PROTECTION OF ATHYMIC (NU/NU) BALB/c MICE AGAINST PLASMODIUM
BERGHEI BY SPLENOCYTES FROM NORMAL (NU/ + ) BALB/c MICE (*)
José J. FERRARONI (1,2), Thomas G. DOUGLASS (2) and Clarence A. SPEER (3)
S U M M A R Y A t h y m i c B A L B / c ( N u / N u ) m i c e d i e d at 7-13 d a y s a f t e r i n o c u l a t i o n ( D A I ) o f P l a s m o d i u m b e r g h e i N K 6 5 , w h e r e a s their h e t e r o z y g o u s ( N u / + ) l i t t e r m a t e s d i e d at 7-8 D A I . N u d e ( N u / N u ) m i c e , r e c o n s t i t u t e d w i t h 2 x 1 07 s p l e n o c y t e s f r o m unin f e c t e d h e t e r o z y g o u s ( N u / + ) l i t t e r m a t e s at 20 d a y s b e f o r e p a r a s i t e i n o c u l a t i o n ( D B I ) , d i e d a b o u t 2 d a y s e a r l i e r t h a n c o n t r o l n u d e m i c e ; n u d e m i c e r e c o n s t i t u t e d at 10 o r 2 D B I l i v e d 2 t o 4 d a y s l o n g e r t h a n c o n t r o l nudes; a n d n u d e m i c e re c o n s t i t u t e d 2 D A I l i v e d e v e n l o n g e r a n d s o m e s u r v i v e d . T h e s e f i n d i n g s i n d i c a t e t h a t P . b e r g h e i N K 6 5 i n d u c e s at l e a s t t w o T - c e l l d e p e n d e n t i m m u n e p h e n o m e n a , o n e s u p p r e s s i v e a n d t h e o t h e r s t i m u l a t o r y . R e c o n s t i t u t i o n o f n u d e m i c e w i t h T-cells f r o m B A L B / c ( N u / + ) m i c e a p p e a r e d t o r e d u c e o r b y p a s s s u p p r e s s i v e T-cell a c t i v i t i e s w h i c h a l l o w e d t h e f o r m a t i o n o f a p r o t e c t i v e i m m u n e r e s p o n s e b y s o m e o f t h e n u d e m i c e . I N T R O D U C T I O N S e v e r a l s p e c i e s of r a t s a n d v o l e s s e r v e as h o s t s f o r t h e N K 6 5 s t r a i n o f P l a s m o d i u m ber ghei i n 'which the i n f e c t i o n is r a r e l y f a t a l 2-l s. H o w e v e r , t h e N.K65 s t r a i n of P . berghei has b e e n f o u n d t o b e 100% lethal f o r a l l s t r a i n s of l a b o r a t o r y m i c e t e s t e d to d a t e 13,25,26,27,33,37. L o n g l a s t i n g i m m u n i t y t o P . berghei N K 6 5 has b e e n a c h i e v e d o n l y b y using p a r a s i t e i n o c u l a t i o n a n d d r u g c u r e ( E L I N G ; F E R R A R O N I & S P E E R )8'1 1. L y m p h o c y t e s f r o m such s o l i d l y i m m u n e a n i m a l s a r e c a p a b l e o n l y o f transfer r i n g s o m e d e g r e e o f r e s i s t a n c e t o P . berghei ( F E R R A R O N I & S P E E R )î 2. A c q u i r e d resistan c e t o P l a s m o d i a h a s b e e n s h o w n t o d e p e n d u p o n b o t h b o n e m a r r o w a n d t h y m u s - d e r i v e d l y m p h o c y t e s 3. H o w e v e r , i n a p r e l i m i n a r y study w e f o u n d t h a t n a i v e a t h y m i c n u d e m i c e i n o c u l a t e d w i t h P . b e r g h e i N K 6 5 s u r v i v e d s i g n i f i c a n t l y lon g e r than their h i r s u t e ( N u / + ) l i t t e r m a t e s . R e cently, W A K I & S U Z U K I3 8 w e r e a b l e t o abro g a t e r e s i s t a n c e t o P . b e r g h e i N K 6 5 b y reconsti t u t i n g a t h y m i c n u d e m i c e w i t h t h y m o c y t e s at t h r e e w e e k s b e f o r e p a r a s i t e i n o c u l a t i o n . I t is still n o t k n o w n h o w s o o n a f t e r re-c o n s t i t u t i o n b y t h y m o re-c y t e s t h a t a t h y m i re-c m i re-c e b e c o m e i m m u n o l o g i c a l l y c o m p e t e n t . T h e e f f e c t s of r e c o n s t i t u t i o n o n b o t h h u m o r a l1 4 a n d cell-m e d i a t e d 1 9-3 2 i m m u n i t y h a v e b e e n d e t e r m i n e d o n l y f o r t h r e e o r m o r e w e e k s a f t e r t h y m i c im p l a n t a t i o n o r i n j e c t i o n o f t h y m o c y t e s . I n o r d e r t o d e t e r m i n e t h e e f f i c a c y o f ma t u r e l y m p h o c y t e s u p o n P . b e r g h e i i n f e c t i o n s in a t h y m i c n u d e m i c e , splenic l y m p h o c y t e s and c u l t u r e - d e r i v e d s p l e e n c e l l s u p e r n a t a n t s f r o m
( * ) This study is based on a dissertation submitted by J. J. Perraroni to the Graduate School of the University of Montana in partial fulfillment to the requirements for the Doctor of Philosophy degree
(1) Universidade do Amazonas/CNPq, Campus Universitário, Departamento de Patologia — ICB — 69.000, Manaus, Ama zonas, Brasil
n a i v e h e t e r o z y g o u s ( N u / + ) m i c e w e r e adopti v e l y t r a n s f e r r e d t o nude m i c e at v a r i o u s inter v a l s b e f o r e o r after p a r a s i t e i n o c u l a t i o n . T h e r e s u l t s o b t a i n e d f r o m these studies a r e r e p o r t e d h e r e i n . M A T E R I A L S A N D M E T H O D S Animals — C o n g e n i c a t h y m i c n u d e ( N u / N u ) B A L B / c m i c e a n d t h e i r h i r s u t e ( N u / + ) l i t t e r m a t e s w e r e r a i s e d i n t h e M i c r o b i o l o g y De p a r t m e n t a n i m a l f a c i l i t y at t h e U n i v e r s i t y of M o n t a n a . A l l m i c e w e r e SJweeks-old at t h e beg inning of e a c h e x p e r i m e n t , e x c e p t f o r experi m e n t N o . 3 i n w h i c h t h e a t h y m i c m i c e w e r e 5-w e e k s - o l d . M i c e 5-w e r e k e p t i n c o v e r e d c a g e s w i t h s t e r i l e s a w d u s t a n d f e d s t e r i l e m o u s e lab c h o w a d l i b i t u m . T e t r a c y c l i n e ( 5 m g / l ) i n ste r i l e w a t e r w a s g i v e n a d l i b i t u m t o t h e m i c e un t i l 10 d a y s b e f o r e p a r a s i t e i n o c u l a t i o n . S w i s s -W e b s t e r o u t b r e d m i c e s e r v e d as h o s t s f o r pa rasite m a i n t e n a n c e . T h e U n i v e r s i t y of M o n t a n a B A L B / c a t h y m i c m o u s e c o l o n y and S w i s s - W e b s t e r m i c e w e r e o r i g i n a l l y o b t a i n e d f r o m the Na t i o n a l I n s t i t u t e of A l l e r g y and I n f e c t i o u s Disea ses, R o c k y M o u n t a i n L a b o r a t o r y , H a m i l t o n , M T .
Parasite, P l a s m o d i u m berghei N K 6 5 w a s ori
g i n a l l y o b t a i n e d f r o m the U n i v e r s i t y of N e w M e x i c o , A l b u q u e r q u e , N M , a n d h a d b e e n main t a i n e d b y w e e k l y b l o o d passages i n S w i s s - W e b s t e r m i c e f o r 5 y r b e f o r e b e i n g u s e d i n this study. P a r a s i t i z e d b l o o d w a s o b t a i n e d f r o m S w i s s - W e b s t e r o r B A L B / c m i c e a n d w a s h e d 3 t i m e s i n p h o s p h a t e b u f f e r e d saline ( P B S ) at 4°C w i t h o u t a n t i c o a g u l a n t . A l l m i c e w e r e inject e d i n t r a p e r i t o n e a l ^ ( I P ) w i t h 107 P. berghei-i n f e c t e d e r y t h r o c y t e s . T h e berghei-i n t e r v a l b e t w e e n b l e e d i n g d o n o r m i c e and i n o c u l a t i n g t h e infect e d b l o o d i n t o r e c i p i e n t m i c e w a s less t h a n 40 m i n . T h e p a r a s i t e m i a w a s d e t e r m i n e d f o r e a c h a n i m a l b y m i c r o s c o p i c e x a m i n a t i o n ( o i l i m m e r s i o n l e n s ) o f c e l l s i n p e r i p h e r a l b l o o d s m e a r s s t a i n e d w i t h G i e m s a . Splenocytes — S p l e e n s w e r e r e m o v e d asep-t i c a l l y f r o m 8 asep-t o 10 w e e k - o l d d o n o r B A L B / c ( N u / + ) m i c e and f o r c e d t h r o u g h a 60-mesh stainless steel s c r e e n i n t o M i n i m a l E s s e n t i a l M e d i u m ( M E M , G i b c o , Santa Clara, C A ) , p H 7.4, c o n t a i n i n g 100 ng/ml ( w / v ) s t r e p t o m y c i n sul f a t e a n d 100 U / m l ( w / v ) p o t a s s i u m p e n i c i l l i n G. A f t e r a l l o w i n g t h e tissue d e b r i s t o s e t t l e f o r 10 m i n , t h e cell s u p e r n a t a n t w a s r e m o v e d and w a s h e d t h r e e t i m e s w i t h M E M . E r y t h r o c y t e s w e r e l y s e d w i t h 0.15 M a m m o n i u m c h l o r i d e , after w h i c h the r e m a i n i n g n u c l e a t e d cells w e r e w a s h e d t h r e e t i m e s i n P B S . N u c l e a t e d c e l l s less t h a n 10 /mi i n d i a m e t e r w e r e f o u n d t o b e 96% v i a b l e as d e t e r m i n e d b y t r y p a n b l u e e x c l u s i o n . E a c h r e c i p i e n t a n i m a l w a s i n j e c t e d I P w i t h 0.2 m l M E M c o n t a i n i n g 2 x 1 07 v i a b l e s p l e n o c y t e s . I n o r d e r t o c o m p e n s a t e f o r t w o m a j o r va r i a b l e s (i.e., s p l e n o c y t e s a n d p a r a s i t e i n o c u l a ) , t w o cell t r a n s f e r e x p e r i m e n t s w e r e p e r f o r m e d . I n t h e f i r s t e x p e r i m e n t , all a n i m a l s r e c e i v e d s p l e n o c y t e s f r o m t h e s a m e p r e p a r a t i o n , w h e r e a s f r e s h i n o c u l a o f P . berghei w e r e o b t a i n e d f r o m m i c e at t h e t i m e o f each p a r a s i t e inocu l a t i o n . N i n e g r o u p s o f 6 m i c e e a c h (3 m a l e s a n d 3 f e m a l e s ) w e r e used. G r o u p s 1-6 a n d 7-9 w e r e B A L B / c a t h y m i c ( N u / N u ) a n d B A L B / c ( N u / + ) , r e s p e c t i v e l y . G r o u p s 1-5 r e c e i v e d sple n o c y t e s at 20, 10 o r 2 d a y s b e f o r e p a r a s i t e ino c u l a t i o n ( D B I ) , at the t i m e o f p a r a s i t e inocu l a t i o n ( D O I ) , o r 2 d a y s after p a r a s i t e inocula t i o n ( D A I ) , r e s p e c t i v e l y . S p l e n o c y t e s w e r e g i v e n t o g r o u p 7 t w o D A I a n d t o g r o u p 8 at D O I . G r o u p s 6 a n d 9 r e c e i v e d n o s p l e n o c y t e s a n d s e r v e d as c o n t r o l s . I n the s e c o n d e x p e r i m e n t , all a n i m a l s w e r e i n o c u l a t e d w i t h the s a m e p a r a s i t e p r e p a r a t i o n , w h e r e a s s p l e n o c y t e s w e r e o b t a i n e d f r o m m i c e at the t i m e of e a c h c e l l t r a n s f e r . F o u r g r o u p s of 7 B A L B / c ( N u / N u ) m i c e (4 m a l e s a n d 3 fe m a l e s ) w e r e used. G r o u p s 1-3 r e c e i v e d spleno c y t e s at t w o D A I , o n e D A I o r at D O I , respec t i v e l y . G r o u p 4 r e c e i v e d n o s p l e n o c y t e s and s e r v e d as a c o n t r o l . Splenocyte supernatants — T h r e e t y p e s of s p l e n o c y t e s u p e r n a t a n t s ( S C S I, 2 a n d 3 ) w e r e o b t a i n e d as f o l l o w s . E i g h t h u n d r e d m i l l i o n s p l e n o c y t e s s u s p e n d e d i n 50 m l M E M contain i n g 5% f e t a l calf s e r u m ( F C S ) w e r e i n o c u l a t e d i n t o e a c h of t h r e e 32 o z B r o c k w a y c u l t u r e flasks a n d t h e n i n c u b a t e d at 37°C, 5% C 02, 95% air. S p l e e n cell c u l t u r e N o . 1 w a s c u l t u r e d 24 h i n t h e p r e s e n c e of 5 / t g / m l C o n c a n a v a l i n A ( C o n A , S i g m a C h e m i c a l C o . , S t . L o u i s , M o . ) ; after 24 h i n c u b a t i o n , 5 / t g / m l C o n A w e r e a d d e d to c u l t u r e N o . 2; c u l t u r e N o . 3 w a s i n c u b a t e d 24 h w i t h o u t C o n A . A f t e r i n c u b a t i o n , t h e superna tant w a s r e m o v e d f r o m e a c h c u l t u r e flask,
cen-t r i f u g e d acen-t 450 g f o r 5 m i n , d i a l i z e d 16 h acen-t 4°C i n 75 1 d i s t i l l e d w a t e r a n d t h e n c o n c e n t r a t e d ~ 20 t i m e s b y 6 h of e v a p o r a t i o n d i a l y s i s . E a c h m o u s e i n t h r e e g r o u p s of 5 B A L B / c ( N u / N u ) m i c e w a s i n j e c t e d I P w i t h 0.5 m l S C S 1, 2 o r 3 ( a p p r o x i m a t e l y the a m o u n t deri v e d f r o m 2 x 107 s p l e n o c y t e s ) at t w o D A I of P. berghei. O n e g r o u p of 5 B A L B / c ( N u / N u > m i c e s e r v e d as an u n t r e a t e d c o n t r o l . I n o r d e r t o d e t e r m i n e the e f f i c a c y of n u d e a n d h e t e r o z y g o u s m i c e t o c l e a r p a r a s i t e s f r o m t h e i r p e r i p h e r a l c i r c u l a t i o n , 0.5 m l of b l o o d c o n t a i n i n g 2.5 x 1 07 P. berghei-inf e c t e d r e d b l o o d c e l l s w a s i n j e c t e d i n t r a v e n o u s l y i n t o each of t w o n u d e m i c e a n d t w o h e t e r o z y g o u s m i c e . T h e p a r a s i t e m i a o f e a c h a n i m a l w a s de t e r m i n e d b y m i c r o s c o p i c e x a m i n a t i o n of b l o o d s m e a r s s t a i n e d w i t h G i e m s a at v a r y i n g inter vals, s t a r t i n g at 2 m i n and e n d i n g after 18 hs. S i g n i f i c a n t d i f f e r e n c e s a m o n g v a r i o u s experi m e n t a l g r o u p s w e r e d e t e r m i n e d b y t h e Stu d e n t ' s t-test at P v a l u e s o f — 0.05. R E S U L T S C o u r s e of P. b e r g h e i infection — F i g u r e 1 s h o w s the m e a n d a i l y p a r a s i t e m i a ( M D P ) up u n t i l the t i m e of d e a t h o f B A L B / c a t h y m i c m i c e i n o c u l a t i o n w i t h P. b e r g h e i at v a r i o u s t i m e s r e l a t i v e t o t h e i r r e c e i v i n g 2 x 107 spleno c y t e s f r o m the s a m e s p l e n o c y t e p r e p a r a t i o n . T a b l e I g i v e s the m e a n d a y o f d e a t h ( M D D ) a n d t h e m e a n p a r a s i t e m i a t h e d a y p r i o r to d e a t h . B e c a u s e t w o o f the a t h y m i c m i c e i n the g r o u p w h i c h r e c e i v e d s p l e n o c y t e s 2 D A I sur v i v e d a n d t h e i r p a r a s i t e m i a n e v e r e x c e e d e d 1% ( T a b l e I I I ) , t h e y w e r e n o t i n c l u d e d i n F i g . 1. T h e M D P a n d the M D D of the a t h y m i c m i c e w h i c h r e c e i v e d s p l e n o c y t e s 20 D B I d i d n o t d i f f e r s i g n i f i c a n t l y f r o m t h o s e o f c o n t r o l n u d e m i c e . I n t h e o t h e r g r o u p s , t h e r e w a s :.a d i r e c t corre l a t i o n b e t w e e n t h e b e n e f i c i a l e f f e c t s of sple n o c y t e s a n d the t i m e of s p l e n o c y t e t r a n s f e r re l a t i v e t o t i m e of p a r a s i t e i n o c u l a t i o n ( F i g . 1 ) . T h e M D P r o s e s l o w e r and the m i c e s u r v i v e d l o n g e r i n n u d e m i c e w h i c h r e c e i v e d s p l e n o c y t e s at 10 D B I , 2 D B I , D O I a n d 2 D A I t h a n i n con t r o l m i c e . S p l e n o c y t e s g i v e n 2 D A I c a u s e d m o r e d e l a y i n r i s e of p a r a s i t e m i a as w e l l as i n d a y of d e a t h . I t w a s i n this g r o u p o n l y t h a t a n i m a l s s u r v i v e d . F i g u r e 2 s h o w s the M D P u n t i l the t i m e of d e a t h o f n u d e a n d h e t e r o z y g o u s ( N u / + ) m i c e w h i c h w e r e i n f e c t e d w i t h the s a m e i n o c u l u m of P. b e r g h e i b u t w h i c h r e c e i v e d s p l e n o c y t e s f r o m d i f f e r e n t p r e p a r a t i o n s at the s a m e t i m e of o n e o r 2 D A I . T a b l e I I g i v e s t h e M D D a n d t h e m e a n p a r a s i t e m i a t h e d a y p r i o r t o d e a t h f o r t h e sa m e m i c e . N o significant d i f f e r e n c e s w e r e obser v e d b e t w e e n c o n t r o l h e t e r o z y g o u s ( N u / + ) m i c e a n d h e t e r o z y g o u s m i c e t h a t r e c e i v e d splenocy tes ( T a b l e I ) . H e t e r o z y g o u s m i c e a l w a y s suc c u m b e d e a r l i e r w i t h a l o w e r p a r a s i t e m i a than n u d e m i c e . B e c a u s e f o u r o f the n u d e m i c e w h i c h r e c e i v e d s p l e e n c e l l s 2 D A I s u r v i v e d and their p a r a s i t e m i a n e v e r e x c e e d e d 1% ( T a b l e I I I ) , their M D P w a s n o t i n c l u d e d i n F i g . 2. A s n o t e d i n t h e p r e v i o u s e x p e r i m e n t , a d i r e c t cor r e l a t i o n e x i s t e d b e t w e e n the b e n i f i c i a l e f f e c t of the t i m e o f s p l e n o c y t e t r a n s f e r and the t i m e of p a r a s i t e i n o c u l a t i o n . T h e g r e a t e s t b e n e f i c i a l e f f e c t w a s o b s e r v e d i n t h o s e m i c e that receiv e d s p l e n o c y t e s at 2 D A I ; t h e i r p a r a s i t e m i a re m a i n e d r e l a t i v e l y l o w ( < 5 % ) f o r 17 D A I and t h e n i n c r e a s e d t o > 85% b y 27 D A I , w h i c h w a s Qilso the" M D D . T a b l e I I I g i v e s t h e i n d i v i d u a l d a i l y para s i t e m i a o f m i c e w h i c h r e c e i v e d s p l e n o c y t e s 2 D A I i n e x p e r i m e n t s 1 a n d 2. I n e x p e r i m e n t 1, p a r a s i t e s w e r e p r e s e n t i n t h e p e r i p h e r a l b l o o d of all m i c e ( e x c e p t m o u s e N o . 2 ) f r o m day six until d e a t h o r r e s o l u t i o n o f the i n f e c t i o n . I n e x p e r i m e n t N o . 2 p a r a s i t e s w e r e usually n o t o b s e r v e d at 9 t o 17 D A I a f t e r w h i c h p a r a s i t e s
r e a p p e a r e d i n the p e r i p h e r a l b l o o d . A f t e r the p a r a s i t e s r e a p p e a r e d i n t h e b l o o d , t h e i n f e c t i o n p r o c e e d e d u n t i l the m i c e d i e d o r the i n f e c t i o n w a s r e s o l v e d . F i g u r e 3 s h o w s the M D P of n u d e m i c e t h a t r e c e i v e d S C S 2 D A I o f Iff7 P . berghei-parasitized e r y t h r o c y t e s . N o s i g n i f i c a n t l y d i f f e r e n c e s occur r e d b e t w e e n the g r o u p s of n u d e m i c e w h i c h r e c e i v e d S C S 1, 2, o r 3 and the c o n t r o l g r o u p . T h e h e t e r o z y g o u s m i c e w e r e m o r e effica cious i n r e m o v i n g i n f e c t e d r e d cells f r o m t h e i r p e r i p h e r a l c i r c u l a t i o n than n u d e m i c e ( T a b l e I V ) . D I S C U S S I O N W A K I & S U Z U K I3 8 f o u n d t h a t a t h y m i c nu de ( N u / N u ) m i c e i n f e c t e d w i t h P . berghei N K 6 5 s u r v i v e d s i g n i f i c a n t l y l o n g e r t h a n t h e i r hirsute ( N u / + ) l i t t e r m a t e s . T h e y a l s o f o u n d t h a t t h e s u r v i v a l t i m e o f n u d e m i c e t h a t h a d b e e n re c o n s t i t u t e d w i t h n e o n a t a l t h y m o c y t e s f r o m hir sute m i c e t h r e e w e e k s b e f o r e p a r a s i t e inocula t i o n w a s n o t s i g n i f i c a n t l y d i f f e r e n t t h a n t h a t of t h e i r t h y m i c c o m p e t e n t l i t t e r m a t e s . B a s e d u p o n t h e s e o b s e r v a t i o n s , t h e s e a u t h o r s suggest e d t h a t n u d e m i c e l i v e d l o n g e r d u r i n g a P .
berghei N K 6 5 i n f e c t i o n b e c a u s e they l a c k e d a T-cell- d e p e n d e n t i m m u n o p a t h o l o g i c r e s p o n s e . T h e results o b t a i n e d i n the p r e s e n t s t u d y indi
c a t e t h a t t h e r e is n o i m m u n o p a t h o l o g i c respon se a s s o c i a t e d w i t h m a t u r e s p l e n i c d e r i v e d T-cells i n m i c e i n f e c t e d w i t h P . berghei N K 6 5 . W e
f o u n d t h a t n u d e m i c e r e c o n s t i t u t e d w i t h sple-n o c y t e s at 20 D B I s h o w e d sple-n o "besple-nificiai o r harm ful e f f e c t a n d t h a t t h o s e r e c o n s t i t u t e d at 10 o r f e w e r D B I o r at 2 D A I s h o w e d a d e c r e a s e in o n s e t o f p a r a s i t e m i a and an i n c r e a s e d s u r v i v a l t i m e , a n d s o m e m i c e e v e n s u r v i v e d . Reconstitu t i o n w i t h n e o n a t a l t h y m o c y t e s p r o b a b l y p r o v i des a g r e a t e r n u m b e r o f T-cell r e c o g n i t i o n and e f f e c t o r f u n c t i o n s t h a n r e c o n s t i t u t i o n w i t h sple-n o c y t e s . R e c o sple-n s t i t u t i o sple-n o f sple-n u d e m i c e w i t h thy
m o c y t e s at t h r e e w e e k s b e f o r e i n o c u l a t i o n m a y a l l o w s u f f i c i e n t t i m e f o r t h e d e v e l o p m e n t of s u p p r e s s o r Tcell a c t i v i t y . S p l e n o c y -tes m a y l a c k a full c o m p l e m e n t o f T-cell s u b s e t s n e c e s s a r y t o g e n e r a t e s u p p r e s s o r T - c e l l a c t i v i t y of a m a g n i t u d e s i m i l a r t o t h a t o b t a i n e d b y thy m o c y t e r e c o n s t i t u t i o n a s o b s e r v e d b y W A K I & S U Z U K I T h u s , P . berghei a p p e a r s t o i n d u c e at l e a s t t w o T-cell d e p e n d e n t i m m u n e p h e n o m e na, o n e s u p p r e s s i v e a n d the o t h e r s t i m u l a t o r y . R e c o n s t i t u t i o n o f n u d e m i c e w i t h s p l e n o c y t e s f r o m p h e n o t y p i c a l l y n o r m a l h e t e r o z y g o u s m i c e a p p e a r s t o r e d u c e o r b y p a s s the s u p p r e s s i v e T-cell a c t i v i t i e s w h i c h a l l o w s t h e f o r m a t i o n of a p r o t e c t i v e i m m u n e r e s p o n s e t o P . b e r g h e i b y s o m e o f t h e n u d e m i c e . S e v e r a l m e c h a n i s m s are p r o b a b l y i n v o l v e d i n t h e a b i l i t y o f s p l e n o c y t e s f r o m t h y m i c c o m p e t e n t m i c e t o p r o t e c t n u d e m i c e against P . berghei N K 6 5 . M a c r o p h a g e s o f n u d e m i c e h a v e a g r e a t e r c a p a c i t y t o p h a g o c y t o s e a n d k i l l c e r t a i n v i r u s e s , b a c t e r i a and y e a s t s t h a n ma c r o p h a g e s o f t h y m i c c o m p e t e n t m i c e ( C A U ' L E Y & M U R P H Y4; C H E E R S & W A L L E R5; E M M E R L I N G e t al. 9; F A U V E & H E V I N1 0;
M O N G E N S E N & A N D E R S E N2* ; N I C K O L & B O N V E N T R E2 3; R O G E R e t a l .2* ) . H o w e v e r , e n h a n c e d a c t i v i t y b y the r e t i c u l o e n d o t h e l i a l s y s t e m ( R E S ) d o e s n o t a p p e a r t o b e respons i b l e f o r the i n c r e a respons e d respons u r v i v a l t i m e a n d p r o -t e c -t i o n a g a i n s -t P l a s m o d i u m a n d B a b e s i a b y m i c e t h a t h a d r e c e i v e d B a c i l l e Calmette-Gue-r i n ( B C G ) o Calmette-Gue-r k i l l e d C o Calmette-Gue-r y n e b a c t e Calmette-Gue-r i u m p a Calmette-Gue-r v u n ( B A Z A R - M A L I K *; C L A R K e t a l . « ; C L A R K , C O X & A L L I S O N7; M U R P H Y2 2; N U S S E N Z -W E I G » ; S M R K O V S K I & S T R I C K L A N D3 1) . I n t h e p r e s e n t study, n u d e m i c e d i d n o t c l e a r m a l a r i a l p a r a s i t e s f r o m their p e r i p h e r a l b l o o d as r a p i d l y as t h y m i c c o m p e t e n t m i c e . T h u s , t h e d e l a y i n o n s e t o f p a r a s i t e m i a i n nonre-c o n s t i t u t e d n u d e m i nonre-c e d o e s n o t a p p e a r t o b e d u e t o e n h a n c e d a c t i v i t y of t h e R E S . R e c e n c y , W A K I & S U Z U K I3 9 r e p o r t e d t h a t n u d e m i c e i n f e c t e d w i t h P. b e r g h e i and c u r e d b y p y r i m e t h a m i n e p r i m e d t h e a n i m a l s s o t h a t a s e c o n d a r y a n t i b o d y r e s p o n s e w a s e l i c i t e d u p o n p a r a s i t e c h a l l e n g e . T h e y conclud-ed, t h e r e f o r e , t h a t at l e a s t o n e p a r a s i t e anti-gen w a s t h y m u s i n d e p e n d e n t a n d t h a t p r i m e d B-cells w e r e t h e d o m i n a n t c e l l s g e n e r a t e d dur-ing a p r i m a r y i n f e c t i o n i n n u d e m i c e . H o w e v e r , B-cells can b e p r i m e d b y t h y m u s d e p e n d e n t a n t i g e n s i n the absence o f T - c e l l s2 9. I m m u n e s e r u m f r o m m i c e d r i v e n t o i m m u n i t y b y re-p e a t e d c y c l e s of P . b e r g h e i i n f e c t i o n and Fan-s i d a r c u r e ( p y r i m e t h a m i n e + Fan-s u l f a d o x i n e ) haFan-s b e e n f o u n d t o suppress p a r a s i t e d e v e l o p m e n t i n n a i v e m i c e , b u t o n l y i f a d m i n i s t e r e d o n a d a i l y b a s i s ( F E R R A R O N I & S P E E R ) 1 2. T w o l i n e s o f e v i d e n c e i n d i c a t e t h a t b o d y i s p r o b a b l y n o t i n v o l v e d i n t h e anti-m a l a r i a l p r o t e c t i o n of n u d e anti-m i c e reconstitut-e d w i t h n o r m a l s p l reconstitut-e n o c y t reconstitut-e s . F i r s t , W A K I & S U Z U K I3 9 f o u n d that n u d e m i c e i m m u n i z e d against P. b e r g h e i w e r e n o t p r o t e c t e d against p a r a s i t e c h a l l e n g e e v e n t h o u g h t h e y d e v e l o p e d s i g n i f i c a n t l e v e l s o f parasite-specific anti-b o d i e s . S e c o n d , w e f o u n d t h a t t h e n u d e m i c e , w h i c h h a d b e e n r e c o n s t i t u t e d w i t h splenocy-tes a n d s u r v i v e d a p r i m a r y P . b e r g h e i infec-t i o n , s u c c u m b e d infec-t o a c h a l l e n g e i n o c u l a infec-t i o n 40 d a y s l a t e r ( a b o u t 10 d a y s a f t e r p a r a s i t e clea-r a n c e o f t h e f i clea-r s t i n o c u l a t i o n ) . T h u s , such clea- re-c o n s t i t u t e d n u d e m i re-c e e v i d e n t l y h a v e n o me-m o r y of their e a r l i e r i n f e c t i o n a n d t h e f a c t o r s i n v o l v e d i n the e l i m i n a t i o n o f t h e p a r a s i t e s in the p r i m a r y i n f e c t i o n a r e s h o r t - l i v e d . C e r t a i n i n v e s t i g a t o r s 15,16,17,35,41 h a v e sug-g e s t e d t h a t i n t e r f e r o n o r i n t e r f e r o n - l i k e subs-t a n c e s m a y b e r e s p o n s i b l e f o r subs-t h é e n h a n c e d r e s i s t a n c e t o b l o o d p r o t o z o a b y a n i m a l s treate d w i t h v a r i o u s n o n s p treate c i f i c i m m u n o p o t treate n t i a t o r s such as e n d o t o x i n , s t a t o l o n , p o l y r i b o i n o -sinic a c i d , N e w C a s t l e D i s e a s e v i r u s ( N D V ) a n d B C G 1.6,7,16,17,24,30,31,34,36. J A H I E L e t a l . i e f o u n d t h a t i n t e r f e r o n i n d u c e r s p r o v i d e d m i c e w i t h s o m e d e g r e e of p r o t e c t i o n against P . ber-g h e i a n d w e r e a b l e t o d e t e c t i n t e r f e r o n i n the s e r u m o f m i c e i n f e c t e d w i t h N D V o r t r e a t e d w i t h s t a t o l o n . N u d e m i c e a r e usually i n f e c t e d w i t h m o u s e h e p a t i t i s v i r u s4 0 and p r e s u m a b l y s u p p o r t l o w g r a d e c h r o n i c i n f e c t i o n s w i t h o t h e r v i r u s e s as w e l l as b a c t e r i a . M a t u r e T-cells p r i m e d b y i n t e r a c t i o n w i t h v a r i o u s in-f e c t i o u s a g e n t s a l r e a d y in t h e n u d e m i c e m a y r e s u l t i n t h e p r o d u c t i o n o f s u b s t a n c e s w h i c h w o u l d n o n s p e c i f i c a l l y p r o t e c t t h e h o s t against P. b e r g h e i . B e c a u s e w e f o u n d t h a t t h e protec-t i o n a f f o r d e d b y s p l e n o c y protec-t e s w a s s h o r protec-t - l i v e d , i n t e r f e r o n d o e s n o t a p p e a r t o b e t h e s u b s t a n c e r e s p o n s i b l e f o r p r o t e c t i n g n u d e m i c e against P. b e r g h e i . H o w e v e r , a s h o r t - l i v e d s u b s t a n c e p r o s s e s s i n g a c t i v i t i e s s i m i l a r t o t h o s e o f inter-f e r o n m i g h t h a v e b e e n p r o v i d e d t o t h e n u d e m i c e b y s p l e n o c y t e s f r o m t h y m i c c o m p e t e n t m i c e . T h e l a b i l i t y o f such an i n t e r f e r o n - l i k e s u b s t a n c e m a y a c c o u n t f o r the f a c t t h a t the g r e a t e s t d e g r e e o f p r o t e c t i o n o f n u d e m i c e against P. b e r g h e i w a s o b t a i n e d w h e n spleno-c y t e s w e r e t r a n s f e r r e d 2 D A I o f P. b e r g h e i .
D u r i n g the o n t o g e n y of the i m m u n e syst e m syst h e a b i l i syst y syst o s u p p r e s s a s p e c i f i c i m m u -ne r e s p o n s e usually d e v e l o p s a f t e r the ability t o r e s p o n d t o s p e c i f i c a n t i g e n2 0. i n a d d i t i o n , s o l u b l e T-cell s u p p r e s s o r s u b s t a n c e s a r e usual-l y n o t p r o d u c e d f o r a b o u t 48 h f o usual-l usual-l o w i n g the i n t e r a c t i o n of T-cells w i t h s p e c i f i c a n t i g e n , w h e r e a s T-cell h e l p e r substances a r e k n o w n t o b e p r o d u c e d as e a r l y as 12 h 1 9>3 2. A g r e a t e r de-g r e e o f p r o t e c t i o n w a s o b t a i n e d i n n u d e m i c e t h a t r e c e i v e d s p l e n o c y t e s at o r o n e a n d . t w o d a y s b e f o r e p a r a s i t e i n o c u l a t i o n ( p r e s e n t stu-d y ) . T h i s i n stu-d i c a t e s t h a t h e l p e r T-cells m a y p l a y an i m p o r t a n t p a r t i n t h e r e s i s t a n c e o i s p l e n i c r e c o n s t i t u t e d n u d e m i c e t o P . b e r g h e i . R E S U M O
Proteção de camundongos atímicos B A L B / c ( N u / N u ) contra P l a s m o d i u m berghei p o r
esplenócitos o r i u n d o s de c a m u n d o n g o s n o r m a i s B A L B / c ( N u / + ) C a m u n d o n g o s a t í m i c o s B A L B / c ( N u / N u ) s u c u m b e m e n t r e 7-13 d i a s a p ó s a i n o c u l a ç ã o ( D A I ) d a c e p a N K 6 5 d e P l a s m o d i u m b e r g h e i , T o d a v i a , seus s i n g e n ê i c o s h e t e r o z i g o t o s ( N u / + ) m o r r e m e m 7-8 D A I . C a m u n d o n g o s n u d e ( N u / N u ) r e c o n s t i t u í d o s c o m 2 x l 07 esple-n ó c i t o s d e c a m u esple-n d o esple-n g o s h e t e r o z i g o t o s siesple-nge- singe-n ê i c o s singe-n o r m a i s singe-n ã o i singe-n f e c t a d o s ( N u / + ) 20 d i a s a n t e s d a i n o c u l a ç ã o a ( D B I ) d o p a r a s i t a , su-c u m b e m 2 d i a s a n t e s q u e o s a n i m a i s su-c o n t r o l e s . C a m u n d o n g o s n u d e r e c o n s t i t u í d o s 10 o u 2 D B I , v i v e m 2-4 d i a s a m a i s q u e o s a n i m a i s c o n t r o l e s e a l g u n s d e l e s s o b r e v i v e m . E s s e s a c h a d o s i n d i c a m q u e a c e p a N K 6 5 d e P . ber-ghei i n d u z , n o m í n i m o , d o i s i m u n o f e n ô m e n o s d e p e n d e n t e s d e l i n f ó c i t o s T ; u m s u p r e s s i v o e o u t r o e s t i m u l a t ó r i o . A r e c o n s t i t u i ç ã o d e cam u n d o n g o s n u d e c o cam c é l u l a s T d e c a cam u n d o n g o s B A L B / c ( N u / + ) p a r e c e r e d u z i r o u " B y -p a s s " a a t i v i d a d e s u -p r e s s o r a d a s c é l u l a s T , o q u a l l e v a à f o r m a ç ã o d e u m a r e s p o s t a i m u n e p r o t e t o r a p o r a l g u n s d o s c a m u n d o n g o s n u d e . REFERENCES
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