experiments indicate the presence of a s u p p r e s s i v e f a c t o r i n t h e serum o f t o l e r a n t l i z a r d s ( 2 5 ) . This factor is n o n - d i a l y s a b l e , h e a t r e s i s t a n t and not a b s o r b a b l e by SRBC (25). The s e r um f a c t o r which m e d i a t e s s u p p r e s s i o n i n v i v o and i n v i t r o was found t o be anti,on specific. T h e r e f o r e , i t seems u n l i k e l y t h a t t h e LZT t o SRBC i n lizards i s e f f e c t e d by a n t i b o d y f e e d - b a c k i n h i b i t i o n . Nonetheless, antib o d i e s may be p r e s e n t i n t o l e r a n t l i z a r d s but t h e s e may be u n d e t e c t e d b e c a u s e o f t h e i r low a ~ f i n i t y o r n o n - a ~ g l u t i n a t i n g n a t u r e . F a i l u r e t o d e t e c t a n t i b o d i e s I n mice t o l e r a n t t o t y p e I I I pneumococc a l p o l y s a c c h a r i d e (HZT) was b e c a u s e o f c o n t i ~ o u s p e r i p h e r a l n e u t r a l i z a t i o n o f a n t i b o d y f o r m e d by r e c i r c u l a t t n g , n o n - d e ~ a d a b l e a n t i g e n ( 2 6 ) . Such a n e u t r a l i z a t i o n o f formed a n t i b o d ~ c a n n o t be e x p e c t e d I n LZT t o SRBC i n l i z a r d s , s i n c e SRBC a r e e a s i l y d e g r a d a b l e and t o l e r a n c e i n d u c t i o n I n v o l v e d v e r y low amount o f SRBC. Low serum a n t i b o d y l e v e l s i n t o l e r a n t l i z a r d s i s l i k e l y t o be due t o r e d u c e d numbers o f a n t i b o d y p r o d u c i n g c e l l s r a t h e r t h a n r e d u c e d p r o d u c t i o n o f a n t i b o d y by i n d i v i d u a l c e l l s b e c a u s e t h e s i z e s o f i n d i v i d u a l p l a q u e s formed by t h e s p l e e n c e l l s f r o m tolex~ant and c o n t r o l l i z a r d s were s i m i l a r . The s p e c i f i c i t y o f t h e t o l e r a n c e o b s e r v e d ( T a b l e 4 , 5 ) shows t h a t LZT t o SHBC i n l i z a r d s i s t h e r e s u l t o f an a c t i v e t s p e c i f i c s u p p r e s s i v e p h e n o menon r a t h e r t h a n a g e n e r a l i n a c t i v a t i o n o f t h e immune s y s t e m . S i m i l a r s p e c i f i c i t y has been shown f o r LZT t o SRBC (7) A ~ phage f d (22) i n mice and t o f l a g e l l t n i n r a t s ( 2 1 ) . In m~ls it is often possible to terminate the unresponsive state by Immunizing t o l e r a n t a n i m a l s w i t h c r o s s - r e a c t i n g a n t i g e n ( 2 ) . The s h a r i n ~ o f some a n t i g e n i c d e t e r m i n a n t s b e t w e e n t h e t o l e r a t e d a n t i g e n ~ d c r o s s reacting anti,on helps to bring about an im~me response to the former (2). Cross-reactivity between SRBC ~r~ RabRBC i n C a l o t e s h a s been shown p r e v i o u s l y i n t e r m s o f T - c e l l h e l p and PFC p r o d u c ~ 4 , 2 5 ) . However, i n t h i s s t u d y , t h e f a i l u r e t o b r e a k t o l e r a n c e w i t h c r o s s r e a c t i n g RabRBC, e i t h e r g i v e n a l o n e ( T a b l e 9) o r a l o n g w i t h t h e t o l e r a t e d a n t i g e n ( T a b l e 4) seems t o i n d i c a t e t h a t t h e c e l l s o f t o l e r a n t l i z a r d s sLre n o t r e a c t i v e t o s h a r e d determinants either. A similar failure to break sheep haemolyeate supern a t a n t - induced t o l e r a n c e (LZT t o SRBC) w i t h c r o s s - r e a c t i n ~ h o r s e e r y t h r o c y t e e (HRBC) i n C3H/He mice has b e e n r e p o r t e d ( 8 ) . However, e x p e r i m e n t s using antigens with different degree of cross-reactivity ( w i t h SRBC) need t o be c a r r i e d out as t h e c h o i c e o f a s u i t a b l e a n t i g e n c o m b i n a t i o n f o r b r e a k i n ~ t o l e r a n c e depends upon t h e optiaaam d e g r e e o f c r o s s - r e a c t i v i t y between a n t i g e n s (1,2). I n l i z a r d s , m a n i f e s t a t i o n o f t o l e r a n c e was e v i d e n t o n l y 3 da~s a f t e r the last tolerizir~ Injection. A s i m l l a ~ i n d u c t i v e p e r i o d was found t o be r e q u i r e d I n o t h e r LZT s y s t e m s ( 4 , 5 , 9 , 1 0 , 2 2 ) . Experimentally induced toler a n c e c a n be e i t h e r t r a n s i e n t o r l o n ~ l a s t i r ~ (1). L i z a r d s made t o l e r a n t t o SRBC a r e r e f r a c t i v e t o c h a l l e r ~ e i n j e c t i o n g i v e n a s l a t e a s 42 d a y s a f t e r t h e l a s t t o l e r i s i n ~ i n j e c t i o n and no breakdown o f t o l e r a n c e was o b served. I n v e s t i g a t i o n s f o r l o n g e r p e r i o d s c o u l d n o t be c a r r i e d out i n l i z a r d s due t o t h e i n m b i l i t y t o m a i n t a i n them i n t h e l a b o r a t o r y c o r d ~ t i o n s beyond two months. However, t h e p r e s e n t f i n d i r ~ a g r e e s w i t h t h e p r e v i o u s r e p o r t s t h a t normal r e s p o n s i v e n e s s i n mice was not r e s t o r e d u p t o s e v e r a l weeks o r months a f t e r t o l e r a n c e i n d u c t i o n ( 7 , 8 ) .
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From t h e f o r e g o i n ~ d i s c u s s i o n , i t i s c l e a r t h a t t h e o b s e r v e d u n r e s p o n s i v e n e s s t o SRBC i n l i z a r d s i s an example o f LZT. The mechanisms o f i n d u c t i o n and m a i n t e n a n c e o f LZT t o SItBC I n l i z a r d s a r e u n d e r i n v e s t i g a t i o n .
ACK~O~ ~G~4~NTS T h i s work was s u p p o r t e d by a r e s e a r c h g r a n t from t h e D e p a r t m e n t o f Atomic Energy o f I n d i a and S. B. was a r e c i p i e n t o f J u n i o r R e s e a r c h F e l l o w s h i p i n t h e scheme. We e x p r e s s o u r g r a t e f u l t h a n k s t o Dr. S. J ~ y a r a m a n for critically reading the manuscript. REFERENCES I.
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So B a s k a r a n d Y R . M u t h u k k - ~ p p a n Department o f Immnnology, S c h o o l o f B i o l o g i c a l S c i e n c e s , Madural KamazaJ U n i v e r s i t y , ~ a d u r a I 625 021, INDIA.
ABSTRACT Adult l i z a r d s p z e t r e a t e d w i t h sublmmuno6en/c d o s e s o f sheep e r y t h r o c y ~ e e (SRBC) and s u b s e q u e n t l y c h a l l e r ~ e d w i t h an o p t t m ~ l dose o f SRBC became t o l e r a n t t o SRBC. I n t r e a t e d l i z a r d s t h e number of plaque-formln~oells (PFC) in the spleen and serum a n t i b o d y l e v e l were r e d u c e d t o 10~ o f t h e r e s p o n s e in n o n - t r e a ted control lizards. This low l e v e l o f r e s p o n s e was due t o t r u e i n d u c t i o n o f t o l e r a n c e and not s i m p l y t o a d e l a y i n t h e o c c u r r e n c e o f peak r e s p o n s e , I n c r e a s e i n t h e c h a l l e n 6 e dose o f SRBC d i d not a l t e r t h e t o l e r a n t s t a t e . A m~nimum p e r i o d o f 3 days was found n e c e s s a r y f o r t h e e s t a b l i s h m e n t o f t o l e r a n c e . The i n d u c e d t o l e r a n c e was antl4~en s p e c i f i c and p e r s i s t e d f o r a t l e a s t 42 d a y s . We d i s c u s s t h e l i k e l i h o o d t h a t t h i s u n r e s p o n s i v e n e s s i s s i m i l a r t o low zone t o l e r a n c e (LET) p r e v i o u s l y r e ported i n o t h e r animal s p e c i e s .
INTRODUCTION Imnmnologieal t o l e r a n c e t o d i f f e r e n t a n t l 6 e n s c a n be I n d u c e d b o t h i n n e o n a t a l and a d u l t ar.lmals by p r i o r t r e a t m e n t w i t h t h e s p e c i f i c a n t i g e n . Serum p r o t e i n s have been used i n a m a j o r i t y o f s t u d i e s d e s i g n e d t o t n v e s ti~te t h e mechanism o f t o l e r a n c e s i n c e t h e s e p r o t e i n s h a t e s e v e r a l p r o p e r t l e s p a r t i c u l a r l y f a v o u r a b l e f o r such s t u d i e s ( 1 ) . S t u d i e s on t h e I n f l u e n c e o f a n t i g e n dose on t h e i n d u c t i o n o f t o l e r a n c e have shown t h a t t o l e r a n c e c a n be induced by a d m i n i s t r a t i o n o f m a s s i v e d o s e s o f a n t i 6 e n ( h J z h
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zone t o l e r a n c e , I~T) (2,3,4,5,6), by r e p e a t e d i n j e c t i o n s o f n o n - i m , ~ n o g e n i c p r e p a r a t i o n s o f a n t i g e n o r by a n t i g e n g i v e n u n d e r n o n - i m m u n i z i n g c o n d l t i o n s (low zone t o l e r a n c e , LZT) ( 4 , 7 , 8 , 9 , 1 0 ) . HZT might r e p r e s e n t a highly artificial e x p e r i m e n t a l s y s t e m t h a t would not l e n d i t s e l f r e a d i l y to generalizatiom in terms of ontogenic events leading to the establishment o f t o l e r a n c e ( 7 ) . Hence, s t u d i e s on LZT have g a i n e d i m p o r t a n c e . The p h y l o g e n e t i c a p p r o a c h t o immunity has been s t r e s s e d m a i n l y b e c a u s e o f i t s potentiality i n p r o v i d i n g i n f o r m a t i o n c o n c e r n i n g t h e mechanism o f lmmunogenesis (11). With r e g a r d t o i m m u n o l o g i c a l t o l e r a n c e , r e p o r t s h a v e a p p e a r e d on HZT t o b o v i n e serum a l b u m i n (~SA) i n t h e a d u l t t o a d , Bufo m a r i ~ u s (12) and HZT t o SRBC and human serum a l b u m i n i n n e o n a t a l c h i c k e n s ( 1 9 ) . L i t t l e i s known a b o u t t . - m - n o l o g i c a l t o l e r a n c e i n n o n - z a ~ z a l i a n s p e c i e s . T h e r e f o r e , t h e p r e s e n t s t u d y on t h e i n d u c t i o n of LZT t o SRBC i n l i z a r d s was made u s i n g s u b t , ~ u n o g e n i c d o s e s o f n a t i v e SRBC.
~THODS Animals Adult l i z a r d s o f b o t h s e x e s w e i g h i n g 40 - 65 g were u s e d . a r d s w e r e housed and m a i n t a i n e d as d e s c r i b e d p r e v i o u s l y ( 1 4 ) .
Liz-
A n t i g e n s Blood was drawn from t h e J u g u l a r v e i n o f a s i n g l e s h e e p i n t o A l s e v e r ' s s o l u t i o n (pH 6 . 1 ) . To o b t a i n SRBC, t h e b l o o d was washed t h r e e t i m e s i n p h o s p h a t e - b u f f e r e d s a l i n e (PBS, pH 7 . 2 ) and r e s u s p e n d e d t o t h e required concentrations shortly before use. R a b b i t blood was o b t a i n e d from t h e m a r g i n a l e a r v e i n . Rat and b a t were b l e d by c a r d i a c p u n c t u r e . R a b b i t , r a t and b a t e r y t h r o c y t e e (RabRBC, RRBC, BRBC) were p r e p a r e d i n t h e same ~ y as SRBC. P o l y v i m y l p y r r o l t d o n e (PVP) o f m o l e c u l a r w e i g h t 360,000 (PVP KgO, T o k y o k a s e i , Tokyo) was u s e d a t a c o n c e n t r a t i o n o f 50 mg p e r ad o f PBS. A s s a y s f o r P l a q u e - f o r m i ~ c e l l and Serum Anttbod~ R e s p o n s e s D i r e c t haemol y s i n p r o d u o i n g c e l l s i n t h e o p l e e n a n d h a e m a g E l u t i n i n (HA) and h a e m o l y s l n (HL) a n t i b o d i e s i n t h e serum were d e t e c t e d by t h e methods d e s c r i b e d p r e v i ously (15). Anti-PVP a n t i b o d i e s were d e t e c t e d by p a s s i v e h a e m a g g l u t t n t n a s s a y a s d e s c r i b e d by Anderson and Blomgren ( 1 5 ) and m o d i f i e d f o r C a / o t e e (Unpubl i s h e d ) , Statistics D a t a from e x p e r i m e n t a l and c o n t r o l g r o u p s were compared u s i ~ Student's t-test. Mean d i f f e r e n c e s were c o n s i d e r e d s i g ~ f i c a n t when P ~0.05. Each gToup o o n t a i n s d a m/hiram o f 5 a n i m a l s ~r~ e a c h p o i n t r e p r e s e n t s t h e mean r e s p o n s e o f a g r o u p . V e r t i c a l b a r s i n f i g u r e s d e n o t e s t a n d a r d e r r o r ( S . E . ) o f t h e mean.
RESULTS I n f l u e n o e o f Route and Dose o f t h e AntiKen on PFC Response L i z a r d s were i n j e c t e d w i t h d i f f e r e n t d i l u t i o n s o f SRBC and s p l e n i c PFC were e s t i m a t e d 7 d a y s a f t e r i n t r a m u s c u l a r ( i . m . ) o r i n t r a c a r d i a c ( i . c . ) o r 14 d a y s a f t e r i n t r . a p e r i t o n e a l ( i . p . ) immnunizations. The r e s u l t s a r e shown i n FIG. 1. The maximum r e s p o n s e t o SfiBC a d m i n i s t e r e d i . p . o r i . m . o c c u r r e d a t a dose o f 6 . 0 x 108 SRBC, whereas 108 e l i c i t e d t h e maximum r e s p o n s e a f t e r i . c .
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297
i~ntzationo SRBC g i v e n by a l l t h e t h r e e r o u t e s p r o d u c e d dose d e p e n d e n t responses. T h i s dose d e p e n d e n c y can be c a t e g o r l s e d as f o l l o w s l noninmmnogenio (103 - 1 0 5 ) , sub-immunogentc (106 - 108, e x c e p t i . e . r o u t e ) , 2°Ptimal09(6°O x 108, e x c e p t i . c , r o u t e ) and s u p r a o p t i m ~ l ( 1 . 2 • 10 ~ and •4 x 1 ). Because o f t h e c o m b i n a t i o n o f e a s e o f i n j e c t i o n and q u i c k r e a c t i o n t i m e , t°m. i m m u n i z a t i o n was p r e f e r r e d i n a l l t h e f o l l o w i z ~ e x periments.
1600
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FIG. I Influence of route and dose of SRBC on the PFC response.
Lizards received
d i f f e r e n t d o s e s o f SRBC by t . p . , i . m . o r i . c . r o u t e . PFC a s s a y was p e r f o r med 7 (l.m., l.c.) and 14 (l.p.) days after li~urdzatlon.
I n d u c t i o n o f Low Zone T o l e r a n c e
To i n d u c e LZT t o SRBC I n l i z a r d s ,
repeated
In~ectlons of subln~unogeD/c doses of SRBC were given. Lizards received i.m. 107 SRBC dally for a period of 10 days. Control lizards received PBS alone. Both groups were challenged with 6.0 x 108 SR3C 10 days after the last
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TABLE 1 Determination of the tolerizing
schedule
Treatment a Number of injections
SRBC dose
Challenge c
Mean PFC/IO 6 W~C + S.E. 12 + 4
(6) d
SRBC
866 _+ 143
(11)
107 SRBC
SRBC
27 + 11
(9)
14
1o 6 s ~ c
s~Bc
9 _+ 6
(8)
12 10
106 SR3C 106 SRBC
SRBC
(5)
SRBC
18 Z 3 24 _+ 9
7
106 s~BC
SEBC
30 _+ 12
(14)
7b
106 SEBC
SRBC
59 + 19
(7)
10.5 SRBC
SRBC
52 + 6
(7)
1o
P~s
P~s
10
P~S
10
10
(10)
a L i z a r d s r e c e i v e d d a i l y i n j e c t i o n s ( i . m . ) o f t h e i n d i c a t e d dose o f SRBC for the period mentioned. Control ani~ls r e c e i v e d PBS a l o n e . h I n d i c a t e d dose o f SRBC were g i v e n on a l t e r n a t e d a y s . c L i z a r d s were c h a l l e n g e d t . m . w i t h 6 . 0 x 108 SRBC 10 d a y s a f t e r i n j e c t i o n and PFC were a ~ u m e r a t e d 7 d a y s l a t e r . d Number o f - n ~ m a l s t e s t e d i s g i v e n i n p a r e n t h e s e s .
the last
injection. PFC a s s a y was c a r r i e d out 7 d a y s a f t e r t h e c h a l l e n g e i n j e c t i o n . Low dose SRBC t r e a t e d l i z a r d s showed a v e r y low ,,,tuber o f PFC ( a b o u t 1096 o f the control response) after challenge (Table !). Likewise, pretreatment w i t h 7, 10, 12 o r 14 i n j e c t i o n s o f e i t h e r 106 o r 105 SRBC r e n d e r e d l i z a r d s unresponsive to a further i~nogenic challenge. Even t h o u g h t h e r e was no o b v i o u s d i f f e r e n c e i n t h e l e v e l o f t o l e r a n c e i n d u c e d by d i f f e r e n t d o s e s o f SRBC, f o r t h e s a k e o f c o n v e n i e n c e , 10 d a i l y i n j e c t i o n s o f 106 SRBC were g i v e n t o induce t o l e r a n c e i n l i z a r d s . I t was r e q u i r e d t o d e t e r m i n e w h e t h e r t h e t o l e r i z i n g i n j e c t i o n s a l o n e induced any ~mmune response. FIG. 2 shows the result of assays for PFC and serum a n t i b o d y (HA and HL) a t i n t e r v a l s u p t o 21 d a y s a f t e r l a s t t o l e r i z i r ~ injection. I m m e d i a t e l y a f t e r t h e l a s t t o l e r i z i n g i n j e c t i o n , t h e r e were a b o u t 300 PFC/106 WBC, which i s a b o u t 30~ o f t h e r e s p o n s e i n c o n t r o l a n i m a l s t o a n o p t i m a l immunogenic d o s e . PFC rmmber and serum a n t i b o d y r e m a i n e d a t t h e 2 1 s t day l e v e l u n t i l 42 d a y s . From t h e s e d a t a a p e r i o d o f 10 d a y s was considered necessary for establishment of tolerance. Hence, i n t h e f o l l o w i n g e x p e r i m e n t s , t h e c h a l l e n g e i n j e c t i o n was g i v e n 10 d a y s a f t e r t h e l a s t tolerizing Injection.
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LOW ZONE TOLERANCE IN LIZARDS
400
1
PFC
1
HA
299
m
HL 300
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m
O
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14 SCHEDULE
21
FIG, 2
P~C and serum a n t i b o d y r e s p o n s e s i n t o l e r a n t l i s a r d s b e f o r e c h a l l e ~ 6 e . PFC s~d serum a n t i b o d i e s ( i n r e c i p r o c a l u n i t s ) were measured on d i f £ e r e n t days a f t e r t h e s t a n d a r d t o l e r t s i ~ schedule. HA - h a e a a ~ l u t i n i n ; HL - H a e m o l y s i n .
R . e s p o n n s An T o l e r a n t L i z a r d s : K i n e t i c s o f FYC stud Serum A n t i b g d ~ Rejponse8 An T o l e r a n t Li s s z d' s To d e t ermine w h e t h e r t h e o b s e r T e d l o v l e v e l o f r e s p o n s e An t o l e r a n t l i z a r d s was due t o a t r u e i n d u c t i o n o f t o l e r a n c e and not s i m p l y t o a d e l l 7 i n t h e o c c u r r e n c e o f a peak r e e p o r m e , t h e f o l l o v i r ~ e x p e r i m e n t was c a r r i e d o u t , C o n t r o l and t o l e r a n t l i z a r d s were c h a / l e ~ e d w i t h 6 . 0 x 108 SRBC and t h e JJmUns r e s p o m l e s v e r e a s s e s s e d on d i f f e r e n t da~s a f t e r c h a l l e n g e . C o n t r o l l i z a r d s shoved h i g h ~ u s b e r o f PFC on d a y ? and h i g h l e v e l s o f HA and HL a n t i b o d i e s on day 14 and 210 r e s p e c t i v e l y (FIG. 3 , 4 ) . ~y c o n t r a s t , t o l e r a n t l l s a r d e shoved low : e s p o n e e s (PFC and s e r ~ a a n t i b o d y ) on a l l da~s t e s t e d (FIG. 3 , 4 ) . I n b o t h c o n t r o l and t o l e r a n t l i z a r d s no 2-ME r e sistant antibodies vere detected, From t h e d a t a i t i s e v i d e n t t h a t t h e obserTed t o l e r a n c e v a s not due t o a s h i f t i n t h e time o f o c c u r r e n c e o f peak r e s p o n s e .
300
LOW ZONE TOLERANCE IN LIZARDS
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2000 --
1000 • 0
• CONTROL O TOLERANT
~0 o 0. I00
I0 3"5
7 DAYS
AFTER
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21
FIG. 3
K i r ~ t t e s o f I~FC r e s p o n s e i n t o l e r a n t l i z a r d s a £ t e r c h a l l e n g e . Tolerant and c o n t r o l l i z a r d s were c h a l l e n g e d i . m . w i t h 6 . 0 x 108 SRBC 10 d a y s after the last tolertztng injection. PFC a s s a y was p e r f o r m e d on d i f f e r e n t days a f t e r c h a l l e n g e .
Vol. 4, No. 2
LOW ZONE TOLERANCE IN LIZARDS
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Serum a n t i b o d y r e s p o n s e I n t o l e r a n t l i z a r d s a f t e r c h a l l e r ~ e . Tolerant and control lizards were challer~ed i@m. with 6.0 • 108 SRBC 10 days after the last tolerizI~ injection. Serum a n t i b o d i e s were measured on d i f f e r e n t days a f t e r c h a l l e D g e .
I n f l u e n o e o f C h a l l e r ~ e Dose To d e t e r m i n e t h e t n ~ l u e n o e o f t h e dose o f c h a l l e n g e a n t i g e n on i n d u c e d t o l e r a n c e t o SRBC, t o l e r t z e d and c o n t r o l l i z a r d s were c h a l l e ~ e d w i t h d i f f e r e n t c o n o e n t r a t i o n s o f SIIBC. PFC a s s aye were p e r f o r m e d 7 and 14 days a f t e r c h - l l e n g e ( T a b l e 2 ) . Only a few ~FC were found i n t o l e r a n t l i z a r d s on d a y s 7 and 14 a ~ t e r c h a l l e ~ e w i t h 6 . 0 x 108 , 1 . 2 x 109 o r 2 . 4 x 109 SRBC. A f t e r c h a l l e ~ i ~ w i t h a low dose ( t 0 6 o e l l s ) o f SRBC, t h e PFC zoeporme i n t o l e r a n t l i z a r d s d i d not d i f f e r l a t h from t h a t o f c o n t r o l s on d ~ 7 ( P > O . 0 5 ) ! however, a s i g n i f i c a n t d i f f e r e n c e was n o t e d on day 14 ( P < 0 . 0 2 5 ) . This ,met be due t o t h e f a c t t h a t a mmxin~a munber o f PFC was e v i d e n t o n l y on day 14 a f t e r low dose c h a l l e n g e . The d a t a i n d t o a t e t h a t t h e induoed t o l e r a n c e was s t r o n g and c o u l d not be b r o k e n s i m p l y by i n o r e & s i ~ t h e o h a l l e n g e dose o f SR~C.
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LOW ZONE TOLERANCE IN LIZARDS
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Specificity of Tolerance Specificity of tolerance was tested by challenging the SRBC-tolerant lizards either with SRBC or other erythrocytes (RRBC, RabRBC, BRBC). The PFC response to SRBC was very low in tolerant lizards; the responses to RRBC, RabRBC and BRBC were not significantly different from that in the respective controls (Table 3)- PFC response to RRBC and RabRBC were not reduced even when these antigens were given to tolerant lizards along with SRBC (Table 4). Another group of control and SRBC-tolerant lizards received i.m. 20 mg of PVP in 0.4 ml PBS and the anti-PVP antibodies were measured on day 7, 14 and 21. The reciprocal titer values were: 667 + 192, 2080 + 481, 1200 + 440 (control lizards) and 629 ~ 81, 1867 ~ 448, I~80 ~ 197 (t~lerant lizards). The means of the titers of the two groups differed by less than 5~, thereby indicating the ability of SRBC-tolerant lizards to respond to an unrelated antigen, PVP.
TABLE
5
Specificity of tolerance
Mean PFC/IO 6 WBC c ~ S.E. Challenge
b
P value Control a
Tolerant a
26 +
7
SRBC
1088 + 559
RRBC
197 (~)+26
124(+)24~
BRBC
184 + 46
162 + 54
<0.5
152 ~ 55 (6)
<0.5
(7) RabRBC
172 + 41 (V)
a
See explanatory notes in Table 2. b c d
Ten days after the last injection both groups of lizards were challenged i.m. with 6.0 x 108 SRBC, 1.0 x 108 RRBC, BRBC or 2.0 x 108 RabRBC. PFC response against the challenge antigen was assessed 7 days after the challenge. Number of animals tested is given in parentheses.
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LOW ZONE TOLERANCE IN LIZARDS
TABLE Specificity
Treatment a
Challenge
Vol. 4, No. 2
4
of t o l e r a n c e Mean PFC/IO 6 WBC c ~ S.E.
b
a n t l - S RBC
a n t i-RRBC
a n t i-EabRBC
Control
SRBC + RRBC
668 + 185
192(± 6 59 -)
N.T.
Tolerant
SRBC + RRBC
68(~)+54*
172+)58~
N.T.
Control
Sl~C , Rab]~C
Tolerant
SRBC + RabEBC
a See b Ten ged 2.0
707(~)205
N.T.
178(~)54
45 + 16"
N.T.
151 + 35
(15)
(15)
explanatory notes in Table 2. days after the last injection both groups of lizards were challenl.m. either with 6.0 • 108 SRBC + 1.0 x 108 RRBC or 6.0 • 108 SRBC + x 108 RabRBC.
e PFC response against challenge antigens were assessed separately 7 days after challenge. d Number of Anlmals tested is given in parentheses; N.T. = Not tested. @
Significantly different from the respective control (P<0.01). D u r a t i o n o f T o l e r a n c e To s t u d y t h e d u r a t i o n o f t o l e r a r ~ e , g r o u p s o f l i s a r d s were c h a l l e n g e d w i t h 6 . 0 • 108 SRBC on d i f f e r e n t d a y s a f t e r t h e t o lerlzing regimen. The PFC assaywas performed 7 days later. An appreciable number of PFC (271 ~ 117/106 WBC) was evident when the lizards were challenged immediately after the last tolerlzing injection (day I). The response was, however, meager when challenged 5, 7 or as late as 42 days after the tolerizing injections ( 4 0 ~ 11, 1 9 ~ 5 and 9 ~ 5, respectively). By c o n t r a s t , t h e PFC r e s p o n s e i n n o n - t o l e r a n t c o n t r o l g r o u p s was u n i f o r m l y high (998 ~ 212),
DISCUSSION The d e g r e e o f inmune r e s p o n s e i s p r o f o u n d l y a f f e c t e d by b o t h t h e r o u t e o f a d m i n i s t r a t i o n and t h e d o s e o f a n t i g e n ( 1 7 , 18, 1 9 ) . Results of t h e p r e s e n t e x p e r i m e n t s ( F I G . 1) showed t h a t a n i n c r e a s e i n SRBC d o s e , i r r e s p e c t i v e o f t h e r o u t e o f i m m u n i z a t i o n , was f o l l o w e d by a n i n c r e a s e i n t h e number o f s p l e n i c PFC i n l i z a r d s , as a l r e a d y r e p o r t e d ( 1 9 ) . The s u p e r i o r i t y o f i . m . r o u t e o v e r i . p . i m m u n i z a t i o n i n l i z a r d s c a n be a t t r i b u t e d t o t h e minimum l o s s o f a n t i g e n due t o p h a g o c y t o s i s , ~ the maintenance of a strong antigenic stimulus by a constant, slow release of antigen from t h e i n j e c t i o n s i t e . Intracardiac immunization also elicited a
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quicker response. Howeverp s i n c e t o l e r a n c e i n j e c t i o n s , t h e i . m . r o u t e was p r e f e r r e d .
induction
305
involved repeated
While the administration of an optimal dose of antigen induces effective I ~ n l t y , an antlgen dose hlgher or lower than the im1,no~enlc range would result in a state of unresponsiveness (20). Tolerance to SRBC in l i z a r d s was i n d u c e d by r e p e a t e d i n j e c t i o n s o f subimmunogenic d o s e s o f SEBC. The s t a n d a r d t o l e r i z i r ~ d o s e , 10 b SRBC, i s 600 t i m e s l e s s t h a n t h e o p t i m a / , imm, nogenic dose (6.0 x 108 SRBC). This finding is in agreement with studies In which a similar prolonged injection schedule of low dose of antigen was used. For example: induction of LZT to fla~ellin in neonatal rats
(21), to SRBC i n neonatal mice ( 6 ) , to BSA (3, 4, 5, 20) and pha~e fd part i c l e s ( 9 , 2 2 ) i n a d u l t m i c e . I n a l l t h e s e s t u d i e s , t h e t o l e r i z i n g dose i s a t l e a s t 100 t i m e s s m a l l e r t h a n t h e o p t i m a l d o s e . Huchet and Feldmann i n duced LZT t o d i n i t r o p h e ~ l c o n j u g a t e d human ~ g l o b u l i n (DNP-HGG) w i t h r e p e a t e d i n j e c t i o n s o f 10, 1 o r 0.1 ~ DHP-SHGG. I n d u c t i o n o f c o m p l e t e t o l e r a n c e r e q u i r e d 10 ) ~ E / i n J e c t i o n , whereas i n j e c t i o n o f 1 o r 0.1 )ag r e s u l t e d in partial tolerance (10). By c o n t r a s t , i n t h e p r e s e n t s t u d y , no s u c h d i s c r i m i n a t i o n was e v i d e n t b e t w e e n t h e d i f f e r e n t d o s e s used t o i n d u c e LZT t o SRBC i n l i z a r d s ( T a b l e 1 ) . F a i l u r e t o a l t e r t h e t o l e r a n t s t a t e by i n c r e a s i r ~ t h e c h a l l e r ~ e d o s e o f a n t i g e n ( T a b l e 2) r u l e s out t h e p o s s i b i l i t y that t h e a n t i g e n t h r e s h o l d l e v e l n e c e s s a r y t o I n d u c e a n lr~nune r e s p o n s e c o u l d be gTeater in tolerant lizards. I t was c o n s i d e r e d p o s s i b l e t h a t t h e t o l e r o ~ e n i c e f f e c t s o f a low dose SRBC t r e a t m e n t might d i m i n i s h w i t h t i m e a f t e r c h a l l e n g e i n j e c t i o n and t h a t a normal r e s p o n s e would d e v e l o p a f t e r a l o n g e r i n t e r v a l t h a n u s u ally required. T h i s p o s s i b i l i t y was r u l e d out s i n c e l i z a r d s e x h i b i t e d no e s c a p e from t h e t o l e r a n t s t a t e when t h e PFC a s s a y was p e r f o r m e d 7, 14 and 21 days after challenge (FIG. 2). Thus, the induced unresponsiveness represents a true tolerant state as shown previously in mlee (8). Tolerant lizards showed very few direct PFC and low levels of 2-ME sensitive antibodies (FIG. 2,3,4 and Table 2). In Lice made tolerant to SRBC, b o t h d i r e c t and i n d i r e c t PFCs were r e d u c e d ( 8 ) . C o n v e r s e l y , mice t o l e r a n t t o DNP-I~GG ( 1 0 ) and pha~e f d p a r t i c l e s ( 9 ) showed marked r e d u c t i o n i n IgG a n t i b o d y r e s p o n s e w i t h normal l e v e l s o f IgM a n t i b o d i e s . I t was s u g g e s t e d t h a t t h e I~G r e s p o n s e i s more thymus d e p e n d e n t t h a n t h e I ~ r e s p o n s e and t h a t t o l e r a n c e c o u l d a f f e c t s p e c i f i c a l l y t h e T - c e l l s which h e l p i n t h e development of the I~ response (4,5,9,10,17). I n l i z a r d s , t h e PFC ( d i r e c t PFC) r e s p o n s e t o SEBC i s thymus d e p e n d e n t ( 2 4 , 2 5 ) and no 2-ME r e s i s t a n t a n t i b o d i e s were i d e n t i f i e d e v e n i n s e c o n d a r y tm-,~ne r e s p o n s e t o SRBC (15)o T h e r e f o r e , i n t h e a p p a r e n t a b s e n c e o f an I ~ r e s p o n s e , t o l e r a n t l i z a r d s e x h i b i t a marked s u p p r e s s i o n o f t h e I ~ 4 r e s p o n s e . The PFC response found 2 days after the last tolerizing ir~eotion was not f o l l o w e d by a p p r e c i a b l e l e v e l s o f serum HA o r HL a n t i b o d y ; a l s o , o n l y a v e r y low amount o f serum a n t i b o d ~ was d e t e c t e d i n t o l e r a n t l i z a r d s a t any time a f t e r c h a l l e n g e (FIG. 2 , 3 ) . I n n e w - b o r n mice made t o l e r a n t t o SP~C (LZT and HZT), F r i e d m a n r e p o r t e d t h e p r e s e n c e o f a p p r e c i a b l e amounts o f a n t i b o d y b e f o r e and a f t e r c h a l l e r ~ e , b u t h e r e t h e nnmber o f e p l e n / c PFC was v e r y low ( 6 ) . Our r e c e n t s t u d i e s show t h a t p a s s i v e t r a n s f e r o f serum from t o l e r a n t t o normal l i z a r d s , s h o r t l y b e f o r e o r a l o n g w i t h t h e a d m i n i s t r a t i o n o f SRBC, s u p p r e s s e d t h e PFC r e s p o n s e o f normal l i z a r d s ( u n p u b l i s h e d o b s e r v e -
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tion). Results of the in vitro experiments indicate the presence of a s u p p r e s s i v e f a c t o r i n t h e serum o f t o l e r a n t l i z a r d s ( 2 5 ) . This factor is n o n - d i a l y s a b l e , h e a t r e s i s t a n t and not a b s o r b a b l e by SRBC (25). The s e r um f a c t o r which m e d i a t e s s u p p r e s s i o n i n v i v o and i n v i t r o was found t o be anti,on specific. T h e r e f o r e , i t seems u n l i k e l y t h a t t h e LZT t o SRBC i n lizards i s e f f e c t e d by a n t i b o d y f e e d - b a c k i n h i b i t i o n . Nonetheless, antib o d i e s may be p r e s e n t i n t o l e r a n t l i z a r d s but t h e s e may be u n d e t e c t e d b e c a u s e o f t h e i r low a ~ f i n i t y o r n o n - a ~ g l u t i n a t i n g n a t u r e . F a i l u r e t o d e t e c t a n t i b o d i e s I n mice t o l e r a n t t o t y p e I I I pneumococc a l p o l y s a c c h a r i d e (HZT) was b e c a u s e o f c o n t i ~ o u s p e r i p h e r a l n e u t r a l i z a t i o n o f a n t i b o d y f o r m e d by r e c i r c u l a t t n g , n o n - d e ~ a d a b l e a n t i g e n ( 2 6 ) . Such a n e u t r a l i z a t i o n o f formed a n t i b o d ~ c a n n o t be e x p e c t e d I n LZT t o SRBC i n l i z a r d s , s i n c e SRBC a r e e a s i l y d e g r a d a b l e and t o l e r a n c e i n d u c t i o n I n v o l v e d v e r y low amount o f SRBC. Low serum a n t i b o d y l e v e l s i n t o l e r a n t l i z a r d s i s l i k e l y t o be due t o r e d u c e d numbers o f a n t i b o d y p r o d u c i n g c e l l s r a t h e r t h a n r e d u c e d p r o d u c t i o n o f a n t i b o d y by i n d i v i d u a l c e l l s b e c a u s e t h e s i z e s o f i n d i v i d u a l p l a q u e s formed by t h e s p l e e n c e l l s f r o m tolex~ant and c o n t r o l l i z a r d s were s i m i l a r . The s p e c i f i c i t y o f t h e t o l e r a n c e o b s e r v e d ( T a b l e 4 , 5 ) shows t h a t LZT t o SHBC i n l i z a r d s i s t h e r e s u l t o f an a c t i v e t s p e c i f i c s u p p r e s s i v e p h e n o menon r a t h e r t h a n a g e n e r a l i n a c t i v a t i o n o f t h e immune s y s t e m . S i m i l a r s p e c i f i c i t y has been shown f o r LZT t o SRBC (7) A ~ phage f d (22) i n mice and t o f l a g e l l t n i n r a t s ( 2 1 ) . In m~ls it is often possible to terminate the unresponsive state by Immunizing t o l e r a n t a n i m a l s w i t h c r o s s - r e a c t i n g a n t i g e n ( 2 ) . The s h a r i n ~ o f some a n t i g e n i c d e t e r m i n a n t s b e t w e e n t h e t o l e r a t e d a n t i g e n ~ d c r o s s reacting anti,on helps to bring about an im~me response to the former (2). Cross-reactivity between SRBC ~r~ RabRBC i n C a l o t e s h a s been shown p r e v i o u s l y i n t e r m s o f T - c e l l h e l p and PFC p r o d u c ~ 4 , 2 5 ) . However, i n t h i s s t u d y , t h e f a i l u r e t o b r e a k t o l e r a n c e w i t h c r o s s r e a c t i n g RabRBC, e i t h e r g i v e n a l o n e ( T a b l e 9) o r a l o n g w i t h t h e t o l e r a t e d a n t i g e n ( T a b l e 4) seems t o i n d i c a t e t h a t t h e c e l l s o f t o l e r a n t l i z a r d s sLre n o t r e a c t i v e t o s h a r e d determinants either. A similar failure to break sheep haemolyeate supern a t a n t - induced t o l e r a n c e (LZT t o SRBC) w i t h c r o s s - r e a c t i n ~ h o r s e e r y t h r o c y t e e (HRBC) i n C3H/He mice has b e e n r e p o r t e d ( 8 ) . However, e x p e r i m e n t s using antigens with different degree of cross-reactivity ( w i t h SRBC) need t o be c a r r i e d out as t h e c h o i c e o f a s u i t a b l e a n t i g e n c o m b i n a t i o n f o r b r e a k i n ~ t o l e r a n c e depends upon t h e optiaaam d e g r e e o f c r o s s - r e a c t i v i t y between a n t i g e n s (1,2). I n l i z a r d s , m a n i f e s t a t i o n o f t o l e r a n c e was e v i d e n t o n l y 3 da~s a f t e r the last tolerizir~ Injection. A s i m l l a ~ i n d u c t i v e p e r i o d was found t o be r e q u i r e d I n o t h e r LZT s y s t e m s ( 4 , 5 , 9 , 1 0 , 2 2 ) . Experimentally induced toler a n c e c a n be e i t h e r t r a n s i e n t o r l o n ~ l a s t i r ~ (1). L i z a r d s made t o l e r a n t t o SRBC a r e r e f r a c t i v e t o c h a l l e r ~ e i n j e c t i o n g i v e n a s l a t e a s 42 d a y s a f t e r t h e l a s t t o l e r i s i n ~ i n j e c t i o n and no breakdown o f t o l e r a n c e was o b served. I n v e s t i g a t i o n s f o r l o n g e r p e r i o d s c o u l d n o t be c a r r i e d out i n l i z a r d s due t o t h e i n m b i l i t y t o m a i n t a i n them i n t h e l a b o r a t o r y c o r d ~ t i o n s beyond two months. However, t h e p r e s e n t f i n d i r ~ a g r e e s w i t h t h e p r e v i o u s r e p o r t s t h a t normal r e s p o n s i v e n e s s i n mice was not r e s t o r e d u p t o s e v e r a l weeks o r months a f t e r t o l e r a n c e i n d u c t i o n ( 7 , 8 ) .
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From t h e f o r e g o i n ~ d i s c u s s i o n , i t i s c l e a r t h a t t h e o b s e r v e d u n r e s p o n s i v e n e s s t o SRBC i n l i z a r d s i s an example o f LZT. The mechanisms o f i n d u c t i o n and m a i n t e n a n c e o f LZT t o SItBC I n l i z a r d s a r e u n d e r i n v e s t i g a t i o n .
ACK~O~ ~G~4~NTS T h i s work was s u p p o r t e d by a r e s e a r c h g r a n t from t h e D e p a r t m e n t o f Atomic Energy o f I n d i a and S. B. was a r e c i p i e n t o f J u n i o r R e s e a r c h F e l l o w s h i p i n t h e scheme. We e x p r e s s o u r g r a t e f u l t h a n k s t o Dr. S. J ~ y a r a m a n for critically reading the manuscript. REFERENCES I.
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