Humoral and cellular aspects of immunity to Trypanosoma musculi in mice
by Bradford Oldham Brooks
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in MICROBIOLOGY
Montana State University
© Copyright by Bradford Oldham Brooks (1979)
Abstract:
Congenitally athymic (nude) mice were not capable of clearing Trypanosoma musculi parasitemia
while their normal, thymus-bearing littermates (NLM) cleared their parasitemia by day 20-24
post-infection (PI). Both nude and NLM mice exhibited an early nonspecific resistance to T. musculi
infection when compared to irradiated (550rads, 60Co), T. musculi-infected mice. Use of trypan blue,
in vivo, to alter the macrophage function of nude and NLM mice was found to abrogate early
nonspecific resistance to T. musculi. Nude mice that received immune or normal spleen cells, immune
or normal thymus cells, or thymus gland grafts were capable of eliminating T. musculi parasitemia.
Passive transfer of immune serum into T. musculi-infected nude mice lowered both the level of
parasitemia and the level of parasite reproduction, but did not generate elimination of the parasitemia.
T. muscui elimination was thus determined to be due to a thymus-dependent cellular mechanism and
not due to a direct antibody effect. Nude mice were shown to be a reliable in vivo model for the assay
of ablastic (parasite reproduction inhibition) activity in immune serum.
A T. musculi population enriched for dividing forms (35%-50% dividing forms) was found to absorb
ablastic activity from Immune serum, while absorption with a nondividing parasite population (<S%
dividing forms) did not appreciably alter the ablastic activity of immune serum. The absorption
experiments provide the crucial evidence needed to substantiate the antibody-nature of ablastin.
Plaque-forming cell (PFC) responses of NLM mice to T-dependent (sheep erythrocytes = SE) or
T-independent (polyvinylpyrrolidone = PVP) antigens were found to be significantly inhibited during
T. musculi parasitemia. PVP-PFC responses of T. musculi-infected nude mice were also significantly
inhibited. However, if NLM mice were primed with SE prior to parasitemia, subsequent development
of SE-specific memory cells and expression of secondary PFC responses to SE were not altered by T.
musculi infection. T. musculi parasitemia was not found to alter delayed hypersensitivity responses of
mice to l-fluoro-2,4-dinitrobenzene. The data imply that the Immune dysfunction of T. musculi
infected mice is at the B cell level and does not require T-cell participation for its development or
expression.
At least one host component requisite for T. musculi elimination was found to have a radiosensitivity
between 350rads and 550rads 60Co. Although 60Co irradiation was found to prevent the development
of acquired immunity to T. musculi, once developed, acquired immunity was found to be
radioresistant. NLM that received immune spleen cells 5 days post-irradiation did not exhibit early
control over parasitemia, but were repaired in their ability to eliminate T. musculi parasitemia.
x i NLM that received immune spleen cells on day 25 post-irradiation exhibited a marked resistance to
T. muscul? infection resulting in elimination of parasitemia 10 days earlier than unirradiated controls.
The adoptive transfer data suggest that the action of immune spleen cells in irradiated mice is not direct
(cytotoxic) but is dependent on collaboration with a minimally radiosensitive host component(s).
Experiments designed to explore the immune status of,postirradiation mice revealed that 30 days
following 550rads °Co the RFC responses of mice to SE were recovered completely whereas RFC
responses to PVR were <20%,of unirradiated controls. Furthermore, doses as small as lOOrads ®Co
were found to significantly inhibit the PVP-PFC responses of mice 30 days following irradiation. The
30-day post-irradiation PFC responses of mice to DNP-Ovalbumin, a T-dependent hapten-carrier
conjugate, were found to be >97% of unirradiated controls, while 30-day post-irradiation responses of
mice to DNP-Ficoll, a T-independent hapten carrier complex, were found to be only <39% of
unirradiated controls. In additional experiments, PFC responses of 30-day post-irradiation mice to
T-independent type 2 antigens (PVP and Type III pneumopolysaccharide= SSSIII) were inhibited
whereas, post-irradiation PFC responses to a T-independent type 1 antigen E_. col i
Lipopolysaccharide or T-dependent antigen (SE) were within normal control values.
Adoptive repair of mice that received 550rads °®Co, 7 days following irradiation with either bone
marrow, spleen, or thymus cells revealed that bone marrow cells completely repaired 30 day
postirradiation PFC responses to PVP whereas, spleen and thymus cell repaired mice had PVP-PFC
responses of 72.8% and 57-9% of normal controls respectively. Collectively these data suggest that B
cell populations can be differentiated by their ability to recover following °®Co radiation; B cell
populations responsive to T-dependent antigens (SE) and T-independent type 1 antigens (E_. col i
Lipopoly-saccharide) recover functionally whereas B cells responsive to T-independent type 2 antigens
(PVP and SSS III) do not recover functional ly. @
1979
BRADFORD OLDHAM BROOKS
ALL RIGHTS RESERVED
HUMORAL AND CELLULAR ASPECTS OF IMMUNITY
TO Trypanosoma mu scu li IN MICE
by
BRADFORD OLDHAM BROOKS
A t h e s i s s u b m i t t e d in p a r t i a l f u l f i l l m e n t
o f t h e r e q u i r e m e n t s f o r t h e degr ee
.
of
DOCTOR OF PHILOSOPHY
in
MICROBIOLOGY
A p p r ov ed :
Chairmafi,
Exami ni ng Comm i t tee,
Head, M a j o r Depar t ment
G r ad u at e Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
May,
1979
ACKNOWLEDGMENTS.
I wi sh t o exp ress my s i n c e r e a p p r e c i a t i o n
his a d v ic e ,
t h ro u g h o u t
Dr .
N. D.
fessional
Dr.
N. D.
Reed f o r
c o n s u l t a t i o n and e s p e c i a l l y f o r h i s empathy and gui d an ce
the d u ra tio n o f
R eed, D r .
this
J'.E-. C u t l e r ,
study.
I would a l s o
and Dr .
J.W.
examples t h ey have p r o v i d e d .
Jacques C h i l l e r
f o r supplying
used in p o r t i o n s o f t h i s
This
to Dr.
Ju tila
like
for
t o thank
t he f i n e pro
Thanks a r e a l s o ext ended t o
t h e DNP-Ovalbumin and DNP- Fi coI I
study.
r e s e a r c h was s u p p o rt e d by Uni ted. S t a t e s P u b l i c H e a l t h S e r ­
v i c e G r an t s No.
Al
12854 and CA 2 4443 .
TABLE OF CONTENTS
:/Y \
l
\
T
A
acknowledgments ;
.
.
.
.
r \ -'-.
.....................
. ....... .. . ; .......... ....... ........ .......................... .
LI ST OF T A B L E S . . . . . . . . .......................................................
LI ST OF F I G U R E S . . . . . . , . . . ; . . . ............................................................................ ..
ii
nr
vi
v iii
ABSTRACT. ................. ................ ................................... ......... ....................... ........................
x
INTRODUCTION........................ .........................................................' ............................... ..
I
MATERIALS AND M E T H O D S . . . . ........................:........................ ............. .. ....................
8
. .
A n i m a l s . " . .................................... ............................................... ....................... . .■.
P a r a s i t e s . ................... .............. .......................... ......; ....................... ; — .
E v a l u a t i o n o f P a r a s i t e m i a . .........................................................
Immune S e r a ......................................................
Thymus G l a n d - G r a f t i n g o f Nude M i c e . > ............ .................. ..
I r r a d i a t i o n o f M i c e ................................................................... . . . . . . . . .
A d o p t i v e T r a n s f e r s ........................ ..................... . . . ; .....................
T rypan B lu e T r e a t m e n t ...................................................................................
A n t i g e n s and i m m u n i z a t i o n s . ................... ......................... ..
J
P l aq u e A s s a y s . . . . . . . . . . . ............................................. ......................
C e l l u l a r Immunol ogi cal A s s a y s . . . ............ ; . , . . . . . . . . . . . . . . . . . .
S ta tis tic s ....
.................................... ..
RESULTS___ _. . . . ......... ......... ............................ ....................................... ..
Thymus•Dependency o f T . muscul i E l i m i n a t i o n ,
From M i c e . ..........; ................................... ...................................; .....................
E f f e c t o f T ry pa n B lu e on t h e E a r l y C o n t r o l o f T . muscul i
I n f e c t i o n in Nude and Normal M i c e . . . ...................
A d o p t i v e R e p a i r o f f . muscul i E l i m i n a t i o n P o t e n t i a l Ih
Nude M i c e : . . . ............ ....................................... .......................................... ..
E f f e c t o f P a s s i v e T r a n s f e r o f Immune Sera on T . muscul i
P a r a s i t e m i a in Nude and Normal M i c e . . . . ............ ..
/ A b s o r p t i o n o f A b l a s t i c A c t i v i t y from Immune Serum............ .. .
.
8
8
10
11
12
12
13 .1 k
14.
16 •
17
17
18
18 .
23
,28
34
42
V
Page
H e m a g g l u t i n a t i o n Assay f o r T_. muscul i S p e c i f i c
Serum A n t i b o d y ................. ................................................................................
E f f e c t o f T \ m u s c u l i P a r a s i t e m i a on D i r e c t and I n d i r e c t
RFC Responses t o SE and D i r e c t RFC Responses t o PVP
in M i c e ................................................................ .................................................
E f f e c t o f T \ m u s c u l i P a r a s i t e m i a on D i r e c t PFG Responses ,
to. PVP in Nude M i c e ................... ..................................................................
E f f e c t o f Ty m u s c u l ? P a r a s i t e m i a on D e l a y e d - T y p e H yp e r ­
s e n s i t i v i t y Responses In M i c e . ................... ............. ..........................
E f f e c t o f V a r y i n g Doses o f y - i r r a d i a t i o n oh T_. m u s c u l i
P a r a s i t e m i a in M i c e ......................................................................................
E f f e c t o f y - i r r a d i a t i o n on A c q u i r e d Immunity t o
Ty muscul I .................................................. ...................................................
E f f e c t o f y - i r r a d i a t i o n and S e l e c t i v e R e c o n s t i t u t i o n
on Subsequent Ty muscul I I n f e c t i o n in M i c e ............................. ..
P o s t - i r r a d i a t i o n PFC Responses o f M ic e t o SE and P V P . . . . . .
PVP-PFC Responses o f Mi ce 30 Days F o l l o w i n g V a r y i n g
Doses o f GOCo I r r a d i a t i o n ........................................................................
P o s t - i r r a d i a t i o n PFC Responses o f Mi ce t o DNP on a
T - d e p e n d e n t o r T - i nd ependent C a r r i e r ........................................... ..
P o s t - I r r a d i a t i o n PFC Responses o f Mi ce t o T - I n d e p e n d e n t
t y p e I o r T - i ndependent t y p e 2 A n t i g e n s . . . . ........................ ....
PVP' O p t i m a l - D a y T i t r a t i o n in 3 0 - d a y P o s t - i r r a d i a t i o n
M ic e and Normal M i c e ............ ..
................. .................................
PVP-PFC Responses in I r r a d i a t e d and S e l e c t i v e l y .
R e c o n s t i t u t e d , M i c e . ....................................... ......................................, . . . .
DISCUSSION. ................... ............................ ..............: . . . .................................................
LITERATURE C I T E D . .............................................................. ..........................
52
54
61
62
64
69
71
78
82
84
86
88
. 89
93
107
vi
LIST OF TABLES
Table
Page
I.
S E - s p e c i f i c PFC responses o f nude, NLM and Nu-TG m i c e . . . .
24
II.
A b s o r p t i o n o f a n t I - SE mouse a n t i s e r u m w i t h DP o r N D P . . . . .
,51
III.
I V.
V.
VI.
V II.
V III.
I X.
X.
XI.
X II.
X III.
U t i l i z a t i o n o f T . mu scul i a n t i g e n - c o a t e d mouse e r y t h r o ­
c y t e s in a p a s s i v e h e m a g g l u t i n a t i o n a s s a y .......... i . . . . . . . .
53
E f f e c t o f X - mu scul i p a r a s i t e m i a on d i r e c t PFC responses
o f mi ce t o PVP and S E . ............................... ............................................
56
E f f e c t o f T . mu scu li p a r a s i t e m i a on d i r e c t and i n d i r e c t
PFC responses o f mi ce t o SE IcCday 14 P I ) /2?(day 35 P l ) . .
58
E f f e c t o f X- mu scu lI p a r a s i t e m i a on d i r e c t and i n d i r e c t
PFC responses o f mi ce t o SE F ( d a y - 7 P l ) / Z ^ d a y | 4 P l ) . . .
,59
E f f e c t o f X- m u s c u l i , p a r a s i tern?a on d i r e c t and i n d i r e c t
PFC responses o f mi ce t o SE H d d y 0 ) / H d a y 21 P l ) . . . . . . .
60
E f f e c t o f T. m u s c u l i p a r a s i t e m i a on d i r e c t PFC responses
o f nude mi ce t o PVP.......... ............................ ........... ..........................: . .
63'
E f f e c t o f X - muscul i p a r a s i t e m i a on d e l a y e d - t y p e h yp er sen ­
s i t i v i t y t o l - f l u o r - 2 , 4 - d ! n i t r o b e n z e n e in m i c e . .......... ......
65
60
E f f e c t o f 5 5 0 r a d s Co i r r a d i a t i o n on t h e p r o t e c t i v e
immuni t y o f mi ce t o X - muscul i .........................................................
70
D i r e c t PFC responses o f mi ce t o PVP and SE 30 days
f o l l o w i n g v a r y i n g doses o f 6OC0 r a d i a t i o n . ............................
83
D i r e c t and i n d i r e c t PFC responses o f mi ce t o DNP on a
T - d e p e n d e n t o r T - i ndep en den t c a r r i e r 30 days f o l l o w i n g
. 550r ad s
-Co r a d i a t i o n . . . . . . ............ ........... ; . . ........... ...................
85
D i r e c t PFC responses o f mi ce t o S S S - 1 I l , I P S , PVP, o r
SE 30 days f o l l o w i n g
I r r a d i a t i o n . .................
87
Vii
Table
XIV.
XV.
Page
PVP o p t i m a l day t i t r a t i o n in 3 0- day, p o s t - i r r a d i a t ion
mi ce and normal m i c e . ..................................................................................
90
D i r e c t PVP-PFC responses o f a d o p t i v e l y . r e p a i r e d mice.
30 days f o l l o w i n g &0 co r a d i a t i o n ..............................................
92
,
:■
■
viii
LIST OF FIGURES
,.
Figure
1.
2.
3.
Page
][. mu scu ii p a r a s i t e m i a i n t h e bl oo d o f NLM, nude,
Nu- TG, and i r r a d i a t e d m i t e ( 5 5 0 r a d s ^ 0C o ) ....................V.............
20
P e r c e n t d i v i d i n g forms in JT. m u s c u i ? p a r a s i t e m i a . i n NLM,
nude, Nu-TG, and i r r a d i a t e d ( 5 5 0 r a d s ° ° C o) m i c e ......................
22
T.
muscuI i p a r a s i t e m i a in t h e p e r i p h e r a l bl ood o f NLM,
NLM/Trypan b l u e , Nude, Nu de /T r y p a n b l u e , and i r r a d i a t e d
( 5 5 0 r a d s ^ C o ) m i c e ............. .......................
27
4.
J\
5.
X* m u s c u i i p a r a s i t e m i a in NLM mi ce r e c e i v i n g immune thymus
c e l l s (NLM/1 T C ) , immune s p l e e n c e l l s (NLM/1 S C ) , o r no
c e l l s ( NLM)........... ........................
33
The e f f e c t o f Abl on X * m u s c u i i p a r a s i t e m i a in, nude and
N L M m i c e ------------ ------------------------ ---------------------------------------------------- . . . .
37
The e f f e c t of. Abl on t h e r e p r o d u c t i v e a c t i v i t y o f
X ' muscui i . in nude and NLM m i c e . ..............................................
39
. 6.
. 7-
muscuI i p a r a s i t e m i a in nude mi ce r e c e i v i n g immune
thymus c e l l s ( N U / I T C ) , normal thymus c e l l s (NU./NTC),
. immune s p l e e n c e l l s ( N U / I S C ) , normal s p l e e n c e l l s .
(NU/NSC) i o r no c e l l s (NUDE)................................................................. 3 ]
8.
The e f f e c t o f
parasitemia
9.
The e f f e c t o f Immune r ec o v e r e d serum on t h e r e p r o d u c t i v e
a c t i v i t y o f _T. muscui i in nude and NLM m i c e . . . . ......................
10.
11.
X-
immune r ec o v e r e d serum on X* m u s c u i ?
i n ’ nude and normal m i c e . , ; . . . . . ......................41
44.
m u s c u i i p a r a s i t e m i a in .nude mi ce m a i n t a i n e d on a regimen
o f A b l , D P - A b s - A b l , NDP-Abs-Abl NMS, o r no s e r u m . . . . ..........
47
P e r c e n t d i v i s i n g forms in X- muscui i p a r a s i t e m i a in nude
mi ce t h a t r e c e i v e d a . r e g i m e n o f A b I , D P - A b s - A b l , NDPAbs-ABL, NMS, o r no s e r u m . .......................■............ ................................
49
ix
Figure
12 .
13.
14.
15.
Page
J . muscul i p a r a s i t e m i a in normal mi ce t h a t r e c e i v e d .
. e i t h e r ' 7 5 0 r a d s , SgOrads, o r 350r ad s 2k hrs p r i o r
t o I . muscul? i n f e c t i o n . . ............ .............................................................
68
I . muscul i . paras i terni a in normal and ,.i r r a d i a t e d (SSOrads
° uQo) mi ce t h a t r e c e i v e d a d o p t i v e t r a n s f e r o f e i t h e r
ITC,. ISC, N I C , NSC, o r no c e l l s . . ........................ ......
73
T.
m u s c u l i p a r a s I t e m i a i n normal mi ce and in i r r a d i a t e d
( 550 rads ^Oq0 ) mi ce t h a t r e c e i v e d a d o p t i v e t r a n s f e r
o f e i t h e r I b d , NSC, !TC, NTC o r no c e l l s on day 25
p o s t - i r r a d i a t i o n . ..................................... .................. ..................... ; ...........
P o s t - i r r a d i a t i o n responses o f mi ce t o PVP o r SE on
v a r i o u s days f o l l o w i n g 550 rads ° ® C o . .......................... ..............
.
77
80
ABSTRACT
Cbngeni t a l l y a t h y m i c . (nude) mi ce wer e n ot c a p a b l e o f c l e a r i n g
Trypanosoma mu scul i p a r a s i t e m i a w h i l e t h e i r n o r ma l , t h ym u s - b e a r i n g
l i t t e r m a t e s (NLM) c l e a r e d t h e i r p a r a s i t e m i a by day 2 0 - 2 4 p o s t - i n f e c t ion
(P I).
Both nude and NLM mi ce e x h i b i t e d an e a r l y n o n s p e c i f i c r e s i s t a n c e
to. T . mu scul I i n f e c t i o n when c o m p a r e d . t o i r r a d i a t e d ( 5 5 0 r a d s , GOco),
T . m u s c u l i - in f e c t e d mice.
Use o f t r y p a n b l u e , in v i v o , t o a l t e r t he
macrophage f u n c t i o n o f nude and NLM mi ce was found t o a b r o g a t e e a r l y
n o n s p e c i f i c r e s i s t a n c e t o T_. muscul I .
Nude mi ce t h a t r e c e i v e d immune
o r normal s p l e e n c e l l s , immune o r normal thymus c e l l s , o f thymus g l an d
g r a f t s wer e c a p a b l e o f e l i m i n a t i n g T . m u s c u l ? p a r a s i t e m i a .
Passive
t r a n s f e r o f immune serum i n t o T . m u s c u l i - i n f e c t e d nude mi ce lowered
both t h e l e v e l o f p a r a s i t e m i a and t h e l e v e l o f p a r a s i t e r e p r o d u c t i o n ,
but d i d not g e n e r a t e e l i m i n a t i o n o f t h e p a r a s i t e m i a .
T \ muscuH_ e l i ­
m i n a t i o n was thus d e t e r m i n e d t o be due t o a t hymus- dependent c e l l u l a r
mechanism and not due t o a d i r e c t a n t i b o d y e f f e c t .
Nude mi ce were
shown t o be a r e l i a b l e in v i v o model f o r t h e assay o f a b l a s t i c ( p a r a ­
s i t e r e p r o d u c t i o n i n h i b i t i o n ) a c t i v i t y in Immune serum.
A T . m u s c u l I p o p u l a t i o n e n r i c h e d f o r d i v i d i n g forms {35%-$0%
d i v i d i n g for ms) was found t o absorb a b l a s t i c a c t i v i t y from immune
serum, w h i l e a b s o r p t i o n w i t h a n o n d i v i d i h g p a r a s i t e p o p u l a t i o n ( < 5%
d i v i d i n g for ms) d i d n o t a p p r e c i a b l y a l t e r t h e a b l a s t i c a c t i v i t y o f
immune serum.
The a b s o r p t i o n e x p e r i m e n t s p r o v i d e t h e c r u c i a l e v i d e n c e
needed t o s u b s t a n t i a t e t h e a n t i b o d y - n a t u r e o f a b l a s t i n .
P l a q u e - f o r m i n g c e l l (PFC) responses o f NLM mi ce t o T - de p e n d e n t .
(sheep e r y t h r o c y t e s = SE) o r T - I n d e p e n d e n t ( p o l y v i n y l p y r r o l Idonb = PVP)
a n t i g e n s were found t o be s i g n i f i c a n t l y i n h i b i t e d d u r i n g JT. musculi
■parasitemia.
PVP-PFC responses o f T \ m u s c u l I - I n f e c t e d nude mice were
also s ig n if ic a n t ly in h ib ite d .
However, i f NLM mi ce w e r e pri med w i t h SE
p r i o r to. p a r a s i t e m i a , subsequent devel opment o f S E - s p e c i f i c memory c e l l s
and e x p r e s s i o n o f secondar y PFC responses t o SE wer e n ot a l t e r e d by
T. musculi i n f e c t i o n .
T . m u s c u l I p a r a s i t e m i a was not found t o a l t e r
d e l a y e d h y p e r s e n s i t i v i t y responses o f mi ce t o l - f l u o r o - 2 , 4 - d i n i t r o b e n ­
zene.
The d a t a i mpl y t h a t t h e immune d y s f u n c t i o n o f T_, muscul i i n f e c ­
t e d m i ce is a t t he B c e l l l e v e l and does not r e q u i r e T - c e l I p a r t i c i p a ­
t i o n f o r i t s devel opment o r e x p r e s s i o n .
A t l e a s t orie h o st component r e q u i s i t e f o r T \ muscul I e l i m i n a t i o n
was found t o have a r a d i o s e n s i t i v i t y between 3.50rads and SSOfads G^Co.
A l t h o u g h &0Co i r r a d i a t i o n was found t o p r e v e n t t h e devel op men t o f ac­
q u i r e d immunity t o T\ m u s c u l i , once d e v e l o p e d , a c q u i r e d immunity was
found t o be r a d i o r e s i s t a n t .
NLM t h a t r e c e i v e d immune s p l e e n c e l l s 5
days p o s t - I r r a d i a t ion d i d n ot e x h i b i t e a r l y c o n t r o l o v e r p a r a s i t e m i a ,
b ut wer e r e p a i r e d in t h e i r a b i l i t y t o e l i m i n a t e T F . mu scu li p a r a s i t e m i a .
xi
NLM t h a t r e c e i v e d immune s p l e e n c e l l s on day 25 p o s t - i r r a d i a t i o n
e x h i b i t e d a. marked r e s i s t a n c e t o 7 \ m u s c u i ? i n f e c t i o n r e s u l t i n g
in e l i m i n a t i o n o f p a r a s i tern I a 10 days e a r l i e r than u n i r r a d i a t e d
controls.
The a d o p t i v e t r a n s f e r d a t a suggest t h a t t h e a c t i o n o f
immune s p l e e n c e l l s in i r r a d i a t e d mi ce is not d i r e c t ( c y t o t o x i c )
but is dependent on c o l l a b o r a t i o n w i t h a m i n i m a l l y r a d i o s e n s i t i v e
h o st component( s ) . .
E x pe ri m en t s desi gned t o e x p l o r e t h e immune s t a t u s o f , p o s t ­
i r r a d i a t i o n mi ce r e v e a l e d t h a t 30 days f o l l o w i n g 550r ads
0Co t h e
RFC responses o f mi ce t o SE were r ec o v e r e d c o m p l e t e l y whereas RFC
responses t o PVP wer e <20%, o f u n i r r a d i a t e d c o n t r o l s .
Furthermore,
doses as smal l as I OOrads
®Co were found t o s i g n i f i c a n t l y i n h i b i t
t h e PVP-PFC responses o f mi ce 30 days f o l l o w i n g i r r a d i a t i o n .
The
3 0 - d a y p o s t - i r r a d i a t i o n PFC responses o f mi ce t o DNP-Ovalbumin,
a T - d e p e n d e n t h a p t e n - c a r r i e r c o n j u g a t e , wer e found t o be >97% o f
u n i r r a d i a t e d c o n t r o l s , w h i l e . 30- d a y p o s t - i r r a d i a t i o n responses o f
mi ce t o DNP-Fi c o l l , a T - i n d ependent h apt en c a r r i e r compl ex, were
found t o be o n l y < 39% o f u n i r r a d i a t e d c o n t r o l s .
In a d d i t i o n a l
e x p e r i m e n t s , PFC responses o f 3Q-day p o s t - i r r a d i a t i o n mi ce t o T i n d ependeht t y p e 2 a n t i g e n s (PVP and Type I I I p neumopol ysacchar ide=
SSSI I I ) we re i n h i b i t e d w h e r e a s , p o s t - i r r a d i a t i o n PFC responses t o
a T - i nd ep en den t t y p e I a n t i g e n . El . c o I i L i p o p o l y s a c c h a r i d e o r
T - d e p e n d e n t a n t i g e n (SE) wer e w i t h i n normal c o n t r o l v a l u e s .
A d o p t i v e r e p a i r o f mi ce t h a t r e c e i v e d SSOrads °®Co, 7 days f o l l o w i n g
i r r a d i a t i o n w i t h e i t h e r bone marrow, s p l e e n , o r thymus c e l l s r e ­
v e a l e d t h a t bone marrow c e l l s c o m p l e t e l y r e p a i r e d 30 day p o s t i r r a d i a t i o n PFC responses t o PVP w h e r e a s , s p l ee n and thymus c e l l .
r e p a i r e d mi ce had. PVP-PFC responses o f 72.8% and 57-9% o f n o rm a l,
controls re sp ective ly.
C o l l e c t i v e l y t h e s e d a t a suggest t h a t B c e l l ,
p o p u l a t i o n s can be d i f f e r e n t i a t e d by t h e i r a b i l i t y t o r e c o v e r f o l ­
lowi ng °®Co r a d i a t i o n ; B c e l l p o p u l a t i o n s r e s p o n s i v e t o T - d e p e n d e n t
a n t i g e n s (SE) and T - i nd ep en den t t y p e I a n t i g e n s (E_. col i L i p o p o l y s a c c h a r i d e ) r e c o v e r f u n c t i o n a l I y whereas B c e l l s r e s p o n s i v e t o , Ti n d ependent t y p e 2 a n t i g e n s (PVP and SSSI I I ) do not r e c o v e r f u n c ­
tio n a l ly.
INTRODUCTION
P a r a s i t i c diseases a f f e c t
latio n ,
represent a s ig n if ic a n t
t o w o r l d devel opment
(21).
up t o one q u a r t e r o f t h e w o r l d ' s popu­
human burden and a r e a m a j o r o b s t a c l e
D e s p i t e r e c e n t concern d i r e c t e d
d e v e l o p i n g n a t i o n s and c u r r e n t a t t i t u d e s o f n a t i o n a l
p a r a s i t i c diseases
factious
has y e t
disease
toward t he
interdependence,
represent a d is p a r a g in g ly neglected area o f
(64).
,.The c u r r e n t e x p l o s i o n o f b i o m e d i c a l
( 21 ) .
to b e n e f i t p a r a s i t i c disease c o ntrol
in-
knowledge
In f a c t ,
in some
a r e a s c e r t a i n p a r a s i t i c d i s e a s e s a r e r e c r u d e s c e n t due t o t h e d e v e l o p ­
ment o f d r u g - r e s i s l a n t s t r a i n s o f p a r a s i t e s and i n s e c t i c i d e - r e s i s ­
tan t vectors
(21).
The e x c r u c i a t i n g l y
and c o n t r o l 1 o f p a r a s i t i c d i s e a s e s
most o f
slow p r o g re ss
is e s p e c i a l l y
the causative .ag en ts are r e l a t i v e l y
most cases a r e
incapable o f
i nd ep en den t
in t h e t r e a t m e n t
f r u s t r a t i n g , in t h a t
s i m p l e organi sms t h a t
life
and a r e
restricted
.
in
to
h i g h l y s p e c i a l i z e d e n v ir on men ts p r o v i d e d by two o r more s u c c e s s i v e
h ost s
(21,64)..
o f t h e h o st
It
is e n t i r e l y
p o s s i b l e t h a t o n l y when t h e mechanisms
immune r esponse t o p a r a s i t e s a r e d e t e r m i n e d and pl aced
into perspective with
the biology of the p a ra s ite
o f p a r a s i t i c d i s e a s e s may become a r e a l i t y
In r e c o g n i t i o n o f t h e g l o b a l
tropical
in itia l
schistosomiasis,
targets of
economic and med ical
intensive
trypanosomiasis ,
th a t control
( 1.7 ) .
d i s e a s e t h e Worl d H e a l t h O r g a n i z a t i o n
d i s e a s e s as
its e lf,
?
i mp ro tan ce o f
(WHO) has s e l e c t e d s i x
research e f f o r t s :
f i Ia ria s is,
le prosy,
and
m alaria,
leishmani-
2
as is
(21j .
some 10 m i l l i o n
affects
(21).
Trypanosomiasis,
The A f r i c a n
people,
in t h e WHO S p e c i a l ' P r og ram m e, . ■
8 m illio n
form o f t h e d i s e a s e ,
mated t o be endemic o y e r
o f whi ch
included
is f e r t i l e ,
in t h e w e s t e r n . hemi sphere.
sleeping sickness,
is e s t i ­
12 m i l l i o n s q u ar e k i l o m e t e r s o f l a n d , most
but abandoned t o t h e ravages o f t h e d i s e a s e
The South Ameri can form o f
the disease,
Chaga1s d i s e a s e ,
( 21 ) .
is u s u a l l y
and may p e r s i s t f o r 20 y e a r s o r more ( 21 ) .
fatal
Current
knowledge o f mechanisms' of. Immunity t o trypanosomes
remains f r a g m e n t a r y and p o o r l y u nd er st oo d
( 60 ) .
whi ch have been s t u d i e d most e x t e n s i v e l y ,
conclusions h a v e .f r e q u e n t ly
been c o n t r a d i c t o r y
of
individual
variation,
(17,2.1,60).
The number and c o m p l e x i t y o f a n t i g e n s
t ry p a n o s o me s, t h e i r a b 11 i t y
and t h e
In t h ose models
l a c k o f a deq uat e
t o undergo c y c l i c a n t i g e n i c
in v i t r o
correlates
but ed t o t h e p a u c i t y o f knowledge concerning, t h e host
responses t o trypanosomes
clin ical
ani mal
(69).
(60).
immunol ogi cal
C o n s t r a i n t s on human, and v e t e r i n a r y
immunoparasi t o l o g y have e l i c i t e d
h o s t - p a r a s i t e systems f o r
have c o n t r i ­
an emphasis on l a b o r a t o r y
immune response c h a r a c t e r i z a t i o n s .
'
..
.
.
The use o f . t h e s e ani mal models al on g w i t h
r e c e n t advances
In
immunology has p r o v i d e d an approach t o u nd er st a n d t he complex mecha­
nisms o f a n t i trypanosome r e s i s t a n c e . . .
The a v a i l a b i l i t y o f
Trypanosoma muscul i-rmouse model, p r o v i d e s a t o o l
for
the
the extension of
.
knowledge o f
' from mi ce
parasite
immune mechanisms
(44).
It
extrapolate
results
in a d is p r o p o r t io n a I
s t u d i e s on a r t i f i c i a l
to n a t u ra l
situations
p o s s i b i l i t y o f applying
in
immunology t o t h e
addition,
tia l
increase
t h e h o st
( 11) .
immune response
(69).
The mouse model o f f e r s
immunity t o t rypansomes.
i n b r ed mouse s t r a i n s
available
to
t he
In
is a . n o n p at h bg e n i t
( 72 ) .
It
is c l o s e l y
( 69 ) .
stercorarian
related
i n d i s t i n g u i s h a b l e from Trypanosoma l e w i s ? ,
r o d e n t trypanosomes
Nosopsyllus f a s c ia t u s
infection
m a in t e n a n c e
( 27)
in n a t u r e .
a l s o been pr o po sed ,
(72).
(i.p .)
t he t y p e - s p e c i e s
L e p t o p s y l l a seg ni s
( 2 7)
that
and
transmit
T r a n s m i s s i o n by way o f c an n a b a l i s m has
b u t n ot a d e q u a t e l y
( 69 ) ,
trypanosome
t o and morphol o­
are the arthropod vectors
is a c h i e v e d by d i r e c t
p e r i tone'al I y
prim arily
in v i r u l e n c e
systems o f t e n p r o ve d i f f i c u l t
investigation of
i n f e c t i v e t o mi ce o n l y
this
host-
t h e most c u r r e n t and i n n o v a t i v e t e c h n i q u e s
Trypanosoma muscu.l I
o f smal l
is a n a tu ra l
t h e s e t e c h n i q u e s can be g r e a t l y enhanced by t h e s u b st a n ­
number o f
g ically
this
Passage o f trypanosomes t hr ough an abnormal
( 35 ) whi ch can s i g n i f i c a n t l y a f f e c t
Furthermore,
in trypanosome e l i m i n a t i o n
s houl d be emphasized t h a t
relationship.
h o st f r e q u e n t l y
involved
investigated
injection of
Parasites d ivid e
( 69 ) •
Laboratory
i n f e c t e d bl ood
in t he p e r i p h e r a l
by m u l t i p l e f i s s i o n o f e p im a st i go t .e s
( 12) ,
intrablood
b ut t h e most
a c t i v e s i t e s o f m u l t i p l e f i s s i o n o f e p i m a s t i g o t e s a r e t h e vasa r e c t a
o f the kidney
( 69 ) and t h e p l a c e n t a
(45).
These p r o t o z o a n h e m o f l a - .
g e l l a t e s produce a s e l f - 1 I m i t i n g p a r a s i t e m i a which
by a s e r i e s o f d i s t i n c t phases.
post-inoculation
(72).
gical
i s . f o l l o w e d , by a phase o f
The l e v e l
between days 8 t o
forms o f
A p r e p a t e n t , p e r i o d o f 3 t o 5 days
.
'
in whi ch d i v i d i n g forms a r e p r e s e n t
parasitemia
bl ood
(Pl)
1st CRISIS
10 Pl w i t h
the r e t i c u lo e n d o t h e lia l
n e t w o r k and ( 2 )
reproduct i o n - i n h i b i t i n g
serum f a c t o r f i r s t
in 1938.
blood.
least
This
is an immunolo­
(72).
two d i s t i n c t
The
t ypes
A b l a s t i n - a thymus- dependent
d e s c r i b e d by T a l i a f e r r o
sp ecifically
IgG]
(23).
c h a ra c te r is tic s of
However,
in t h e T^.
Iew isi-
r a t m o d e l , a b l a s t i n can not be absorbed from serum usi ng homologous
trypanosomes
( 1 8 ) . . I n v e s t i g a t i o n o f t h e a b s o r b a b i l i t y o f a b l a s t i n in
t h e T_. muscul i - m6 Use m o d e l . has p o t been r e p o r t e d . ' The i.nabi I i t y t o . •
absor b o u t a b l a s t i n a c t i v i t y
from serum usi ng homologous trypanosomes
has r a i s e d q u e s t i o n s as t o t h e a n t i b o d y n a t u r e o f t h i s
(18).
where
Reviews o f a b l a s t i n and i t s
(8,18,23,5 6).
.
trypanosomes t o c l e a r a n c e by
A b l a s t i n has t h e p h y s i co c h e m i c al
an. i mmu no gl ob ul in ,
'
( I ) T r y p a n o c i d a l A n t i b o d y - a t h ym u s - i n d e p e n ­
dent antibody t h a t p u rp o rte d ly s e n s i t i z e s
( 66 )
■
in t h e p e r i p h e r a l
t o as t h e " 1 s t C R IS I S"
is t h e o r e t i c a l l y m e d i at e d by a t
(72):
increasing
t h e concommitant c l e a r i n g o f d i v i s i o n a l
t h a t has. bgen r e f e r r e d
of. serum f a c t o r s
rapidly
o f p a r a s i t e m i a becomes s t a b i l i z e d , , u s u a l l y
t h e p a r a s i t e from t h e p e r i p h e r a l
event
is c h a r a c t e r i z e d
K en d a l l
(44),
serum f a c t o r
properties are a v a ila b le e ls e ­
in h i s o r i g i n a l
description of
.
5
'X* m u s c u l i , noted t h e
lack o f d e t e c t a b le a g g l u t i n a t i n g antibody
responses t o T_. muscul i .
Subsequent
in v e s t ig a t io n s , o f the e xisten ce
o f an a g g l u t i n a t i n g a n t i b o d y response t o _T. m u s c u l i have produced
conflicting
results
(12,69).
Precipitating
a n t i g e n s and c e l l u l a r a n t i g e n s o f
reported
mu scu li
antibodies
have been r e c e n t l y
(24).
By days
14 t o . 16 Pl
a "2nd C R IS I S" o c cu rs whi ch r e s u l t s
e l i m i n a t i o n o f t h e p a r a s i t e from t h e p e r i p h e r a l
24 P I .
t o bot h exo­
in the
bl ood by days 20 t o
A l t h o u g h t h e 1st CRISIS, is a n t i b o d y me d i at e d
(72),
t he
mechanism r e s p o n s i b l e f o r t h e 2nd CRISIS does n ot depend on a d i r e c t
(trypanocidal)
antibody e f f e c t ,
c e l l u l a r mechanism ( 7 1 ) .
the recent
t o be dependent o n . a
The immunity t h a t f o l l o w s
i n f e c t i o n was t ho ug ht by most
However,
but appear s
investigators
report of m u lt ip lic a t iv e
t h e vasa r e c t a o f ki d n ey s o f
r e c o v e r y from
( 69 ) .
t o be a b s o l u t e
in fe c tiv e parasites
12 m o n t h - r e c o v e r e d mice whi ch were
immune to homologous c h a l l e n g e has r a i s e d
i n t e r e s t i n g q u e s t i o n s about
t h e n a t u r e and m a in t e n a n c e o f t h e r e s i s t a n c e t o r e i n f e c t i o n
by t h e se mi ce
tic a lly
is g e n e r a l l y c o n s i d e r e d n on pat ho gen i c ;
IxlO-Ym l.
This
low l e v e l
i n c r e a s e d by i r r a d i a t i o n
tomy ( 3 8 ) ,
shown
( 70 ) .
X* mu scul i
seldom exceed
in
pregnancy
(45),
(37)»
para sitem ias
o f p a r a s i t e m i a can be drama­
RES b lo c k ad e
and T c e l l d e p r i v a t i o n
(38),
( 68 ) .
splenec­
SublethaI
6
doses o f b a c t e r i a ]
hance I .
muscul i
( 23 ) o r c o r t i c o s t e r o i d s
endotoxin
infection.
Furthermore,
t h e a t t a c h m e n t o f TV m u s c u l i a n t i g e n s
of
i n f e c t e d mi ce r e s u l t s
sequent anemia
(39)r
In
usi ng
transfer of
immunity
t hymect omi zed mi ce
ponse r e q u i s i t e
musculI
( 69)
r e s i s t a n c e t o T. m u s c u l i has been r e p o r t e d
or
immune serum ( 6 9 ) •
in a d u l t -
immune r e s ­
i r r a d i a t e d mi ce a g a i n s t T.
i n f e c t i o n have n o t been i n v e s t i g a t e d .
musculI
l ead t o a s u p p r e s s i o n o f h o s t
( 2)
o r mi t og e n s
i n f e c t e d mi ce have been r e p o r t e d
sheep e r y t h r o c y t e s
(2).
immune
S p l een c e l l s
1 1 p op o l y sa cc h ar i d e
from
t o be h y p o r e s p o n s i ve t o
( 2 ) and t o t h e mi t og en s p h y t o h e m a g g l u t i n i n ,
ca n a va l in A, and E_. col i
from
in normal mi ce t han
t o p r o t e c t nude mi ce o r
responses t o v a r i o u s a n t i g e n s
maximal
Adoptive or passive
Those components o f t h e h o st
Trypanosome i n f e c t i o n s
T.
t h e nonpat ho-
and o t h e r r o d e n t t ryp a n o so me s.
is more e f f i c i e n t
( 68 ) .
that ;
to the surface o f ery th ro c y te s
I t may be a p p r o p r i a t e t o q u e s t i o n
t o mice o f
Immune c e l l s
r e c e n t work, i n d i c a t e s
i n c r e a s e d e r y t h f o p h a g o c y t o s i s and sub­
g e n i c c l a s s i f i c a t i o n o f T. mu scu li
Transfer
( 56 ) a l s o en­
( 2) .
con-
S u pp r e ss io n was
d u r i n g peak p a r a s i t e m i a and waned as t h e donors r ec o v er e d
in fectio n
(2).
Recent work i m p l i e s
t h a t s o l u b l e subst ances
d e r i v e d from T^ m u s c u l ? a c t d i r e c t l y on B- Iymp ho cyt es
activating
s u p p r e s s o r T c e l l s o r macrophages
TV muscuI i
induced s u p p r e s s i o n on a n t i g e n p r i m i n g ,
r a t h e r t han by
( 3 ) • . The e f f e c t o f
d evel opment and
f u n c t i o n o f memory c e l l s
and ori secondar y
immune responses t o
v a r i o u s a n t i g e n s has n ot been i n v e s t i g a t e d .
In o r d e r t o f u r t h e r d e f i n e t h o se mechanisms r e s p o n s i b l e f o r t he
control
and e l i m i n a t i o n o f Ti. mu sou I i, in m i c e ,
t o c h a r a c t e r i z e t h e host
response t o T .
t h e i r phenotypical Iy normal,
47+),
it
m u s c u l ? in nude mi ce
thymus-bearing
litterm ates
in s e l e c t i v e
(nu/nu),
( NLM: . n u / + o r
i r r a d i a t e d mi ce. ( ^ C o ) , . and t r y p a n b l u e - t r e a t e d mi Ce.
models wer e a l s o u t i l i z e d
These,
r e c o n s t i t u t i o n experiments
d esi gn ed t o d e t e r m i n e t h o s e c e l l u l a r and humoral
h o st
has been my approach
immune r esponse t h a t a r e r e q u i s i t e f o r
components o f t h e
the control
and e l i m i n a ­
t i o n o f %. muscul? p a r a s i t e m i a .
In a d d i t i o n
studies
d irectly
t o t ho se s t u d i e s d e s c r i b e d above,
suggest ed by r e s u l t s o b t a i n e d f r o m ,
to,
background
t he J .
m u s c u l { - mouse model
immunol ogi cal
but not p e r t a i n i n g
have been p r e s e n t e d .
l i t e r a t u r e p e r t i n e n t to these studies w i l l
The
be p r e s e n t e d
in ah a b b r e v i a t e d form in t h e r e s u l t s and d i s c u s s i o n s e c t i o n s o f t h i s
thesis.
These d a t a wer e
and cogency t o t h e f i e l d
I n c l u d e d due t o t h e i r p r o v a c a t i ve n a t u r e
of
immunology.
MATERIALS AND METHODS
An imal s
BALB/c mi ce o f both sexes r a i s e d
from Cumberland View Farms
Congenitally
(C linton,
,
in .our l a b o r a t o r y o r purchased
TN) wer e Used f o r most e xp e r i m e n t s
t h y m u s - d e f i c i e n t mi ce (nu/n.U) , h e r e a f t e r d e s i g n a t e d nude,
and t h e i r n o r m a l , t h y m u s - b e a r i n g
litterm ates
(NLM:
nu/+ or + / + ) ,
here­
a f t e r d e s i g n a t e d NLM, w e re produced f rom h e t e r o z y g o u s s t o c k o b t a i n e d
from a l i n e b ei ng made co n ge n i c w i t h BALB/c mice by c r o s s - i n t e r c r o s s
mating.
Nude and NLM mi ce used in t h e s e s t u d i e s were d e r i v e d from
h e t e r oz yg o us p a r e n t s o f t h e 12 t h . a n d
All
mi ce wer e 6 t o
l|th
10 weeks old, a t
generation.
the beginning o f experiments.
Groups o f mi ce w e re c a r e f u l l y age and sex matched w i t h i n each i n d i v i dual
experiment.,
■
Al I mi ce wer e m a i n t a i n e d on Wayne L a b - B l o x
C h ic ag o ,
Co.,
IL)
Paxton,
and a c i d i f i e d . w a t e r .
(A llied M ills;
Inc.
S a n i c e l I b e d d i n g ( P a x t o n P r ocessi ng
(L) was r o u t i n e l y s t e r i l i z e d
b e f o r e use.
Parasites
The P a r t i h i c o
C ollection,
R ockville;
sites o r ig in a lly
Culture.
I I s t r a i n o f T . muscul i
(American Type C u l t u r e
MD) was used t h r o u g h o u t t h ese s t u d i e s .
r e c e i v e d wer e t r a n s p o r t e d
C u ltu re supernatant f l u i d
passaged t w i c e t hr o u g h s u b l e t h a I I y
Par a- ;
in a D i p h a s i c Blood Agar
and c e l l s were removed and ..sub­
irrad iated
( SSOrads, ^ Co) m i ce.
.
On day 8 Pl
o f t h e second subpassage, mi ce wer e b l e d
A l seven's c i t r a t e v ia
the r e t r o - o r b i t a l plexus.
c e n t r i f u g e d a t 450g a t 4 C, f o r
and.the p e l l e t
resuspended
10 m i n u t e s ,
t h e ends o f whi ch w e re f l a m e s e a l e d ,
dry
ice bath.
until
Used.
T\. muscul i
I n f e c t e d bl ood was
the supernate discarded
1: 3 in A l s e v e r ' s g l y c e r o l
g l y c e r o l i zed bl ood was t hen t r a n s f e r r e d
into c h ille d
into
(51).
Infected
I ml o r 5 ml ampoules,
and q u i c k - f r o z e n
in a m e t h a n o l -
s t a b i l a t e m a t e r i a l was m a i n t a i n e d a t -70 C
Al I c r y o p r e s e r v a t i o n p ro ce d u r es were c a r r i e d o u t under
aseptic conditions.
u
To produce a s t a n d a r d
o f T.
Inoculum o f 3x10^ T\ muscu11, an ampoule
m u s c u l i s t a b i l a t e was a l lowed t o thaw a t
le th a l Iy
i r r a d i a t e d mi ce r e c e i v e d 0 .2 ml o f
injection...
room t e m p e r a t u r e .
thawed s t a b i l a t e v i a
On days 6 t o 7 P I t h e s e mi ce wer e b l ed
sever's c i t r a t e .
Subi.p.
into c h ille d A l-
7 \ muscul I ' i n f e c t e d bl ood was washed t h r e e times, in.
chilled
p hosphat e b u f f e r e d s a l i n e suppl emented w i t h 2%. normal mouse
serum.
The c o n c e n t r a t i o n o f trypanosomes
mined u si ng a N e u b a u e r r r u l e d c o u n t i n g
luting
buffer
(73)•
3x10^ trypanosomes/ ml
chamber and a f o r m a l i z e d d i ­
The suspensi on was a d j u s t e d
o f buffered s a lin e .
standard
inoculum o f 3x10^ T . m u s c u l i
via
injection.
i.p .
In t h e p r e p a r a t i o n was d e t e r ­
to a c o n cen tratio n o f
Mice were i n f e c t e d w i t h , a
in 0 . 1 ml o f b u f f e r e d s a l i n e
I n j e c t i o n s wer e p er f or med usi ng a I ml d i s p o -
s a b l e s y r i n g e and a 23 ga n e e d l e .
.
10
To produce a p r e p a r a t i o n o f %. muscul i
s u i t a b l e f o r use in t he
absorbtion o f vario us T\ m u s c u l!-s p e c i f i c a n t i s e r a ,
subpassaged
days
12 t o
saline
i n t o sub l e t h a l I y
14 P I ,
( 7 4) «
( 5 5 0 r a d s , ^®Co)
i n f e c t e d mi ce wer e b l e d
s t a b ! I a t e s were
I r r a d i a t e d mice.
into c h ille d
On
heparinized
trypanosomes wer e s e p a r a t e d from t h e bl ood components
by d i f f e r e n t i a l
centrifugation
f rom t h e bl ood o f
(25.) •
P a r a s i t e populations obtained
i r r a d i a t e d mi ce c o n t a i n e d 315% t o 50% d i v i d i n g forms
Evaluation of Parasitem ia
Each m o u s e . I n each e x p e r i m e n t a l
v a r i o u s days a f t e r T .
group was b l e d from t h e t a i l
muscul i ' . i n f e c t i o n .
on
A s i n g l e drop o f b l o o d Wds
used t o make a t h i n - b l o o d smear u si ng a b e v e l - e d g e d m i c ro s c o p e s l i d e
(VWR S c i e n t i f i c ) .
f i x e d , in methanol
stained
T h i n - b l o o d smears wer e a l l o w e d t o a i r
f o r f i v e minutes.
F i x e d smears w e r e . t h e n Giemsa-
f o r 20 mi n u t es u si ng a 10% Giemsa s o l u t i o n
Giemsa s o l u t i o n / 2 0 0 ml d i s t i l l e d
d i s t i l l e d w a t e r and b l o t t e d d r y .
d ry and were
HgO).
(20 ml t i f s t o c k
S l i d e s wer e t hen dipped
in .
S l i d e s wer e e v a l u a t e d on a Z e i s s
l i g h t mi cr os co pe usi ng t h e 40x o b j e c t i v e .
T h i r t y , h i g h power f i e l d s
wer e examined f o r each m o u s e . f o r each day t he p a r a s i t e m i a was moni­
tored.
L e v el s o f p a r a s i t e m i a f o r each mouse wer e r eco rd ed as t he
number o f organisms p e r h i g h power f i e l d
Parasite
( 0 / . H PF ) .
r e p r o d u c t i o n was a l s o m o n i t o r e d t h r o u g h o u t t h e d u r a t i o n
of parasitem ia.
The. trypanosome p o p u l a t i o n was c l a s s i f i e d
i n t o two
g roups:
sites
I)
Nondi vi . di ng p a r a s i t e s
(parasites
( T r y p b m a s t i g o t e s ) ; 2)
l ess t han 25 ym in l e n g t h ,
tigotes with m u ltip le o rg a n e lle s ).
percent d iv id in g
forms
Dividing para­
epimastigotes,
o r trypomas-
R e p r o d u c t i v e a c t i v i t y was r e p o r t e d
(%DF= no. o f d i v i d i n g trypansomes counted
t o t a l no. o f trypanosomes counted
.
P a r a s i t e m i a s wer e m o n i t o r e d e v e r y 2 t o 5 days f o r t h e . d u r a t i o n o f t he
infection.
Immune Sera
To o b t a i n v a r i o u s s p e c i f i c
T.
-
m u scu li-infected, T .m u s c u l!-re c o v e re d ,
.
-------------
'
the r e t r o o r b i t a l
ture fo r
I hr,
—
vated a t
;---------:-----
plexus.
A ll
■
to c l o t a t
Unl ess o t h e r w i s e s t a t e d *
t ypes o f T\
In t h e se s t u d i e s :
all
s e r a we re i n a c t i ­
Three
AbV- llA b l a s t i c 11 a n t i s e r u m o b t a i n e d on day 18
;
'
r ec o v e r e d serum o b t a i n e d on day 28 Pl
T. musculI
i.p .;
an i n i t i a l
I n j e c t i o n o f 3x10
i .p .;
'
f rom mice t h a t
(2)
I RS-
L'
T.
m u s c u l i and two a d d i t i o n a l
Ia.'
injection of parasites.
Immune
.
r e c e i v e d 3x l 04
H I S- Hyperimmune serum from mi ce t h a t
o f 3x 1O^ %. m u s c u l I • , a t one week i n t e r v a l s ,
in itia l
12 h r s , a n d . c e n t r i f u g e d
storage at -7 0 ° C ..
from mi ce t h a t had r e c e i v e d 3x10^ T_. muscul i
from t h e
room tempera­
m u s c u l i - s p e c i f i c a n t i s e r a were p r e p a r e d and used
(I)
( 3)
sera,
o r normal mi ce wer e bl ed v i a
blood was l e f t
56°C f t i r 30 m i n u t e s b e f o r e f i n a l
d ifferen t
control
.
s t o r e d a t 4 ° C f o r an a d d i t i o n a l
f o r serum c o l l e c t i o n .
Pl
immune s e r a o r normal
received
injections
subsequent t o r ec o v e r y
12
When a b s o r p t i o n o f a n t i s e r a was n e c e s s a r y ,
I x l O ^ trypanosomes was
4°C f o r 30 m i n u t e s .
a concentration of
added p e r ml o f a n t i s e r a ,
and i n cu b at e d a t
A n t i s e r a were r ec o v e r e d f o l l o w i n g
centrifugation
and absorbed two more t i m e s .
in p a s s i v e t r a n s f e r e x p e r i m e n t s u t i l i z i n g
(nude and NLM m i ce)
r e c e i v e d 3x10
I . muscul i
antisera,
i . p.
at
recipients .
the. i n i t i a t i o n
o f t h e e x p e r i m e n t and wer e m a i n t a i n e d on an a n t i s e r u m r e g i m e n , o f
0.25 ml o f a p p r o p r i a t e a n t i s e r u m i n t r a v e n o u s l y ( i . v . )
f o r the d u ra tio n o f the experiment.
normal mouse serum in p l a c e o f
As c o n t r o l s ,
e v e r y f i v e days
some mi ce r e c e i v e d
immune serum.
Thymus G l a n d - G r a f t i n g o f Nude Mice
Nude mi ce 4 t o 6 weeks, o l d w e re a n e s t h e s i zed w i t h sodium p e n t a barbitol
(Abbott L a b o r a t o r i e s ) ,
g r a f t e d w i t h thymus g l a nd s f r o m , 4-
d a y - o l d . B A L B / c mi ce u si ng t h e t e c h n i q u e e s t a b l i s h e d by Dukor e t a l .
( 22 ) ,
and a l l o w e d t o c o n v a l e s c e a t
l e a s t 42 days b e f o r e t h e i r use in
experiments.
To assess ..thymic f u n c t i o n
gland-grafted
nude (Nu-TG)
hemolysis-in-gel
erythrocytes
assay
( 53 )
r e p r e s e n t a t i v e NLM,
and nude mi ce were assayed by t h e
5 days a f t e r
thymus
localized
i mmu ni zat i on w i t h sheep
(SE).
I r r a d i a t i o n o f Mi ce
Mi ce r e c e i v e d v a r y i n g doses o f y - i r r a d i a t i o n
in c e r t a i n e x p e r t -
13
merits d e s c r i b e d
was u t i l i z e d
A P i c k e r M- MbO w i t h a ^ 0 Co. source
in t h e s e s t u d i e s .
in t h e s e e x p e r i m e n t s .
s o u rce was 7 0 r a d s / m i n u t e .
ments were used t o
A p pr o x i m a t e d e l i v e r y
P l a s t i c holders w ith
r a t e o f t he
individual
compart ­
i m m o b i l i z e mi ce d u r i n g exposure t o t h e ^ C o source
S ki n t o s o u rce d i s t a n c e was 70 cm.
Adoptive T ra n s fe rs
Single c e ll
of J .
cell
suspensi ons were p r e p a r e d from t h e s p l e e n o r thymus
m u s c u l { - r ec o v e r e d o r normal mi ce ( 7 4 ) .
In a d d i t i o n ,
suspensi ons o f bone marrow wer e p r e p a r e d from normal mi ce ( 74 ) .
Al I c e l l s w e re washed once
in c h i l l e d
phosphat e b u f f e r e d s a l i n e ,
suspended t o t h e a p p r o p r i a t e c o n c e n t r a t i o n , and i n j e c t e d
total
single
volume o f 0 .5 ml
i.v .
t o 0.75 ml dependi ng on t he n a t u r e o f
re­
in a
the c e l l
preparation.
C e r t a in experiments
r e q u i r e d thymus c e l l - r e c o n s t i t u t e d
nude mice
D i s p e r s e d thymus c e l l s w e re o b t a i n e d from thymus g l an ds o f 5- d a y - o l d
BALB /c donors by f o r c i n g
scr eens
t h e g l an ds
(Ambac I n d u s t r i e s ,
In c.,
t hr ough 80 mesh s t a i n l e s s
D e tro it,
Ml)
in c h i l l e d
b u f f e r e d s a l i n e c o n t a i n i n g 2% normal mouse serum.
a t e d and assayed f o r v i a b i l i t y
steel
phosphate
C e l l s w e re enumer­
by a t r y p a n b l u e e x c l u s i o n t e s t
( 10) ;
O
subsequently,
1x 10° v i a b l e thymus c e l l s w e r e i n j e c t e d i . v .
r e c i p i e n t nude mouse.
ThymUs c e l l - r e c o n s t i t u t e d
nude mi ce
i n t o each
(Nu-TC)
in e x p e r i m e n t s 21 days a f t e r c e l l
wer e used
Recipients o f c e lls
prepared
transfer.
in t h i s manner wer e nude m i c e , NLM,
o r NLM whi ch had r e c e i v e d sub l e t h a l
I r r a d i a t ion
( 5 5 0 r a d s , ^®Co)
in some e x p e r i m e n t s , mi ce r e c e i v e d 3x 10^ T \
previously.
t he t i m e o f c e l l
2k hrs
muscul i
at
transfer.
T rypan Bl ue T r e a t m e n t
I n e x p e r i m e n t s d es ig n ed t o d e t e r m i n e t h e r o l e o f macrophages
the e a r ly
control
was u t i l i z e d
m i ce .
and f i n a l
elim ination of J .
t o a l t e r macrophage f u n c t i o n
Al I mi ce r e c e i v e d an I n i t i a l
I.p .
blue
in s a l i n e
24 hr s b e f o r e T. m u s c u l i
blue
in s a l i n e
i.p .
regimen o f
(30-34,46)
try pan blue
o f NLM and nude,
i n j e c t i o n o f 4 . 0 mg o f t r y p a n
i n f e c t i o n and 1 . 0 mg o f t r y p a n
3 hrs b e f o r e T. m u s c u l i
3x 10** %. m u s c u l I i . p .
musculi,
in
infection.
A fter
receiving
t r y p a n b l u e - t r e a t e d mice were m a i n t a i n e d on a
1.0 mg t r y p a n b l u e in s a l i n e , s u b c u t an e o u sl y ( s . c . ) , e v er y
72 hrs f o r t h e d u r a t i o n o f t h e e x p e r i m e n t .
-
A n t i g e n s and I mmu ni zat i on s
Sheep e r y t h r o c y t e s
(SE)
and burrow e r y t h r o c y t e s
f rom t h e C ol o r ad o Serum C o . , D e n v e r , CO.
( I0)
(BE) wer e o b t a i n e d
Mi ce wer e g i v e n a p r i m a r y
i n j e c t i o n o f 0.25 ml o f a 10% suspensi on o f washed e r y t h r o c y t e s
(SE o r BE) v i a
t he
lateral
wer e r e q u i r e d ,
a sim ilar
ta il
vein.
When secondary
(2°)
Injections
i n j e c t i o n o f e r y t h r o c y t e s was g i v e n 21 days
15
follow ing
1°
the
injection.
P olyvinylpyrrolidone
New Y o r k ,
NY)
(PVP)
used f o r
i mmu ni zat i on
(GAP C o r p o r a t i o n ,
had an a v e r a g e m o l e c u l a r w e i g h t o f 3 6 0 , 0 0 0
PVP K90 by m a n u f a c t u r e r ) .
PVP K90 was d i s s o l v e d
s a lin e at a concentration o f
I yg / m l
(designated
in pho sp hat e b u f f e r e d
and 0.25 U9 was
injected
in 0.25
' ml .
Type I I I
pneumococcal
by D r . P h i l l i p J . B a ke r ,
polysaccharide
Laboratory o f M icro b ial
I n s t i t u t e s o f A l l e r g y arid I n f e c t i o u s
t hose e x p e r i m e n t s
to SSS-I I I ,
(S S S -IIl)
re q u irin g I the
Diseases,
in duction o f
I mmuni ty, N a t i o n a l
NI H,
-
t ..p.
'
s u b s e q u e n t l y c h a l l e n g e d w i t h 0 . 5 yg S S S - 111
B e t h e s d a , MD.
low dose t o l e r a n c e
iriice wer e pri med w i t h 0 . 0 1 yg S S S - 1 11
<
was k i n d l y donated
after
the
S S S -IIl
s p e c i f i c antibody
last SSS-III
Lipopoiysaccharide
Dr. J.A.
i.p .
In s a l i n e and
in s a l i n e .
In o t h e r
I.p .
in
responses w e r e . d e t e r m i n e d 5 days
In jectio n .
(LPS)
e x t r a c t e d f ro m IE. col l was suppl led by
Rudbach, D epar t ment o f M i c r o b i o l o g y ,
M i s s o u l a , MT.
( 7)
•
e x p e r i m e n t s mi ce r e c e i v e d o n l y a P dose o f 0 . 5 yg o f S S S - I I I
saline.
In
Mice wer e g i v e n
U n i v e r s i t y o f Montana,
1 . 0 yg L P S . i n s a l i n e
i.v .
and t h e . m a g n i ­
t ude o f t h e L P S - s p e c i f i c a n t i b o d y r esponse was d e t e r m i n e d f i v e dayS
following
i m m u n iz at i on u s i n g t h e s l i d e m o d i f i c a t i o n o f t h e J e r n e
plaque technique.
Both 2 , l f - d i n i t r d p h e n o l - o v a . l b u m i n (DNP-Ova)
and 2 , 4 - d i n i t r o p h e n o l -
16
Ficoll
(DNP-Ficoll)
wer e g e n e r o u s l y p r o v i d e d by Dr.
D epar t ment o f M e d i c i n e ,
D en v e r,
CO.
National
1° dose o f DNP-Fi col I
( Gl bco)
i.p .
in 0 . 2 ml o f s a l i n e
i m mu n i za t i o n w i t h DNP-Ova o r f i v e days a f t e r
Ficol I ,
and Research C e n t e r ,
Mi ce r e c e i v e d a 1° dose o f 400 yg o f DNP-Ova in 0 . 2 ml
o f Compl ete F r e u n d ' s A d j u v a n t
yg
Jewi sh H o s p i t a l
Jacques C h i l l e r ,
O t h e r mi ce r e c e i v e d a 5
i.p .
E i g h t days a f t e r
i m m u ni z a t i on w i t h DNP-
PFC responses were d e t e r m i n e d u si ng T N P - I i n k e d SE ( 4 1 ) .
P l a q u e Assays
Plaque forming c e l l s
(PFC)
s p e c i f i c f o r BE, SE, PVP, S S S - I I I ,
LPS,
o r TNP wer e d e t e c t e d by a s l i d e m o d i f i c a t i o n o f t h e l o c a l i z e d hemoly­
sis-in-gel
technique
(53).
Routinely,
t h e magni t ude o f t h e PFC r e ­
sponse t o each a n t i g e n was d e t e r m i n e d f i v e days f o l l o w i n g
i m m u ni z at i on w i t h
t h e e x c e p t i o n o f DNP-Ova.
ponses wer e d e t e r m i n e d e i g h t days f o l l o w i n g
the
last
DNP-Ova a n t i b o d y r e s ­
immunization.
The p r o c e d u r e f o r c o a t i n g S E. w i t h PVP has been d e s c r i b e d e l s e ­
where ( 4 8 ) .
To d e t e c t
t h e m ag ni t ud e o f .PFC responses a f t e r
t i o n w i t h DNP-Ova o r D N P - F i c o l l ,
(Eastman Kodak) was l i n k e d
(40).
Mostly
mostly d i r e c t
indirect
(I gM)
2 , 4 , 6- t r i n i t r o b e n z e n e s u l f o n i c a c i d
t o SE usi ng
( I gG )
immuniza­
t he R i t t e n b e r g - P l a t t
technique
p laques w e re o b t a i n e d w i t h DNP-Ova w h i l e
pl aq ues wer e o b t a i n e d w i t h DNP - Fi co l I usi ng t h i s
technique.
In t h o se p l a qu e e x p e r i m e n t s t h a t
required
the d e t e c t io n o f
in-
17
d i r e c t plaque
( I gG )
used t o f a c i l i t a t e
form ation,
r a b b i t a n t i - m o u s e immunogl obul i n was
i n d i r e c t plaque formatio n
(48).
Counts o f
pl aq ues we re r ecor ded as t h e d i f f e r e n c e between t h e t o t a l
indirect
number o f
p la q u e s and t h e number o f d i r e c t p l a q u e s .
C ellu lar
Cell-mediated
Pi)
Immunol ogi cal Assay
Immune competence o f T_. muscul i - 1n f e c t e d
(day 14
mi ce was d e t e r m i n e d by measur i ng d e l a y e d - t y p e h y p e r s e n s i t i v i t y
responses t o
l-flu o ro -2,4-d in itro b en zen e.
Individual
responses, o f
i n f e c t e d and u n i n f e c t e d mi ce wer e e v a l u a t e d usi ng an e a r s w e l l i n g
assay d e s c r i b e d by Moorehead e t a l .
(58).
was made w i t h a S c h n e l l t p s t e r m i c r o m e t e r
Germany)
and was r e p o r t e d as p e r c e n t
Measurement o f e a r s w e l l i n g
( H. C.
K r o e p l i n GMBH, Hessen,
I n c r e a s e o v e r a p p r o p r i a t e con­
( 58 ) .
tro ls
S tatistics
Results o f experiments
in which RFC responses, wer e d e t e r m i n e d ,
wer e p r e s e n t e d as t h e a r i t h m e t i c mean o f P FC /s pl ee n o r PFC/ 10^ spl een
cells.
cent
R e s u l t s o f e a r s w e l l i n g assays wer e p r e s e n t e d as t h e mean p e r ­
i n c r e a s e , o v e r a p p r o p r i a t e normal
differences
only
if
controls.
In bot h
instances,
between a r i t h m e t i c means were j udged t o be s i g n i f i c a n t
p v a l u e s were < . 0 5 as assessed by t he S t u d e n t ' s
t test.
RESULTS
Thymus
dependency o f TT. muscul i e l i m i n a t i o n
from mice
As a p r e l i m i n a r y s t e p
volved
in %. m u s c u l ? e l i m i n a t i o n from m i c e ,
competence in t h e
or. i r r a d i a t e d
o f 3x10
Il
in­
t h e i mp o rt a n c e o f thymus
immune r esponse t o T . m u s c u l i was d e t e r m i n e d .
Groups o f nude m i c e ,
Pl
in t h e e l u c i d a t i o n o f t h e mechanisms
thymus g l a n d - g r a f t e d nude mice
( 5 5 0 r a d s , ^ C o ) . NLM mi ce wer e
T\ r o u s c u I i •
( N u - T G ) , NLM m i ce ,
i n f e c t e d by i . p .
injection
E v a l u a t i o n o f 0/HPF and %DF was begun on day 4
and r e p o r t e d as an a r i t h m e t i c mean f o r each t r e a t m e n t group f o r
each day and p a r a s i t e m i a was m o n i t o r e d .
t h a t by day 4 Pl
The r e s u l t s
I)
show
a l l . m i c e demo nst rat ed d e t e c t a b l e p a r a s i t e m i a s , w i t h a
r em a r k a b l y h i g h p a r a s i t e m i a b ei ng obser ved
d i a t e d mi ce wer e dead by day 10 P I ,
350 0/ HPF .
(Figure
As e a r l y as day 10 Pl
between t h y m u s - b e a r i n g
Nu-TG mi ce c l e a r e d
in
exhibiting
i r r a d i a t e d mice.
Irra ­
p a r a s i t e m i a as hi gh as
t h e r e was a d i s c e r n i b l e d i f f e r e n c e
(NLM and Nu-TG) mi ce and nude m i c e .
NLM and
t h e p a r a s i t e m i a from t h e blood by day 24 P I .
,In
marked c o n t r a s t , nude mice n o t o n l y m a i n t a i n e d a h i g h e r . p a r a s i t e m i a ,
b ut a l s o s u s t a i n e d t h i s
h i g h e r p a r a s i t e m i a f o r as long as day 85 P I ,
the extend o f experim ental
observation.
ser ved t h r o u g h o u t nude i n f e c t i o n s
mice,
D i v i d i n g p a r a s i t e s were ob­
( F i g u r e 2) w h i l e
%DF was m a r k e d l y reduced by day 16 Pl
in NLM and Nu-TG
(Figure 2 ).
In o r d e r t o assess t h e g e n e r a t i o n o f thymus f u n c t i o n
in Nu-TG
19
Figure
I.
T.
muscui i
parasitemia
i n t he bl ood o f NLM, nude, Nu-TG,
and i r r a d i a t e d m i ce ( 5 5 0 r a d s ,
6n
Co).
Each v a l u e r e p r e s e n t s
t h e a r i t h m e t i c mean o f two e x pe ri m en t s w i t h no f e w e r than
s i x mi ce i n any e x p e r i m e n t a l gro up .
ORGANISMS PER HIGH POWER FIELD
Irradiated
Nude
Nu-TG
4
6
8
K) 12 14 16 18 20 22 24 40
50
DAYS POST-INOCULATION
21
F i g u r e 2.
Percent d i v i d i n g
forms
in T . m u s c u i ? p a r a s i t e m i a
nude, Nu-TG, and i r r a d i a t e d
in NLM,
( 5 5 0 r a d s , ^0Co) m i c e .
Each
v a l u e r e p r e s e n t s t h e a r i t h m e t i c mean o f two e x p e r i m e n t s
w i t h no f e w e r t han s i x mi ce in any e x p e r i m e n t a l
-
"
'
...
' '
'
gro up .
\
% DIVIDING FORMS
r IRRADIATED
NUDE
DAYS POST-INFECTION
23
mice,
r e p r e s e n t a t i v e NLM, Nu-TG, and nude mi ce were assayed f o r t h e i r
a b ility
(SE).
t o respond t o t h e t hymus- dependent a n t i g e n ,
F i v e days f o l l o w i n g
sheep e r y t h r o c y t e s '
i m m u n iz at i on w i t h SE, s p l e e n s were assayed
f o r S E - s p e c i f i c PFC u si ng t h e l o c a l ! z e d - h e m o l y s i s - i n - g e l
The PFC responses
(Table
I)
assay
(53).
o f nude and NLM mice wer e 3^5 P F C /s p l ee n
and 2 5 , 5 6 2 / s p l e e n r e s p e c t i v e l y .
Thymus g l a n d - g r a f t e d
sponses w i t h
These o b s e r v a t i o n s e s t a b l i s h e d
16,000 PFC/spleen.
thymus f u n c t i o n was g e n e r a t e d
nude mi ce r e ­
that
in Nu-TG m i ce .
These d a t a s u p p o r t t h e concept t h a t
t he c l e a r a n c e o f T \ musculi
p a r a s i t e m i a from t h e bl ood o f mi ce i s a t hymus- dependent e v e n t .
A l­
though nude mi ce wer e not c a p a b l e o f e l i m i n a t i n g T_. muscul i p a r a s i t e ­
mia,
t h e y w e re c a p a b l e o f e x e r t i n g some c o n t r o l
t e m i a when compared t o
irradiated
(550rads,
over th e e a r l y p a r a s i ­
^Co)
m i ce .
E f f e c t o f T rypan Bl ue on t h e E a r l y c o n t r o l o f T \
I n f e c t i o n i n . N u d e and Normal Mi ce
muscul i
Because nude mi ce a r e known t o e x h i b i t a p a r a d o x i c a l l y h igh l e v e l
o f a c t i v a t e d macrophage a c t i v i t y
and d e m o n s t r at e an e a r l y non­
s p e c ific control
o f T\
was
th a t e a rly nonspecific control
investigated
may be due, a t
least
musculi
( 13)
in p a r t ,
infection
(Figure
I),
the p o s s i b i l i t y
o f T_. muscul i
in mice
t o macrophages.
t h e use o f t r y p a n b l u e t o a l t e r macrophage f u n c t i o n
nude mi ce and NLM mi ce a l l o w s an e v a l u a t i o n o f t he
(30-34,46)
of
i mp or t an ce o f macro-
24
T ab le.!.
"
SE-speci f i c
RFC response o f nude, NLM and Nu-TG m i c e 3
D i r e c t SE-PFC/Sp.leeh
1 . Group.
Nude
345
25,562
NLM
Nu-TG
b
- 16, 00 0
3 M ic e wer e immunized w i t h 0 . 2 5 ml o f a 10% suspensi on o f SE i . v .
Mice, wer e a s s a y e d . f o r . SE-PFC f i v e days a f t e r . i m m u n i z a t i o n .
Results
a r e ex p r es s ed as t he number o f d i r e c t ( I gM) P F C / S p l ee n .
b Nu-TG m i t e were
immunized 10 weeks a f t e r
thymus g r a f t i n g .
25
phages
in t h e e a r l y n o n s p e c i f i c c o n t r o l
of I .
Groups o f nude mi ce and NLM mi ce wer e
muscul i
injected
o f trypan blue
in s a l i n e 24 hrs b e f o r e T. m u s c u l i
of
i.p.
trypan blue
as w e l l
as
w i t h 4 . 0 mg
i n f e c t i o n and 1 . 0 mg
i r r a d i a t e d mice
and a p p r o p r i a t e c o n t r o l s wer e i n f e c t e d by i . p .
These
(550rads, ^Co)
i n j e c t i o n o f 3x10^
T r yp a n b l u e - t r e a t e d mi ce w e r e m a i n t a i n e d on a regimen o f
1 . 0 mg o f t r y p a n b l u e s . c .
ment .
i.p .
in s a l i n e 3 hrs b e f o r e T. m u s c u l ? i n f e c t i o n .
trypan b l u e - t r e a t e d mice,
%. m u s c u l I .
parasitemia.
e v e r y 72 hr s f o r
t h e d u r a t i o n o f t he e x p e r i ­
E v a l u a t i o n o f 0/ HPF f o r each mouse in each group was begun on
day 4 Pl
and d a t a wer e r eco rd ed as group means ( a r i t h m e t i c )
f o r each
day t h e p a r a s i t e m i a was m o n i t o r e d .
By day 4 Pl
The r e s u l t s
all
mi ce wer e found t o have d e t e c t a b l e p a r a s i t e m i a s .
( F i g u r e 3)
show t h a t as e a r l y as day 6 Pl
treated
nude mi ce
sitemia
t h a t was s t r i k i n g l y
obser ved
in
sim ilar
e x h i b i t e d a h igh
to the high le v e l
i r r a d i a t e d mi ce ( F i g u r e 3 ) •
t ed nude mi ce
temia,
( N ud e/ Tr yp an b l u e )
(Nude), w h ile m aintaining
e v e n t u a l l y d e v el op ed hi gh
in t h e i r d e a t h by day 30 P I .
trypan bluelevel
o f para­
o f parasitemia
U n t r e a t e d , _T. muscul i - i n f e c an e a r l y c o n t r o l
over p a r a s i­
le v e ls o f p a rasitem ia t h a t culminated
T rypan b l u e - t r e a t e d ,
u n i n f e c t e d nude
mi ce wer e obser ved t o l i v e up t o 45 days w h i l e m a i n t a i n e d on t h e t r y ­
pan b l u e regimen d e s c r i b e d .
Furthermore,
by day 8 Pl
t h e r e was a
marked d i f f e r e n c e between t r y p a n b l u e - t r e a t e d NLM mi ce (NLM/ Trypan blue)
26
A
;
■■■
•
■
f::-
F i g u r e 3.
T . mu s c u I i
parasitemia
NLM/Trypan b l u e ,
(550rads,
..
'
in the p e rip h e ra l
Nude, N ud e/ Tr yp an b l u e ,
^®Co) m i c e.
-
:'
bl ood o f NLM,
and
irradiated
Each v a l u e r e p r e s e n t s
the a r i t h ­
m e t i c mean o f two e x p e r i m e n t s w i t h no f e w e r t han s i x mi ce
in each e x p e r i m e n t a l g r o u p .
27
--------• ---------NLMZTRYf=AN BLUE
------- e --------- NUOE/TRYPAN BLUE
------- o --------- NLM
PER
HIGH-POWER FIELD
(O/HPF)
------- o --------- NUDE
----- A ------- ^ Z N L M ______
DAYS POST-INOCULATION
28.
and t h e i r
untreated,
infected controls
e x h i b i t e d a l oss o f e a r l y c o n t r o l
( NLM) .
c h a r a c t e r i z e d by an a c u t e r i s e
l e v e l s o f p a r a s i t e m i a by day 8 P I . • However,
an e q u a l l y a c u t e d e c r e a s e
in t h e f i n a l
NLM/Trypan b l u e mice
in
by d a y . 12 PI . t h e r e was
in p a r a s i t e m i a f o l l o w e d by a 10 day d e l a y
clearance of parasites
from t h e p e r i p h e r a l
bl ood o f
NLM/Trypan b l u e m i ce .
These d a t a s u p p o r t t h e h y p o t h e s i s t h a t
control
part,
o f T \ muscu11 p a r a s i t e m i a
the e a r l y n o n s p e c ific
in mi ce may be due, a t
least
to. macr ophages. . T r e a t m e n t b f both nude and NLM mice, w i t h
pan b l u e was found t o a b l a t e t h e e a r l y c o n t r o l
sitem ia.
over T\
muscul I
in
try-
.
para­
T r yp an b l u e t r e a t m e n t d i d not p r e v e n t t h e devel opment o f
acquired
immunity, and t h e subsequent d e c r e a s e
in p a r a s i t e m i a
in NLM/
T r y p a n b l u e m i ce . '
. . Adoptive Repair o f T . musculi E lim in a t io n
P o t e n t i a l In Nude Mice
W i t h t h e t hymus-dependency o f T . m u s c u l i e l i m i n a t i o n from mice
firm ly established,
a d o p t i v e t r a n s f e r e x p e r i m e n t s wer e d esi gn ed t o
identify
populations capable o f re p a ir in g
ten tial
t ho se c e l l
In nude m i c e .
transfers
Previous
in normal mi ce
(Jl)
investigations
and In a d u l t
e l i m i n a t i o n po-
in volving adoptive
thymectomized mi ce (68) were
c o m p l i c a t e d by t h e r e c i p i e n t ' s own thymus competence making
p r e t a t io n o f the r e s u lts d i f f i c u l t .
-
The aval l a b i l i t y
inter­
o f nude mice
29
a l l o w e d a more c r i t i c a l
evaluation of
the
influence o f various c e ll
p o p u l a t i o n s on J .
mu scu li
parasitemia.
Groups o f I .
muscul!
I n f e c t e d nude and NLM mi ce wer e
either
immune ( see m a t e r i a l s
8
. (.1x10 ,
i . v . ) at
the time o f
nude mi ce r e c e i v e d
time o f
at
infection.
the time o f
The r e s u l t s
and methods)
o r normal
i n f e c t i o n .. . O t h e r
immune o r normal
spleen c e l l s
muscul ? i n f e c t e d
thymus c e l l s
( 1x10®,
NLM c o n t r o l s were a l s o g i v e n
infection.
injected with
i.v .)
immune thymus c e l l s
E v a l u a t i o n o f 0/HPF was begun on day 4 P I .
( F i g u r e s 4 and 5 ) show t h a t a l l
cell
populations
In vesti­
g a t e d wer e a b l e t o g e n e r a t e T. m u s c u l i e l i m i n a t i o n p o t e n t i a l
m i ce .
the
Nude mi ce t h a t
in itia l
r e c e i v e d no c e l l s
parasitem ia,
s it e m ia t h a t culminated
tia l
In t h e i r d e at h by day 13 P I .
either
t han nude mi ce t h a t
crease
9 PL
in
population
20 days, e a r l I e r
Immune thymus c e l l s o r normal
received
in c r e a s in g para­
Immune spl een
investigated.
r e c e i ve d . i mm u n e s p l e e n c e l l s wer e a b l e t o e l i m i n a t e
p a r a s i t e m i a by day 20 P I ,
over
in g e n e r a t i n g Ti. m u s c u l i e l i m i n a t i o n p o te n ­
in nude mice t han a n y . o t h e r c e l l
mi ce t h a t
in nude
exhibited e a rly control
b ut e v e n t u a l I y d e v e l o p e d . a n
c e l l s were more e f f i c i e n t
a t the
r e c e i v e d normal
immune s p l e e n c e l l s
t he
t han nude mi ce r e c e i v i n g
thymus c e l l s ,
spleen c e l l s .
( F i g u r e 5)
Nude
and 15 days e a r l i e r
NLM c o n t r o l s
that
a l s o showed a d r a m a t i c de­
l e v e l s o f par as I tern I a w i t h e l i m i n a t i o n o f Ti. muscul I by day
30
.■
§
. ■
•
I
■
v ir
ti-j
F i g u r e 4.
T.
muscul i p a r a s i t e m i a
in nude m i ce r e c e i v i n g
thymus c e l l s
( N U / I T C ) , normal
thymus c e l l s
(NU/NTC),
spleen c e l l s
( N U / I S C ) , normal
spleen c e l l s
( N U/ NS C) , o r no
cells
(NUDE) .
' experim ental
immune
Each v a l u e r e p r e s e n t s t h e a r i t h m e t i c mean
o f two e x p e r i m e n t s w i t h no f e w e r than s i x mi ce
•
immune
g r ou p.
in any
.
'
V
56
DAYS POST-INFECTION
32
F i g u r e 5-
T_* muscui i p a r a s i t e m i a
thymus c e l l s
o r no c e l l s
mean o f
(NLM/ITC),
( NLM) .
i n NLM mice r e c e i v i n g
immune s p l e e n c e l l s
Each v a l u e r e p r e s e n t s
immune
( N LM / I SC ) ,
the a rith m e tic
two e x p e r i m e n t s w i t h no f e w e r than s i x mice in
any e x p e r i m e n t a l
g r ou p.
ORGANISMS/HIGH POWER FIELD
NLM
30
DAYS P O S T -IN F E C T IO N
40
34
These d a t a c l e a r l y
i n d i c a t e t h a t nude mi ce a r e
m i n a t i n g T \ rnuscul?, but t h a t a d o p t i v e t r a n s f e r o f
populations o f c e lls
i n capable o f e l i ­
immune o r normal
o b t a i n e d f rom t h e s p l e e n o r thymus o f NLM mice
a re capable o f ge n e ra tin g
rnusculi
elim ination
potential
in nude,
m i ce.
E f f e c t o f P a s s i v e T r a n s f e r o f Immune Sera on
T \ rnusculi p a r a s i t e m i a i n Nude and NLM mi ce
It
has been suggested t h a t
rnusculi
parasitemia
the
in itia l
is a n t i b o d y m e di a t ed
t o as t h e 1st C R I S I S ,
(72).
dependent a b l a s t i c serum f a c t o r
referred
(67-72).
ant ibody, and a thymus-
P r e v i o u s r e p o r t s have i n ­
18 days a f t e r T .
rnusculi
infection.
h e r e a f t e r be d e s i g n a t e d a b l a s t i c a n t i s e r u m
indicate
tected
This ev e n t,
t h a t bot h a b l a s t i n and t r y p a n o c i d a l , a n t i b o d y a r e found
serum o f mi ce
w ill
o f Ty
is t ho u gh t t o be t h e r e s u l t o f t h e c o o p e r a t i v e ,
a c t i o n o f a t h ym u s - i n d e p e n d e n t t r y p a n o c i d a l
dicated
immune c o n t r o l
t h a t by day 28 Pl
in mouse serum ( 6 9 ) .
designated
only the tryp an o cid al
Day 18 Pl
(A bl).
serum
Other reports
a n t i b o d y can be de­
Day 28 Pl mouse serum w i l l
h e r e a f t e r be
immune r ec o v e r e d serum ( I R S ) .
In o r d e r t o d e f i n e t h o se mechanisms r e s p o n s i b l e f o r
immune c o n t r o l
o f T_. rnuscul i by t h e h o s t ,
w e re d esi gn ed t o exami ne t h e e f f e c t s
parasitemia
in t h e
of
the
in itia l
p a s s i v e t r a n s f e r e x pe ri m en t s
immune s e r a on Ty rnusculi .
i n nude mi ce and t h e i r NLM c o n t r o l s .
35
Groups o f JT. mu s c u l i
0 . 2 5 ml o f e i t h e r Abl o r
i n f e c t e d nude mi ce and NLM c o n t r o l s
I RS ( i . v . )
a t the time o f
i n f e c t e d w i t h normal mouse serum (NMS)
infection.
s er ved as c o n t r o l s .
( F i g u r e 6)
t h e e x t en d o f e x p e r i m e n t a l
show t h a t as e a r l y as day 8 Pl
between nude .mice t h a t
n o t r e c e i v e Abl
noticeable
r e c e i v e d Abl
(Nude, Nude/ NMS).
in %. m u s c u l i
and nude mi ce t h a t d i d
Abl
ance was d e l a y e d by 10 d ays.
.
The e f f e c t o f Abl was e s p e c i a l l y
infection.
both 0/ HPF and %DF
In s p i t e o f t h i s cbnr
in p a r a s i t e m i a and r e p r o d u c t i v e a c t i v i t y ,
i n f e c t i o n s o f NLM m i ce.
on %DF in NLM m i ce;
The r e s u l t s
t h e r e was a d i f f e r e n c e
I n f e c t i o n s o f nude mi ce;
mi ce wer e dead by day 26 P I ,
muscul I
(i.v .)
observation.
(Nude/Abl)
w e re m a r k e d l y reduced t h ro u g h o u t t h e
spi cu ou s r e d u c t i o n
Mice
M ic e were
m a i nt a i n e d , on a regimen o f 0.25 ml o f a p p r o p r i a t e a n t i s e r u m
e v e r y f i v e days f o r
received
a l s o produced a l t e r a t i o n
Nude/Abl.
o f T.
0/HPF was reduced and o v e r a l l
F u r t h e r m o r e , Abl
clear­
had a d r a m a t i c e f f e c t
r e p r o d u c t i v e a c t i v i t y was not d e t e c t e d a f t e r day
8 Pl . ( F i g u r e 7 ) .
Nude mice t h a t
r e c e i v e d an I RS regimen a l s o showed a r e s t r i c t e d
p a r a s i t e m i a as e a r l y as day 8 Pl
duction
(Figure 8 ),
in s p i t e o f
in p a r a s i t e m i a , Nude/ I RS were dead by day 20 P I .
r e c e i v e d ah I RS regimen e x h i b i t e d a d ec re as e d
accompanied by a 10 day d e l a y
pheral
but
blood.
level
this
re­
NLM mice t h a t
of parasitemia
in T . m u s c u l i . c l e a r a n c e from t h e p e r i ­
In. marked c o n t r a s t t o Nude/ Abl
and NLM/Abl mi ce
(Figure
36
I
i'
-
Figure 6
The e f f e c t o f Abl on %. mu scu li
NLM m i c e .
Each v a l u e r e p r e s e n t s
parasitemia
t h e a r i t h m e t i c mean o f
two e x p e r i m e n t s w i t h no f e w e r than s i x mice
e x p e r i m e n t a l g r ou p.
i n nude and
in each
ORGANISMS/HIGH POWER FIELD
NUDEi
NLM
NLM /NM S
N U D E /A b !
N L M /A b i
DAYS POST-INFECTION
,
38
'
:
V1
F i g u r e 7«
'
4
■.
-
.
-
:
'
:
The e f f e c t , o f . Abl on t h e r e p r o d u c t i v e a c t i v i t y o f J_.
mu scu Ii
in nude and NLM m i c e .
Each v a l u e r e p r e s e n t s
t he a r i t h m e t i c mean o f two e x p e r i m e n t s w i t h no f e w e r
t han s i x mi ce
in each e x p e r i m e n t a l
■ "
gr oup.
.
'
.
x
% DIVIDING FORMS
NUDE/NM S
NUDE
NUDE/Ab!
3
12
16
20
24
DAYS POST-INFECTION
28
32
36
40
F i g u r e 8.
The e f f e c t o f
immune r e c o v e r e d serum on T. muscul I
p a ra s I tern I a in nude and NLM m i c e .
Each v a l u e r e p r e s e n t s
t h e a r i t h m e t i c mean o f no f e w e r than s i x m i ce
e x p e r i m e n t a l group;
in each
ORGANISMS/HIGH POWER FIELD
NUDE
N U D E /IR S
DAYS POST-INFECTION
N U D E /N M S
42
7)., Nude/ I RS and NLM/IRS mi ce d i d n o t e x h i b i t an o bv io u s r e s t r i c t i o n :
o f p a ra s ite reproductive a c t i v i t y
(Figure 9 ).
These d a t a c o n f i r m t h e u s e f u l I ness, o f t h e nude mouse as an _m_ ■
v i v o model f o r t h e assay o f a b l a s t in a c t i v i t y .
ac tiv ity
in
I RS is
f e r e n t techniques
The l a c k o f a b l a s t i c
in agr eement w i t h e a r l i e r o b s e r v a t i o n s u s i n g d i f ­
(69).
It
i s n o t e w o r t h y t h a t Abl o r
were a b l e t o p r o f o u n d l y m o d i f y T\ mu scul i
parasitemia
IRS regimens
in nude m i ce ,
b ut were n ot a b l e t o p r o v i d e T.. m u s c u l ? e l i m i n a t i o n
potential
mice,
in g e n e r a l
nor p r e v e n t t h e i r d e a t h .
ment w i t h t h e concept t h a t
peripheral
that
blood o f mi ce
fin al
This o b servation
is
t o nude
agree­
e l i m i n a t i o n o f %. m u s c u l i from t he
is dependent on a c e l l u l a r mechanism and
t h i s mechanism is n o n f u n c t i o n a l , in nude m i ce.
Absorption o f A b l a s t i c A c t i v i t y
Immune Serum
from
The a b l a s t i c f a c t o r produced by T . m u s c u l i - i n f e c t e d mi ce has been
a h i s t o r i c a l l y , p e r p l e x i n g compound.
p h ys i c o c h e m i c a l I y s i m i l a r
ever,
its
to
i mmu no gl ob ul in , s p e c i f i c a l l y
its
the ant I body-nature o f a b l a s t in.
lity
has been r e p o r t e d
e lic itin g
How­
and t he
a n t i g e n s have c a s t doubt on
S i n c e abI as t i n
is proposed t o
r e p r o d u c t i o n ^ w i t h o u t d e s t r o y i n g trypanosomes
that dividing
t o be
IgG j.'
honabsorbab.M i t y w i t h homologous trypansomes . ( 8 )
lack o f c h a r a c t e r i z a t i o n o f
h ib it
A blastin
( 23 ) ,
in-
.
the p ossibi­
trypanosomes, were t h e sour ce o f t h e e l i c i t i n g
•
43
F i g u r e 9*
The e f f e c t o f
immune r e c o v e r e d serum on t h e r e p r o du c ­
t i v e a c t i v i t y o f JT. muscul ? in nude and NLM m i c e .
value represents
Each
t h e a r i t h m e t i c mean o f two e xp e r i m e n t s
no f e w e r t han s i x mi ce
in each e x p e r i m e n t a l
g r ou p.
CO 4 0
N U D E /N M S
NUDEy
Q
10
N L M /IR S
DAYS PO ST-INFECTIO N
a n t i g e n seemed p l a u s i b l e .
mu scu ii
If
enriched f o r d i v i d i n g
this
is t h e c as e ,
a .p o p u la t io n o f X.
forms s houl d p r e s e n t a more e f f i c i e n t
an tig en p re p a ra t io n f o r absorption o f a b l a s t i c antiserum.
p a r a s i t e populations
p o p u l a t i o n was d e s i g n a t e d DI VI DI NG POPULATIONa D P ) ..
p o p u l a t i o n s were o b t a i n e d from T .
16 Pl
N DP ) .
(this
Dividing
(35%"50% DF) w e re o b t a i n e d from i r r a d i a t e d
( 5 5 0 r a d s , ^ C o ) , Tv m u s c u i ? - i n f e c t e d mi ce on day 16 Pl
site
.
(this
parasite
Nondividing
para­
m u s c u i i - i n f e c t e d NLM on day
p a r a s i t e p o p u l a t i o n was d e s i g n a t e d NONDIVIDlNG POPULATION=
Serum c o n t a i n i n g a b l a s t i c a c t i v i t y was o b t a i n e d from Tv m u s c u i i -
i n f e c t e d NLM on day 18 Pl
assay o f a b l a s t i c a c t i v i t y
(A b l).
Nude mi ce were used as an in v i v o
( s ee p r e v i o u s s e c t i o n ) .
Groups o f Tv m u s c u i I - i n f e c t e d nude mi ce r e c e i v e d 0 . 2 5 ml o f A b l ,
Abl
absorbed w i t h DP ( D P - A b s - A b l ) , Abl
NMS, o r no serum a t
t he t i m e o f
absorbed w i t h NDP ( N D P - A b s - A b l ) ,
infection.
Nude mi ce we re then mai n­
t a i n e d on a regimen o f 0.25 ml o f a p p r o p r i a t e , a n t i s e r u m e v e r y f i v e days
for
the d u ra t io n o f experim ental
and %DF was begun on day 4 P I .
t h a t as e a r l y as day 8 Pl
mi ce t h a t
observation.
The r e s u l t s
.
.
(Fi gures 10 and 11)
show
t h e r e was a dichot omy between, groups o f nude
r ecei ve d. Abl o r NDP-Abs-Abl
.
Q u a n t i t a t i o n o f 0/HPF
and groups o f nude mi ce t h a t
.
c e i v e d D P - A b s - A b l , NMS o r no serum r eg im en .
t r e a t e d mi ce e x h i b i t e d
low p a r a s i t e m i a s
low l e v e l s o f p a r a s i t e
reproductive a c t i v i t y
■.
■
Abl and NDP-Abs-Abl
(Figure
10). and r em a rk a bl y
(Figure
11)
t h ro u g h o u t
re-
46
•A
. v .
■■
Figure
10.
I.
niuscul i
parasitemia
regi men o f Abl
NDP-Abs-Abl
.■4. .
■
<
in nude mi ce m a i n t a i n e d oh a
( A b l / N u d e ) , DP-Abs-Abl
( D P- Ab s- Ab l/ Nu de)
.(NDP-Abs-AbI / N u d e ) , NMS (NMS/Nude, o r no
serum ( N u d e ) .
T . mu s c u l i
a ls o monitored
(NlM)..
i n f e c t e d NUM c o n t r o l s were
Each v a l u e r e p r e s e n t s
the
a r i t h m e t i c mean o f t h r e e e x p e r i m e n t s w i t h no f e w e r
t han s i x mi ce
i n each e x p e r i m e n t a l
group.
ORGANISMS/HIGH POWER FIELD
NUDE
,* D P -A b s -A b l/N U D E
NMS/N U D E
N D P -A b s -A b l/N U D E
A b l/N U D E
DAYS POST-INFECTION
48
Figure
11
Percent D iv id in g
nude mi ce t h a t
DP-Abs-Abl
Forms
in T_. muscul i p a r a s i t e m i a
r e c e i v e d a regimen o f Abl
in
( Ab l / N u d e)
( D P - A b s - A b l / N u d e ) , NDP-Abs-Abl
(NDP-Abs-
A b I / N u d e ) , NMS ( NMS/ Nude) , o r no serum ( N u d e ) .
T.
m u s c u l i - ?n f e c t e d NLM c o n t r o l s we re a l s o m o n i t o r e d .
Each v a l u e r e p r e s e n t s t h e a r i t h m e t i c mean o f t h r e e
e x p e r i m e n t s w i t h no f e w e r than s i x mice
experim ental
gr o up .
in each .
% DIVIDING FORMS
NM S/NUDEf
^.NUDE
" D P -A b s - A b !/NUDE
... • NDP -A b s -Ab!/N U D E
--------A b l/N U D E
DAYS POST-INFECTION
' 50
■
the
infection.
A f t e r day 20 Pl Abl
d isplayed gradual
sulted
that
increase
and NDP-Abs-Abl
treated
nude mice
in p a r a s i t e m i a and %DF t h a t e v e n t u a l l y
in t h e i r d e at h by day 26 P I .
re­
In marked c o n t r a s t , nude mice
r e c e i v e d D P - A b s - A b l , NMS, o r no serum regimen m a i n t a i n e d con­
s p i c u o u s l y h i g h l e v e l s o f bot h 0/HPF and %DF t h r o u g h o u t e x p e r i m e n t a l
observation.
Nude mi ce t h a t
w h i l e nude mi ce t h a t
received e i t h e r
w e re dead by day 26 P I .
mu scul i
r e c e i v e d NMS we re dead by day 20 P I ,
Control
DP-Abs-Abl o r no serum regimen
NLM mi ce e x h i b i t e d a t y p i c a l
parasitem ia w ith extensive reduction
in %DF by day 14 Pl
e l i m i n a t i o n o f t h e p a r a s i t e from t h e p e r i p h e r a l
As an a d d i t i o n a l
bl ood by day 20 P I .
(SE)
antiserum.
Ab­
u n r e l a t e d a n t i s e r u m w i t h e i t h e r DP o r NDP had no .
a p p r e c i a b l e e f f e c t on h e m a g g l u t i n a t i o n t i t e r s
(Table
and
c o n t r o l , e i t h e r DP o r NDP p a r a s i t e p r e p a r a t i o n s
w e re used t o absor b mouse a n t i - s h e e p e r y t h r o c y t e
sorption of th is
Tv
o b t a i n e d a g a i n s t SE
11).
These d a t a p r e s e n t d i r e c t e v i d e n c e t h a t a d i v i d i n g
preparation
activity
(35%-50%DF)
from day 18 Pl
form-enriched
o f Tv m u s c u l i can be used t o absor b a b l a s t i c
antiserum
(A b l).
Abl w i t h a n o n d i v i d i n g p a r a s i t e p o p u l a t i o n
diminish a b l a s t i c a c t i v i t y .
Furthermore, absorption of
(<5%DF)
These o b s e r v a t i o n s
d i d n ot a p p r e c i a b l y
su ggest t h a t a b l a s t i n
is an a n t i b o d y t h a t can be adsorbed o n t o homologous t ry p a n o s o me s.
addition,
these r e s u lt s p rovide
sponsible fo r
the
i n d i r e c t evidence t h a t antigens
re­
i n d u c t i o n o f a b l a s t i n p r o d u c t i o n a r e found more
In
51
Table
II.
A b s o r b t i o n o f a n t i - SE mouse a n t i s e r u m w i t h DP o r NDP
T r e a t m e n t Group
Anti-SE
(1° response)a
HA U t e r c
256 .
Anti-SE ( I 0 response)3
DP-absorbed
256
A nti-SE ( 1 ° response)3
NDP-absorbed
256
0
NMS
Anti-SE
(2° response)^
1,024
Anti-SE
(2°
response)^
1,024
Anti-SE (2° response)^
DP-absorbed
1,024
A n t i - S E ( 2 ° r esp o ns e) ^
NDP-absorbed
I ,024
a S i x mi ce r e c e i v e d 0 . 2 5 ml o f a 10% suspensi on o f SE i . v . and were
b l e d seven days l a t e r .
Serum was p o ol ed and s p l i t i n t o t h r e e a l i ­
q u o t s:
one a l i q u o t was absorbed w i t h DP; a second a l i q u o t was ab­
sorbed w i t h NDP; t h e t h i r d a l i q u o t was not ab sor bed.
k S i x mi ce r e c e i v e d 0 . 2 5 ml o f a 10% suspensi on o f SE i . v . on day 0,
day 14, and day 2 1 ,
M ic e wer e b l e d f i v e days a f t e r t h e l a s t immun­
ization.
Serum was pool ed and s p l i t i n t o t h r e e a l i q u o t s :
one a l i ­
qu ot was absorbed w i t h DP; a second a l i q u o t was absorbed w i t h NDP;.
t h e t h i r d a l i q u o t was n ot ab so rb ed .
c R e c i p r o c a l o f t h e h i g h e s t d i l u t i o n o f serum showing c l e a r l y r ecog­
nizable agglutination p a tte rn s .
Identical 2-fold s e ria l dilutions
in RBS wer e made in m i c r o t i t e r p l a t e s .
52
readily
iri a d i v i d i n g
p a r a s i t e p o p u l a t i o n than
in a n o n d i v i d i n g
.
p a ra s ite population.
,
i ■
H e m a g g l u t i n a t i o n Assay f o r T% m u s c u l ! - s p e c i f i c
Serum. A n t i b o d y
To measure TV m u s c u l j - s p e c i f I c
antibody,
■
.
"
'
coated
in t h e f o l l o w i n g manners
Co i r r a d i a t i o n
2k hrs p r i o r t o i . p .
On day 12 Pl mi ce were b l e d
c i t r a t e v ia the r e t r o o r b l t a l
plexus.
injec­
into c h ille d
C o n c e n t r a t i o n o f con­
t a m i n a t i n g trypanosomes was reduced t o <10% usi ng d i f f e r e n t i a l
fugation
(25).
E r y t h r o c y t e s wer e washed two t imes
t hen resuspended t o a 2% c o n c e n t r a t i o n .
e r y t h r o c y t e p r e p a r a t i o n was p la ce d
Serum t o be t e s t e d was s u b j e c t e d
PBS.
200 p i
NLM
•
t i o n o f 3x10^* T \ muscul i .
A lsever's
Red c e l l s
60
mi ce r e c e i v e d SSOrads,
•
a passive hemagglutina-.
t ion assay u si ng m i c r o t i t e r p l a t e s was d e v e l o p e d .
w i t h T . m u s c u l i a n t i g e n w e re o b t a i n e d
•
25 p i
in each w e l l
in c h i l l e d PBS and
of this
antigen-coated
of a m icroti ter p late.
to a s e r ie s o f 2 - f o l d
o f eaqh d i l u t i o n was p i p e t t e d
c en tri­
dilutions
i n t o s e p a r a t e we I I s .
in.
A fter
t he a d d i t i o n of. e r y t h r o c y t e s and s e r a t o t h e w e l l s , m i c r o t i t e r p l a t e s
wer e g e n t l y a g i t a t e d
serum.
to
i n s u r e t horough m i x i n g o f e r y t h r o c y t e s ahd
P l a t e s w e re a l l o w e d
t o i n c u b a t e f o r 2k hrs a t
room tempera­
t u r e a f t e r w h i ch e a s i l y d i s t i n g u i s h a b l e a g g l u t i n a t i o n p a t t e r n s were
apparent.
The r e s u l t s
(Table
I Il)
show t h a t h i g h e s t serum t i t e r s were
53
Table
III.
U t i l i z a t i o n o f T . muscul i a n t i g e n co at e d mouse e r y t h r o ­
c y t e s in a p a s s i v e h e m a g g l u t i n a t i o n a s s a y .
Sour ce o f
antigen-coated
erythrocytes.
NLM/D-18 Pl
NLM/D-18 P l ,
DP-absorbed
NLM/D-28 Pl
NLM/D-28 P I ,
DP-absorbed
NLM/Hyperimmunized
PHA t i t e r 3
Source o f
antiserum
,
I r r a d i a t e d , T . musculi
i n f e c t e d mi ce ( D- 12 P i )
256
'
4
M
512
M
Il
8
1,024
:
'
.I
N u d e / D - 18 Pl
0
11
N u d e / D - 2 8 Pl
0
11
N u - T C / D - 2 8 Pl
2
Il
NMS
0
M
NLM/Hyperimmunized
0
N.LM/Hype r immun I zed
0
. Ir r a d ia te d , uninfected,
mice
T . m u s c u l i - i n f e c t e d mice
NLM/Hyperimmunized
0
Normal mi ce
,
3 R e c i p r o c a l o f t h e h i g h e s t d i l u t i o n o f serum showing a c l e a r l y recog
nizable ag glutination p attern .
Identical 2-fo ld s e ria l dilutions
w e re made i n m i c r o t i t e r p l a t e s o f each pool o f serum.
54
a c h i e v e d w i t h serum o b t a i n e d from mi ce t h a t had r e c e i v e d a t o t a l ' o f
th ree.!,
mu scu li
infections
(NLM/ Hyper i mmuni zed) . . Serum o b t a i n e d
f rom X* m u s c u I i - i n f e c t e d NLM mi ce on day 18 Pl
28 Pl
(NLM/ D-28 P I )
.
had t i t e r s
( N LM / D - 18 P i )
o f 256 and 512 r e s p e c t i v e l y .
and day
In n o t e ­
w o r t h y c o n t r a s t , " !_. muscul I - 1n f e c t e d nude mi ce d i d not produce a
detectable ag g lu tin in
(Nude/D-28 P i ) .
response a t day 18 P I
Furthermore,
and Methods s e c t i o n ) , T \
( N u d e / D - 18 P i )
thymus c e l l - r e c o n s t i t u t e d
on day 28 P I .
.
To i n v e s t i g a t e t h e s p e c i f i c i t y o f t h i s a s s a y ,
absorbed w i t h a d i v i d i n g p o p u l a t i o n o f T \ m u s c u l i
Absorbed Abl
(NLM/D-28 P | ,
absorption.
irradiated
i nfected
( see M a t e r i a l s
m u s c u l ? - i n f e c t e d nude mi ce (N u - T C / D - 28 P I )
produced b a r e l y d e t e c t a b l e t i t e r s
tested.
o r day 28
( N L M / D - 18 P I ,
DP-Absorbed)
DP-Absorbed)
some a n t i s e r a were
(DP)
b e f o r e being
and absorbed
showed a d r a m a t i c r e d u c t i o n
I RS
in t i t e r
after
No a g g l u t i n a t i o n was obser ved w i t h e r y t h r o c y t e s from
un in fected mice,
(day 12 P l ) . ,
u n i r f a d i a t e d mi ce t h a t wer e T .
o r normal mice
(Table
musculi -
111).
E f f e c t o f T . mu scuI i P a r a s i t e m i a on D i r e c t and I n d i r e c t PFC Responses
o f M ic e t o SE and D i r e c t PFC Responses t o PVP in Mi ce
Mahy p a r a s i t i c
ab ility
Infections
l ead t o t h e d e p r e s s i o n o f
t o respond t o v a r i o u s a n t i g e n s a n d . m i t og e n s
Du ri ng T *
mu scul i
p a r a s ite m ia the a b i l i t y
the host's
(1,1 5 ,2 6 ,5 2 ,6 5 ).
o f mice t o respond t o un­
55
re la te d antigens
is d r a m a t i c a l l y d e p r e s s e d .
d icate that soluble,
Recent
investigations
in­
p a r a s i t e - d e r i v e d subst ances a c t d i r e c t l y on B
cells
t o produce t h i s
h y p o r e s p o n s i ve
of I *
m u s c u l i - induced h yp or esp on si ven es s on a n t i g e n p r i m i n g ,
ment and f u n c t i o n o f memory c e l l s
immune s t a t u s
(2 ,3 ).
and on t h e secondary
The e f f e c t
.
develop­
immune response
has n o t been c h a r a c t e r i z e d .
In o r d e r t o b e t t e r u nd er st a n d t h e
musculi - In fe c te d mice,
immune system d y s f u n c t i o n
e x p e r i m e n t s were desi gned t o e v a l u a t e t he d i r e c t
RFC. response t o a T - independent a n t i g e n ,
antigen,
SE ( 14)
NLM r e c e i v e d an o p t i m a l l y
and a T - de p e n d e n t
T.
muscul i - i n f e c t e d
Immunizing dose o f e i t h e r PVP o r SE (see
M a t e r i a l s and Methods s e c t i o n )
IV)
PVP ( 4 8 ) ,
in T_. muscul i - i n f e c t e d NLM m i c e .
received s i m i l a r
in T .
i.v .
on day .14 P I .
i mm u n iz at i on s on t h e same day.
show t h a t T. muscul i - i n f e c t e d NLM mi ce
U n i n f e c t e d NLM
The r e s u l t s
(Table
(Group ; I ; Tab lei IV)
had a.
s i g n i f i c a n t l y . l o w e r number o f d i r e c t P V P - s p e c i f i c and d i r e c t S E - s p e c i - .
f i c PFC when compared t o normal
d a t a show t h a t T.
muscul i
both a . T - i ndep en den t
Additional
(PVP)
controls
parasitemia
These
induces h y p o r e sp o ns iveness to
and T - d e p e n d e n t
(SE) a n t i g e n
in m i ce .
e x p e r i m e n t s were desi gned t o d e t e r m i n e t h e e f f e c t o f
T„ m u s c u l i p a r a s i t e m i a on S E - p r i m i n g ,
and on secondar y
(Group 2; .Tabl e I V ) .
devel opment o f SE-memory c e l l s ,
immune responses t o SE.
d e s i gn s wer e used:
(I)
Three d i f f e r e n t experimental
M ic e r e c e i v e d a p r i m a r y
i n j e c t i o n o f SE d u r i n g
TabielV. Effect of T. musculi parasitemia on direct PFC responses of mice to PVP
a '
b
and SE .
C
C
PVP-Specific PFC.
SE-Specific PFC
Treatment Group
6
PFC/.Spleen
I. T. musculi-infected
I ( d a y 14 PI)
2. Uninfected control
3,987
■4.8
*
*
9,270
6
PFC/10
PFC/Spleen
13,088
.
*
41.6
PFC/10
18.5
*
34,375
164.5
a
Mice were immunized with.0.25 ug of PVF i.v.
b
Mice were immunize with 0.25 ml of a 10% suspension of SE i.v.
c
:
Arithmetic mean for 12 mice determined five days after immunization. .
*
.
'
Indicates a significant (p <.05) difference between infected and uninfected groups using
the Students t test.
57
T . mu scu lI
after I .
parasitemia
(day 14 P I )
musculi e l i m i n a t i o n
a primary
(day 35 P I )
i n j e c t i o n o f SE b e f o r e
i n j e c t i o n o f SE d u r i n g
(Table V I ) ;
( 3)
SE a f t e r X -
infection
(day 0 P i )
musculi e l i m i n a t i o n
experimental
design,
sim ilarly
i n j e c t i o n o f SE
( 2)
infection
musculi
Mi ce r e c e i v e d a p r i m a r y
muscu11
(Table V);
musicui i
a secondar y
day as
and a secondary
M ic e r e c e i v e d
(day - 7 P i )
infection
and
(day 14 P i )
i n j e c t i o n o f SE on t he same
and a secondar y
(day 21 P I )
in je c tio n of
(Table V I I ) .
In each
.
immunized u n i n f e c t e d NLM mi ce served
as c o n t r o l s .
The r e s u l t s
( T a b l e V)
show t h a t
c e i v e d SE i n j e c t i o n s on day 14 Pl
significantly
l ower
( P < . 05)
(P<,05)
controls
(Group 2 ; T a b l e V ) .
had
PFC r e ­
In a d d i t i o n , T\
(Groups 3 and 5 ; T a b l e V)
had s i g n i f i ­
l ower d i r e c t PFC responses t o SE t han u n i n f e c t e d NLM
(Groups 4 and 6 ; T a b I e V)..
Ho wever ,
-7 Pi)
(Group I j T a b l e V)
r e c e i v e d a s i n g l e p r i m i n g dose o f SE on
e i t h e r day 14 Pl o r day 35 P I
cantly
and day 35 Pl
d i r e c t and i n d i r e c t S E - s p e c i f i c
sponses than, u n i n f e c t e d c o n t r o l s
m u s c u l i - i n f e c t e d mi ce t h a t
m u s c u l I - ?n f e c t e d NLM t h a t r e ­
i f m i c e , w e r e S E - primed b e f o r e JT. m u s c u l i
and then r e c e i v e d a second
IjTable V I) ,
infection
i n j e c t i o n o f SE on day 14 Pl
(day
(Group
t h e i r d i r e c t and i n d i r e c t S E - s p e c i f i c PFC responses d i d
n ot s i g n i f i c a n t l y
(P>.05)
d iffer
2 ; T a b I e V i ) , ■ These r e s u l t s
from u n i n f e c t e d NLM c o n t r o l s
suggest t h a t T \ muscul,i
(Group
p a r a s i t e m i a does
Table V.
.
E f f e c t of T. musculi parasitemia on d i r e c t and i n d ir e c t RFC responses of mice
to SEa 1° (day 14 P l ) / 2 ° (day 35 Pi)
S E - s p e c i f i c PFCZlO^b
S E - s p e c i f i c PFC/Spleen*3
T r e a t m e n t Group
Di f e e t
I . T. musculi - in fe c te d
1 ° (day 14 P l ) / 2 u (day 35 P I )
79,225
2.
22,754
Indirect
Dir e c t
Indirect
174.0
188,025
376.0
*
Uninfected control
.
4 14 , 8 7 1
• *
77.0
1,421.0
637
ND
1.8
ND
2,009
ND
9 .1
ND
5. T . m u s c u l i - i n f e c t e d
I v (day 35 P D
185,125
ND
450.0
6 , . Uni n f e c t e d c o n t r o l
220,875
3. T. m u s c u l i - in fe c te d
. T 0 (day 14 P I )
4.
Uninfected control
a Mice were
'
.
-
ND
*
ND
813.0
ND,
immunized w i t h 0. 25 ml o f a 10% suspensi on o f SE T . v .
b A r i t h m e t i c mean f o r
JU
.
. '
12 mi ce d e t e r m i n e d f i v e days a f t e r
.
immunization.
■
I n d i c a t e s s i g n i f i c a n t (p < . 0 5 ) d i f f e r e n c e between
as d e t e r m i n e d by S t u d e p t 1s t t e s t .
i n f e c t e d arid u n i n f e c t e d groups
Table V I .
E f f e c t of I . muscull parasitemia on d i r e c t and i n d ir e c t RFC responses of mice
to SEa I cH d a y - 7 Pl ) / 2 ° (day 1.4 PI) .
SE-specif i c
PFC/Spleen*3
T r e a t m e n t Group
Di r e c t
I.
T. m u s c u li-infected
T°(day -7 PI ) /2°(d ay
2.
Uninfected control
3.
T. m u s c u li-infected
T 0 (day - 7 P I )
507,173
.15,554
561,045
.
Indirect
34.20
1 ,3 8 3
50.00
1,574
14 Pi ).
530
ND
0.57
*
ND
.*
ND
7.13
ND
T. m u s c u li-infected
T 0 Cday 14 P I )
19,062
ND
22.30
ND
Uninfected control
38,359 .
Uninfected control
5.
a M i c e wer e
b
15,051
D irect
1,624
4.
6.
Indirect
S E - s p e c i f i c PFCZlO6b
immunized w i t h 0. 25ml
A r i t h m e t i c mean o f
*
*
ND
67.24
ND
o f a 10% suspensi on o f SE i . v .
12 mi ce d e t e r m i n e d f i v e days a f t e r
immunization.
*
I n d i c a t e s a s i g n i f i c a n t (p < . 0 5 ) d i f f e r e n c e between
as d e t e r m i n e d by S t u d e n t ' s t t e s t .
i n f e c t e d and u n i n f e c t e d groups
in
ID
Table V I I .
E f f e c t of T\ musculi parasitemia on d i r e c t arid i n d ir e c t RFC responses of mice
to SEa 1° (day 0 P I ) / 2 ° (day 21 P t ) .
S E - s p e c i f i c PFC/Spleen*3
T r e a t m e n t Group
I.
S E - s p e c i f i c PFCZlO6b
'
Di r e c t
Indirect
D irect
T. m usculi-infected
(day 14 P I )
44,500
308,593
89.2
704
Uninfected control
77,624
207,890
146.9
409
1.1
ND
Indirect
T ° (day 0 P I ) / 2 °
2.
3.
Ti m u scu li-in fected
555
ND
T ° (day o P I )
4.
5.
*
Uninfected control
T.
m usculi-infected
1,380
ND
7.2
ND
118,000
ND
204.8
ND
T d Cday 21 P i )
6.
*
Uninfected control
M i c e r e c e i v e d 0 . 2 5m l
k A r i t h m e t i c mean f o r
271,875
of
ND
897.6
ND
10% su sp en si on o f SE i . v .
12 mi ce d e t e r m i n e d f i v e days a f t e r
immunization.
I n d i c a t e s a s i g n i f i c a n t (p < . 0 5 ) . d i f f e r e n c e between i n f e c t e d and u n i n f e c t e d groups
as d e t e r m i n e d by S t u d e n t ' s t t e s t .
61
not s i g n i f i c a n t l y a l t e r S E - s p e c i f , | c devel opment o r subsequent ex­
p r e s s i o n o f secondar y
immunity t o SE i f S E - p r i m i ng o c cu rs b e f o r e
in­
fection.
I f mice, r e c e i v e d a p r i m i n g
.muscul i
in fectio n
(day 0)
and t hen r e c e i v e d a second
a f t e r e lim in a tio n of parasitemia
responses a r e d i m i n i s h e d
fected controls
i n j e c t i o n o f SE oh t h e day o f T.
(day 21 P i ) ,
i n j e c t i o n o f SE
d ir e c t SE-specif i c
(Group l ; T a b l e Vl I ) when compared t o u n i n ­
(Group 2 ; T a b l e V I I ) ,
w hile
in d ire c t SE-specif i c
responses a r e augmented when compared t o u n i n f e c t e d c o n t r o l s .
reduction
(Groups
in PFC responses o r a u g m e n t a t i o n o f
indicate that
s i t e m i a , . S E - s p e c i f i c memory c e l l
condary
immunity
C ollectively
T.
PFC
Direct
i n d i r e c t PFC responses
I and 2 ; T a b I e Vl I ) was n ot d e t e r m i n e d t o be s i g n i f i c a n t
These r e s u l t s a l s o
muscUlI
if
S E - p r i m i ng occurs p r i o r
is not s i g n i f i c a n t l y
these data
(Tables
p a r a s ite m ia s i g n l f i c a n t Iy
IV-VI I ) pro vid e evidence th at
(p < ; 0 5 )
Inhibits
an tig en prim ing,
musculi
■
infection,
immunity a r e
h ot s i g n i f i c a n t l y a l t e r e d .
.■
to para­
altered.
devel opment and e x p r e s s i o n o f secondary
■
( p > . 05).
devel opment and e x p r e s s i o n o f se­
i f mi ce a r e pri med b e f o r e o r on t h e same day o f
memory c e l l
PFC .
■
E f f e c t o f T \ muscul i. Par as I t e m i a on D i r e c t RFC
Responses t o PVP in Nude N i c e
62
Recent
i n v e s t i g a t i o n s propose t h a t T_. muscul?
induced a l t e r a t i o n s
o f t h e immune system o f mi ce a r e due t o a d i r e c t e f f e c t o n . B c e l l s
r a t h e r t han t h e a c t i v a t i o n o f s u p p r e s s o r T c e l l s o r macrophages
To r u l e o u t t h e p o s s i b i l i t y o f ! , c e l l
particip atio n
in _T. m u s c u l ?-
induced h y p o r e s p o n s i v e n e s s , e x p e r i m e n t s wer e desi gn ed t o
d i r e c t PFC responses t o a T - i nd ependent a n t i g e n
i n f e c t e d nude m i c e.
vigorous
(4,5)
optim ally
(47).
T \ m u s c u l i - ?n f e c t e d nude mi ce r e c e i v e d an
nude mice s er ved as normal
. The r e s u l t s
(Table V I I I )
had a s i g n i f i c a n t l y
(p < . 0 5 )
particip atio n
S im ilarly
immunized
controls.
i n d i c a t e t h a t T . m u s c u l i - i n f e c t e d nude
l ower d i r e c t P V P - s p e c i f i c PFC response
t han u n i n f e c t e d nude c o n t r o l s .
that T c e ll
in T . m u s c u l ?-
T - c e l I d e p r i v e d mice
immunogenic dose o f . PVP on day 14 P I .
uninfected
(PVP)
investigate
P r e v i o u s s t u d i e s have shown t h a t PVP induces a
immune response in both a r t i f i c i a l l y
and nude mi ce
(2,3).
These d a t a p r o v i d e d i r e c t e v i d e n c e
is n ot
r e q u is it e fo r the
p r e s s i o n o f t h e h y p o r e sp o ns I v e s t a t u s o f 7 \
i n d u c t i o n o r ex­
m u s c u l I - I n f e c t e d mi ce.
E f f e c t o f %. muscul? P a r a s i t e m i a on Del ayed H y p e r s e n s i t i v i t y
Responses in Normal Mice
A l t h o u g h t h e humoral
mi ce has been r e p o r t e d
immune d y s f u n c t i o n o f T \ m u s c u l i - i n f e c t e d '
(2 ,3 ),
t he
in te g r ity of cell-m ediated
o f %. m u s c u l ? - i n f e c t e d mi ce had not been
investigated
p e r i m e n t s w e re desi gned t o assess t h e c e l l - m e d i a t e d
responses
t o d a t e . . Ex­
immune responses
63
Table V I I I .
E f f e c t o f T_. muscul I p a r a s i t e m i a on d i r e c t RFC responses
o f mi ce t o PVP. a
T r e a t m e n t Group
I,
T. musculi - in fe c te d
nude mi ce (.day 14 P I )
PFCZSpleenb
4, 451
PFCZio6b
10.4
*
2.
U n i n f e c t e d nude mi ce
1 1, 7 9 9
58.7
a M ic e wer e immunized w i t h 0 . 2 5 Hg o f PVP I . v.
^ A r i t h m e t i c mean f o r s i x mi ce d e t e r m i n e d f i v e days a f t e r
* Indicates a s ig n if ic a n t
i m mu n i z a t i o n .
(p < , 0 5 ) d i f f e r e n c e between i n f e c t e d and
u n i n f e c t e d groups as d e t e r m i n e d by S t u d e n t ' s t t e s t .
64
o f T\ muscuj_i_-infected mice, by measur i ng d e l a y e d - t y p e h y p e r s e n s i t i v i t y
responses t o 1 - f I u o r - 2 , 4 - d ! n i t r o b e n z e n e
( DNFB).
Individual
o f T\ m u s a j j ^ - i n f e c t e d .'were d e t e r m i n e d on day 14 Pl
s w e l l i n g assay t h a t has been p r e v i o u s l y d e s c r i b e d
responses
usi ng an e a r
(58).
Mi ce were
p a i n t e d w i t h 0 . 2 ml o f DNFB (0.5%). on t h e abdomen on each o f two
consecutive
d ays.
T h i c k n e s s o f both e a rs o f a l l
and r ecor ded f o u r days a f t e r
the la s t p a in tin g .
mi ce was measured
A f t e r b a s e l i n e mea­
surements wer e p e r f o r m e d , mi ce wer e c h a l l e n g e d w i t h 0 .2ml
( 0. 2%)
on t h e d o r s a l
s i d e o f each e a r .
o f DNFB
E a r t h i c k n e s s was measured
24 hrs a f t e r c h a l l e n g e and exp ress ed as t h e mean % i n c r e a s e
in e a r
thickness f o r
(Table
show t h a t J \
tiv ity
The r e s u l t s
IX)
m u s c u l i - I n f e c t e d mice produced d e l a y e d - t y p e h y p e r s e n s i ­
responses t h a t w e re n ot d r a m a t i c a l l y d i f f e r e n t
controls.
(e.g.
12 mi ce d e t e r m i n e d on. day 18 P I .
.
from u n i n f e c t e d
. These d a t a p r o v i d e e v i d e n c e t h a t c e l l - m e d i a t e d
delayed-type h y p e rs e n s itiv ity )
par as i t e m i a i n . m i c e .
Furthermore,
w i t h t h e concept t h a t
the
remains
immunity
.
i n t a c t d u r i n g JJ musculi
these observations a re co n sisten t
immune system d y s f u n c t i o n
i n . Ty m u s c u l i -
I n f e c t e d mi ce is due t o a d i r e c t e f f e c t on B c e l l s and does not
v o l v e T c e l l s o r macrophages.
E f f e c t o f V a r y i n g Doses o f y - i r r a d i a t i o n on
T . mu scul i P a r a s i t e m i a In Mice
in­
65
Table
IX.
E f f e c t o f T . mu scu li p a r a s i t e m i a on d e l a y e d - t y p e h y p e r ­
s e n s i t i v i t y t o 1 - f I u o r o - " 2 , 4 - d i n i t r o b e n z e n e in m i ce .
% increase over b a s e lin e
T r e a t m e n t Group
thickness
I .
2.
T. m u s c u l i - in fe c te d
DNFB-t r e a t e d
( d a y . 14 P I ) ,
T. m u s c u li-infected
v e h i c l e alone
(day
69.6 %
3,
Uninfected,
DNFB-treated
4.
Uninfected,
v e h i c l e alone
14 P I ) ,
■■
2,0 %
86.4 %
6.6 %
a M ic e wer e p a i n t e d w i t h Q.02ml o f DNFB ( 0. 5%) on t h e abdomen on each
o f two c o n s e c u t i v e d ays.
T h i c k n e s s o f both e ar s o f a l l mi ce was
measured and r ecor ded f o u r days a f t e r t h e l a s t p a i n t i n g .
A fter,
b a s e l i n e measurements we re p e r f o r m e d , mi ce wer e c h a l l e n g e d w i t h
0 . 0 2 ml o f DNFB ( 0. 2%) on t h e d o r s a l s i d e o f each e a r .
Ear t h i c k n e s s was measured Zk hrs a f t e r c h a l l e n g e and exp ress ed as
t h e a r i t h m e t i c mean % i n c r e a s e i n e a r t h i c k n e s s f o r 12 mi ce d e t e r ­
mined on day 14 P I .
P r e v i o u s s t u d i e s , have shown t h a t X- o r n e u t r o n
dram atically
servation
t h e host
test
i n c r e a s e subsequent T. muscul? p a r a s i t e m i a .
implies t h a t
t h e r e a r e r a d i o s e n s i t i v e components
immune response t o T. m u s c u l i .
this
h y p o t h e s i s and t o e s t a b l i s h
irrad iatio n
n e c e ss a r y t o
e it h e r 750rads,
hrs p r i o r
to
induce t h i s
55Qr ads, o r 350 ra d s
mus c u I i
'
of
A n
.
/ :
'
'
Co i r r a d i a t i o n ,
t i o n apd 200 d a y s . Pl
M ice.that
t han T .
m i ce.
'
but wer e not
i mpl y t h a t
mined t o l i e
12)
(0/HPF)
show t h a t mice
By day 10 P I ,
-
infected
75Qrads o r
'
l i ve d . 14 days p o s t - i r r a d i a ­
observation)
t h e i r p ar as I terni a b y . da y 27 Pl.;
there
respectively.
d i d not d e v e l o p f u l m i n a n t
7 days
later
controls.
is a r a d i o s e n s i t i v e h o s t component,
Immune e l i m i n a t i o n o f T . musculi
Furthermore,
24
uCo i r r a d i a t i o n devel op ed f u l ­
(e x t e n t o f experim ental
m u s c u l I - i n f e c t e d normal
r e q u is it e f o r the
Co
C o n t r o l mi ce wh ich r e c e i v e d 7 50 f a d s o r . 550rads
but c l e a r e d
These d a t a
'
_T. muscul i - j n f e c t e d ,
(Figure
r e c e i v e d 350r ads ^^Co i r r a d i a t i o n
parasitem ias,
60
( w ho le body)
Evaluation of parasitem ia
p a r a s i tern I a by day. 6 P I .
550r ads mi ce we re d ead.
in
B a i b / c mi ce r e c e i v e d
u Co i r r a d i a t i o n
r e c e i v e d e i t h e r 750r ad s o r 550r ads
m i n an t T .
involved
'
aberration.
The r e s u l t s
T h i s ob­
l ower t h r e s h o l d dose o f
i n f e c t i o n w i t h 3x10^ T.. muscul i ..
was i n i t i a t e d bn day 4 P I .
can
Exp er i men ts w e re desi gned t o
the
u n i r r a d i a t e d m i ce s e r ve d as c o n t r o l s .
that
irradiation
p a r a s i t e m i a from
t h e r a d i o s e n s i t i v i t y o f t hi s, component was d e t e r ­
between 350r ad s and 5 5 0 r a d s .
.
F i g u r e 12.
T\
muscul i
parasitemia
e i t h e r 750rads,
IT. muscul i
in normal mice t h a t
received.
SSOrads, o r SSOrads. 25 hr s p r i o r
infection.
Each v a l u e r e p r e s e n t s
a r i t h m e t i c mean o f 8 m i c e .
the
to
THE EFFECT OF buCo IRRADIATION ON
T. MUSCUU INFECTION IN MICE
130-
120
-
QilO-
•
■
A
O
UJ IOO-
^
7 5 0 RAD
5 5 0 RAD
3 5 0 RAD
NORMAL CONTROL
80-
60-
OC 2 0 -
8
IO
12
14
16
18 20 22 24 26 28
30 32 34 36 38 4 0 42
DAYS POST- INOCULATION
69
E ffect of
Irrad iatio n
on A c q u i r e d
Immunity t o
muscul i
A f t e r documenting t h e d r a m a t i c e f f e c t o f y - i r r a d i a t i o n on t h e '
;
d e v e l o p m e n t . o f a c q u i r e d immunity in t h e
muscul jr-mouse m o d e l , e x p e r ­
iments' w e re d esi gn ed t o
established
investigate
immunity-to
t h e e f f e c t s o f y - i r r a d i a t i o n on
musculi ^
In a d d i t i o n ,
i r r a d i a t i o n on t h o se mechanisms r e s p o n s i b l e f o r
T \ muscul i
the e f f e c t s o f y -
the s e q u e s tra tio n of
j n t h e vasa r e c t a o f t h e ki d n e y s o f mi ce w e re a l s o
investi
L
gated.
M i ce wer e i n f e c t e d w i t h 3x10
d e v e l o p a normal
parasitem ia.
One group o f I .
(day 30 P I )
60
Co on day. 30. PT .
receive y - ir r a d ia t io n
m u s c u l [ - r ec o v er e d mi ce t h a t
r e c e i v e d a second
and we re a l l o w e d t o
AT I mi ce c l e a r e d p a r a s i t e m i a by day
21 PI . . Some mi ce r e c e i v e d . 5 5 0 raids
r ec o v e r e d mi ce t h a t d i d h o t
T \ muscuI i
T \ muscul i - .
ser ved as c o n t r o l s .
r e c e i v e d 550 ra d s ^®Co
inoculum o f T . m u s c u l ? ( 3 x 1 0 ^ ,
i.p .)
day 31 P I , w h i l e a n o t h e r group t h a t
r e c e i v e d SSOrads
d i d n ot r e c e i v e a second
inoculum ser ved as c o n t r o l s .
u nder goi ng a p r i m a r y T\
musculi
muscul i
p a r a s i t e m i a wer e a l s o
(day 30 P I )
on
but
Mi ce
i n c l u d e d as .
controls, .
The r e s u l t s
( T a b l e X): show t h a t o v e r 58% o f t h e mi ce t h a t had
e l i m i n a t e d T . m u s c u l ? p a r a s i t e m i a from t h e p e r i p h e r a l
PI,
retained
bl ood on. day 21
m u s c u l ? in t h e vasa r e c t a o f t he k i d n ey s f o r a t
50 days p o s t - e l i m i n a t i o n ,
least
Subsequent y - i . r r a d i a t i o n o f t h e s e mice d i d
not cause a r e l a p s e o f T . m u s c u l i p a r a s i t e m i a
in t h e p e r i p h e r a l
blood.
60
Table X.
The E f f e c t o f 550 ra d s
o f mi ce t o T . m u s c u l i .
Co i r r a d i a t i o n bn t h e p r o t e c t i v e
Immunity
THE EFFECT OF 550r 60co IRRADIATIO N ON THE PROTECTIVE IM MUNITY OF MICE TO T. musculi.
N o. o f M ice Positive fo r
1 ° T m usculf Challenge
a
D ay 0
xy
D ay 3 0
D ay 0
-
D ay 0
D ay 3 0
2 ° T. muscu/i Challenge ®
Between Days 3 1 —71
C
N o. o f M ice Positive fo r
T. musculi in K idney
Between Days 3 1 —71
-
0
3 /5
D ay 31
0
3 /6
0
4 /6
6 /6
6 /6
D a y 31
D ay 31
3 3x10^ T . mu scu lI
b
T. musculi in Peripheral B lood
i.p .
^ A r i t h m e t i c mean o f 90 h i g h power f i e l d s
o f G i e m s a - s t a i n e d t h i n blood smear s.
c A r i t h m e t i c mean o f 90 h i g h power f i e l d s o f G i e m s a - s t a i n e d k i d n e y
s me ar s.
.6 0
= SSOrads
Co i r r a d i a t i o n .
impression,
71
Mice t h a t
r e c e i v e d a secondar y T. muscul? c h a l i e n g e a f t e r y - i r r a d i a t i o n
w e re r e f r a c t ! I e t o r e i n f e c t i o n .
These d a t a p r o v i d e d i r e c t e v i d e n c e t h a t y - i r r a d i a t i o n does not
a lte r
t h o se h o st mechanisms r e s p o n s i b l e f o r
musculi
In t h e vasa r e c t a o f t h e k i d n e y .
t h a t acquired
immunity t o I .
t he s e q u e s t r a t i o n o f T_.
In a d d i t i o n ,
m u s c u l i , once d e v e l o p e d ,
these data
imply
is. q u i t e r a d i o ­
resistant.
E f f e c t o f y - I r r a d i a t i o n and S e l e c t i v e R e c o n s t i t u t i on ..
o f M ic e on Subsequent T \ m u s c u l i I n f e c t i o n
In o r d e r t o f u r t h e r d e f i n e t ho se components r e q u i s i t e f o r
immune e l i m i n a t i o n o f T \ m u s c u l I from m i c e ,
i n whi ch T \
ous s y n g e n e i c c e l l
wh ol e body.
f o l l o w e d .by s e l e c t i v e
populations.
F i v e days a f t e r
8
injections of e ith e r
IxlO
immune thymus c e l l s ,
normal
addition,
pair.
re c o n stitu tio n with v a r i ­
Mi ce r e c e i v e d 550r ads ^ C o
irrad iatio n ,
thymus c e l l s
Z1
7\
musculI
irrad iatio n
groups o f mi ce r e c e i v e d
normal
i.v .
spleen c e l l s ,
o r no a d o p t i v e t r a n s f e r .
i.p .
In
t h e day o f a d o p t i v e r e ­
T \ m u s c u l i - i n f e c t e d mi ce were i n c l u d e d as con­
Evaluation o f parasitemia
The r e s u l t s
in mice t h a t had r e c e i v e d
immune s p l e e n c e l l s ,
mi ce r e c e i v e d 3x10
U nirradiated,
tro ls.
e xp e r i m e n t s w e re conducted
m u s c u l i . p a r a s i t e m i a was. m o n i t o r e d
sublethal y - ir r a d ia t io n
t he
(Figure
13)
( 0/ HPF) was begun on day 4 P I ,
show mi ce t h a t
r e c e i v e d SSOrads ^ C o and
72
U
Figure
13*
T.
mu s c u l i
6bCo)
•
parasitemia
mi ce t h a t
Sn normal and i r r a d i a t e d
(550rads,
received adoptive tr a n s fe r o f e it h e r
.
immune thymus c e l l s
( ' S z D5- 1TC and Normal
c o n t r o l - I TC) ,
immune s p l e e n c e l l s
( ' S / D5“ ISC and Normal
control-SC ),
normal
( s ^ b 5 “ NTC) , normal s p l e e n c e l l s
thymus c e l l s
( - S z D5-NSC) , o r no c e l l s .
NLM and i r r a d i a t e d mi ce r e ­
c e i v e d a d o p t i v e t r a n s f e r on day 5 p o s t - i r r a d i a t i o n .
Each v a l u e r e p r e s e n t s
s ix mice.
t h e a r i t h m e t i c mean o f a t
least
73
A
LU
>
or
LU
O
CL
X
CD
NO RM AL CONTROL
A
^ D S -N S C
□
- ^ 0 5 - IS C
■
NORM AL C O N T R O L -SC
•
IM M U N E CONTROL
O
A , ,
♦
¥
•
NO RM AL C O N T R O L -!T C
IRRADIATION DELAY 5 ADOPTIVE REPAIR
X
\
CO
S
CO
Z
S
O
i
i
i
i
I
i
i
6 8 IO 12 14 16 18 20 22 24 26 28 30 32
DAYS POST-INOCULATION
wer e
i n j e c t e d f i v e days
thymus c e l l s
o r normal
la te r with e ith e r
spleen c e l l s
immune thymus c e l l s , normal
c o u l d not c o n t r o l
sitem ia;
t h e s e mi ce devel op ed e x t r e m e l y h i g h
day 6 Pl
and w e r e dead by day 10 P I .
immune s p l e e n c e l l s
b ut by day 10 Pl
l e v e l s o f p a r a s i t e m i a by
I r r a d i a t e d mi ce t h a t
e x h i b i t e d a l oss o f e a r l y c o n t r o l
in itiated
in t h e e l i m i n a t i o n o f 7 \
T_. musculj_ p a r a ­
a vigorous
received
over parasitemia ,,
immune response t h a t c u l m i n a t e d
m u s c u l ? from, t h e p e r i p h e r a l
t h e same day as e l i m i n a t i o n o f "T, muscul.i
in normal
bl ood by day 18 PI,
controls^
These d a t a p r o v i d e d i r e c t e v i d e n c e t h a t a d o p t i v e t r a n s f e r o f
mune s p l e e n c e l l s
fectively
trol
r e p a i r T / muscuTI
potential
normal
in
the
ten tial
in m i ce .
irrad iatio n
60
Co)
can e f ­
o r normal
Furthermore,
immune thymus c e l l s ,
s p l e e n c e l l s were not c a p a b l e o f r e ­
irrad iatio n -in d u ced defect
in I .
muscul?
e l i m i n a t i o n po­
Additional
e x p e r i m e n t s wer e d esi gn ed t o i n v e s t i g a t e t h e p o s s i b i ­
t h a t donor
immune s p l e e n c e l l s may a c t
components
potential
in t h e p o s t - i r r a d i a t i o n
in m i c e .
were a l l o w e d
muscul I
in c o l l a b o r a t i o n w i t h host
r e p a i r o f T. musculi e l i m i n a t i o n
To d e t e r m i n e t h e v a l i d i t y , o f t h i s
t o r e c o v e r 25 days f o l l o w i n g
b e f o r e a d o p t i v e t r a n s f e r of . v a r i o u s c e l l
T.
(550rads,
e l i m i n a t i o n p o t e n t i a l , b ut n o t e a r l y con­
i r r a d i a t e d mice.
thymus c e l l s ,
pairing
lity
f i v e days a f t e r
im-
parasitem ia.
. Mi ce r e c e i v e d
irrad iatio n
p r o p o s a l mice
(550rads,
60
Co)
p o p u l a t i o n s , and subsequent
I.v .
injection of e ith e r
1x10
8
75
immune s p l e e n c el I s ,
thymus c e l l s ,
normal
spleen c e l l s ,
immune thymus c e l l s ,
o r no c e l l s on day 25 p o s t - i r r a d i a t i o n .
normal
M ic e a l s o r e ­
ft
c e i v e d 3x10
%. mu scu li
i.p .
m u s c u l i - i n f e c t e d mi ce were
t h e day o f a d o p t i v e r e p a i r .
i n c l u d e d as c o n t r o l s .
s i t e m i a was begun on day. 4 P I .
The r e s u l t s
mi ce a d o p t i v e l y
o r normal
( F i g u r e . 14)
spleen c e l l s
from T. mu scu li
in
adoptive tr a n s fe r of c e l l s .
spleen c e l l s
reduction
irradiated
thymus c e l l s
d i d n ot d i f f e r
that
r e c e i v e d no
received
immune
i n t he i n f e c t i o n ,
b ut a l s o
in t h e d u r a t i o n o f p a r a s i t e m i a when
compared t o T \ m u s c u l i - i n f e c t e d normal
transferred
controls
in
n ot o n l y e x h i b i t e d a pr ofound
in TeVels o f p a r a s i t e m i a e a r l y
These d a t a c l e a r l y
irrad iatio n
C o n v e r s e l y , mi ce t h a t
25 days p o s t - i r r a d i a t i o n
e x h ib it e d a dram atic decrease
muscul? p a ra s ite m ia
immune thymus c e l l s * normal
25 days f o l l o w i n g
parasitemia
Q u a n t it a t io n o f para­
.
indicate that J .
repaired with
Normal T .
indicate that
controls.
immune s p l e e n c e l l s
adoptively,
i n t o mi ce 25 days, p o s t - i r r a d i a t i o n can produce a s t a t e o f
enhanced r e s i s t a n c e t o T. mu scul i
m u s c u l i - i n f e c t e d normal
controls.
p a r a s i t e m i a when compared t o T\
Both e a r l y c o n t r o l
mechanisms and
e l i m i n a t i o n mechanisms appear ed t o be enhanced by a d o p t i v e t r a n s f e r o f
immune s p l e e n c e l l s
are
25 days p o s t - i r r a d i a t i o n :
in agr eement w i t h t h e p r o po sal
co lla b o ra te with a minimally
t h a t donor
In a d d i t i o n ,
t h ese d a t a
immune s p l e e n c e l l s may
r a d i o s e n s i t i v e p o p u l a t i o n o f h ost c e l l s
76
:
:ty
Figure
14.
T.
muscu I i. paras I tern i a in normal mice and
( 5 5 0 r a d s , ^^Co) mi ce t h a t
e ith er
immune o r normal
irradiated
received a d o p tiv e t r a n s f e r o f
thymus c e l l s o r
s p l e e n c e l l s on day 25 p o s t - i r r a d i a t i o n .
represents
in
t h e a r i t h m e t i c mean o f a t
immune o r normal
Each v a l u e
l e a s t s i x mi ce.
DAYS POST-IN FECTION
ORGANISMS/HIGH POWER FIELD
78
t h a t have r e g a i n e d f u n c t i o n by day 25 p o s t - i r r a d i a t i o n .
P o s t - i r r a d i a t i o n RFC Responses o f Mi ce
t o PVP and SE
To f u r t h e r
60
whol e body
i n v e s t i g a t e t h e immune s t a t u s o f mice a f t e r
Co i r r a d i a t i o n ,
receiving
PFC responses t o PVP and SE w e r e d e t e r -
mined on v a r i o u s days p o s t - i r r a d i a t i o n .
Mi ce r e c e i v e d 5 5 0 r a d s ,
i r r a d i a t i o n w h o l e body.
20,
On days
10,
d i a t i o n mi ce r e c e i v e d an o p t i m a l l y
PVP.
15,
Co
2 5, 30 and 60 p o s t - i r r a ­
immunogenic dose o f . e i t h e r SE or
PFC responses wer e d e t e r m i n e d f i v e days p o s t - i m m u n i z a t i o n .
The r e s u l t s
di chot omy
presented
in F i g u r e
15 c l e a r l y
I n d i c a t e a conspicuous
in r e c o v e r y o f PFC responses t o SE and PVP p o s t - i r r a d i a t i o n .
By day 30 p o s t - I r r a d i a t t o n , S E - s p e c i f i c
PFC, responses o f
t e d mi ce w e re o v e r
normal
s es ,
60
Conversely,
100% o f u n i r r a d i a t e d
on day 30 p o s t - I r r a d i a t i o n
w e re o n l y
18% o f u n i r r a d i a t e d
thermore,
this
ab ility
normal
These d a t a
550r ad s ^ C o
SE-PFC r e s p o n ­
PV P -specific
responses
PVP-PFC r esp on ses.
i r r a d i a t i o n was s t i l l
i mpl y t h a t d i s t i n c t
of sublethal
in
a p p a r e n t 60
(or
s u b p o p u l a t i o n s o f B c e l l s mi gh t be
lack o f a b i l i t y )
irradiation.
t o r e c o v e r from
Heterogeneity o f B c e lls
.
their classific atio n
Fur­
irradiation..
c h a r a c t e r i z e d by t h e i r a b i l i t y
the e f f e c t s
Co i r r a d i a ­
d i f f e r e n c e between SE and PVP r e s p o n s i v e B c e l l s
to recover f u n c t io n a f t e r
days f o l l o w i n g
control
control
60
■
and
v
i n t o d i s c r e t e s u b p o p u l a t i o n s has r e c e i v e d a g r e a t
79
Figure
15.
Post-irradiation
responses o f mi ce t o PVP o r SE on
v a r i o u s days f o l l o w i n g
represents
55. 0rads, ^ C o .
t h e mean o f a t
f i v e days f o l l o w i n g
o f e ith e r antigen.
least
Each v a l u e
12 . mice d e t e rm i n e d
i m m u ni z at i on w i t h o p t i m a l
doses
R F C R E S P O N S E O F M IC E V A R Y IN G DAYS
A F T E R 5 5 0 ^. (W H O L E B O D Y ) 6 0 C o
IO O -
A SRBC Specific RFC/IO
• PVP Specific PFC/ IO
-J >
60-
------ — •
—
IO
15
20
25
e - ---------
30
35
DAYS P O S T -IR R A D IA T IO N
40
60
81
deal
of a tte n tio n
in t h e p a s t two y e a r s
Gershon and Kondo ( 28)
(29,40,43,49,50,54,55,62).
d e m o nst r at ed t h a t some B c e l l s
require T cells
t o become t o l e r a n t , w h i l e a second s u b c l a s s o f B c e l l s
without T c e ll
adoptively
p articipation.
transferred
cells
P l a y f a i r and Purves
in
lim iting
d ilu tio n s,
become t o l e r a n t
(59),
u tilizin g
.
found t h a t sy­
nergy between thymus and s p l e e n o r bone marrow c e l l s was not p r o p o r ­
tional
to the
absence o f I
r e a c t i v i t y , o f e i t h e r s p l e e n o r bone marrow c e l l s
cells.
From t h i s o b s e r v a t i o n
populations of B c e l l s ,
one o f w h i c h ,
in t he
t h ey proposed two sub­
B j , d i d n ot r e q u i r e T c e l l
t o g e n e r a t e an immune r e sp o ns e, whereas t h e second s u b p o p u l a t i o n ,
did
require T c e ll
studies,
G o rcz yn ski
to sep arate B c e l l s
differences
h e l p t o g e n e r a t e an immune r esponse.
and Feldman
(29)
help
Bg,
. In o t h e r
have used v e l o c i t y s e d i m e n t a t i o n
i n t o two p o p u l a t i o n s which showed d i s c e r n i b l e
in t h e i r a b i l i t y
i n dep en den t a n t i g e n s .
t o respond t o thymus- dependent and thymus-
Jenni ngs and R i t t e n b e r g
( 41)
have used s e v e r a l
t h ym u s - i n d e p e n d e n t and thymus- dependent hapt en c a r r i e r complexes
in
a d d i t i o n and syner gy e x p e r i m e n t s t o f u r t h e r s u b s t a n t i a t e t h e e x i s t e n c e
of d is tin c t B cell
( 62)
subpopulations,
In a d d i t i o n , Q u i n t a n s and Kaplan
.
have shown a d i f f e r e n c e between a d u l t and newborn B c e l l s o f mi ce
in t h e i r a b i l i t y
irrad iatio n .
to q u ic k ly
r e c o v e r f rom t h e e f f e c t s o f s u b l e t h a l
82
PVP-PFC Responses o f M i ce 30 days F o l l o w i n g
V a r y i n g Doses o f °®Co I r r a d i a t i o n
Based on o u r
g a t i o n s by o t h e r s
to. f u r t h e r
a b ility
in itia l
investigate B c e ll
to recover f o llo w in g
tomy in r e c o v e r y ,
60
(Figure
(29,40-43 ,49,50,54,55,6 2),
t h e t h r e s h o l d amount o f
rads o f
observation
60
and r e c e n t
sublethal
6o
in t h e i r
Co i r r a d i a t i o n .
required
B a l b / c mi ce r e c e i v e d e i t h e r
in vesti­
e x p e r i m e n t s wer e desi gned
subpopulations t h a t d i f f e r
Co i r r a d i a t i o n
Co i r r a d i a t i o n .
15)
to
To d e t e r m i n e
induce t h i s
dicho­
550rads, 3 5 0 ra d s ,o r
100
Mi ce r e c e i v e d 0 , 2 5 pg o f PVP o r 0 . 2 5 ml o f
I
a 10% suspensi on o f SE I , v .
30 days f o l l o w i n g
irrad iatio n .
PVP-
s p e c i f i c and S E - s p e c i f i c PFC responses we re d e t e rm i n e d f i v e
immunization.
The r e s u l t s
^ C o can s i g n i f i c a n t l y
n i z e d 30. days a f t e r
(Table X I)
(p < . 0 5 )
irrad iatio n .
ses were above normal
control
55Qr ads, 3 5 0 r a d s , 2 0 0 r a d s , o r
These d a t a
capable o f
B cells
i mpl y t h a t
inactivating
little
as I OOrads
l ower PVP-PFC responses o f mi ce immu­
Conversely,
S E - s p e c i f i c PFC respon­
responses 30 days f o l l o w i n g e i t h e r
IOOrads
relatively
cells
show t h a t as
days p o s t -
60
Co.
low doses
( I OOr ad s)
and induce a d e f e c t
60
of
Co a r e
in t h e r e c o v e r y o f
c a p a b l e o f r espondi ng t o t h e T - i nd ependent a n t i g e n ,
PVP.
In
60
550r ads
,
marked c o n t r a s t ,
even a f t e r
Co, B c e l l s
capable o f
d ing t o SE f u l l y
r e c o v e r f u n c t i o n by day 30 p o s t - i r r a d i a t i o n .
respon­
83
T a b l e XT.
D i r e c t RFC r e s p o n s e s o f m i ce t o PVPc and SEc* 30 days f o l ­
lowing v a r y i n g doses o f
0 Co r a d i a t i o n 9 .
T r e a t m e n t Group
Normal
control
D i r e c t PVP- PFC/ 106 ± SDb
312.00 ± 48.7
550rads
11.46 +
350r ads
3 4 . 6 0 ± 16.1
200r ads
40.90 ± . 21.0
10Orads
165.70 ± 6 8 .4
9 Mi ce r e c e i v e d
5.0
•k
A'
*
.*
D i r e c t S E - P F C / I O 6 ± SDb
200.96 ±
48.6
106,90 ±
6 5 -3
204.00 ±
36,5 .
298,90 ±
. 6.9
318.50 ±
87.4
Co r a d i a t i o n whol e body
k A r i t h m e t i c mean o f f i v e mi ce d e t e r m i n e d f i v e days a f t e r
i mmuni zat ion
C M ic e r e c e i v e d 0 . 2 5 yg o f PVP i . v .
^ M ic e r e c e i v e d 0 , 2 5 ml o f a 10% suspensi on o f SE i . v .
I n d i c a t e s s i g n i f i c a n t (p < . 0 5 ) d i f f e r e n c e from normal
as d e t e r m i n e d by S t u d e n t ' s t t e s t .
control
values
84
P o s t - i r r a d i a t i o n RFC Responses o f Mi ce t o DNP
on a ! - d e p e n d e n t o r ! - i n d e p e n d e n t C a r r i e r
Additional
'DNP,
linked
e x p e r i m e n t s wer e conducted
to e it h e r a !-dependent c a r r i e r
i ndep en den t c a r r i e r
(DNP-.Ficol I )
gate the d i f f e r e n t i a l
tions.
mal dose o f e i t h e r
diation.
i r r a d i a t i o n on B c e l l ,subpopul a -
Co wh ol e body.
Mi ce r e c e i v e d an o p t i ­
i n d i r e c t PFC responses o f DNP-Ovalbumin
day 8 and day 5 p o s t - i r r a d i a t i o n
immunized mice and
immunized mi ce wer e d e t e r m i n e d on
respectively,
!he results
p r o v i d e e v i d e n c e t h a t mi ce r ec o v e r e d t h e i r a b i l i t y
DNP-Ovalbumin 30 days p o s t - i r r a d i a t i o n .
In marked c o n t r a s t , t he DNP-
r esponse o f mi ce had n ot r e c o v e r e d 30 days f o l l o w i n g
tion,
! h e r e was a s i g n i f i c a n t
(p < . 0 5 )
in v i v o exp osu re t o SSOrads ^ C o .
agreement w i t h
cells
r e d u c t i o n o f D N P- F i c o l I PFC
can be c l a s s i f i e d
s u b p o p u l a t i o n s based on t h e i r a b i l i t y
I
irra d ia ­
i r r a d i a t e d mi ce when compared t o u n i r r a d i a t e d c o n t r o l s ,
! h e s e d a t a suggest t h a t B c e l l s
that B
(!able
t o respond t o
Ficoll
responses o f
irra ­
immunized u n i r r a d i a t e d mi ce ser ved as c o n t r o l s ,
d i r e c t PFC responses o f D N P - F i c o l l
X II)
o r . a T~
D NP - F ic o l I o r DNP-Ovalbumin 30 days f o l l o w i n g
S im ilarly
D i r e c t and
60
(DNP-Ovaibumin)
was used as an immunogen t o . i n v e s t i ­
effect of ^Co
Mi ce r e c e i v e d 550r ad s
in which a s i n g l e h a p t e n ,
t h e B^ B^ h y p o t h e s i s
are
I m p ar t ed
into d is t in c t
to fu n c tio n a l Iy recover a f t e r
F u rt h e r m o r e t h e s e d a t a a r e
(.59)
in .
and suggest t h e p o s s i b i l i t y
in t h e i r a b i l i t y
t o f u n c t i o n a l I y r ec o v e r
Table X I I .
D i r e c t and i n d i r e c t RFC responses o f m i ce t o DNPa on a T - i ndep en den t c a r r i e r
30 days f o l l o w i n g 550r ad s
Co r a d i a t i o n .
, 6d
D i r e c t PFC/10
T r e a t m e n t Group
101.64
^^Co/DNP-OvaI bumin
Normal
c o n t r o l /DNP-Ova I bum in
I n d i r e c t PFCZlO6d
'
67.45
140.3,4
control
' 97.95 %
1 4 3 - 36
60CoZDNP-Fi cdl I
13.9.40 *
ND
Normal
35 8. 71
ND
control/DNP-FicolI
% Normal
38.70 %
a 2 , 4 - d i n i trophenol
6 DNP-Ovalbumiri- 400 yg
in 0 . 2 ml o f F r e u n d ' s Compl ete A d j u v a n t
■
C D N P -F i c o l 1- 5 yg
i.p .
.
in' s a l i n e
i.p .
^ A r i t h m e t i c mean o f f i v e m i ce d e t e r m i n e d e i t h e r 5 days
(DNP-Ovalbumin) f o l l o w i n g i m m u n i z a t i o n .
(DNP-Ficoll)
o r e i g h t days
.
v
■;
f o l lowi ng . ^ C o
86
-
i r r a d i a t l o n , whereas
cells
•
■
are not.
P o s t - i r r a d i a t i o n PFC Responses o f Mice, t o
T-Independent
I and ! - i n d e p e n d e n t
2 antig ens
H o s i e r , Mond and G o l d i n g s
a n t i g e n s can be d i v i d e d
type 2 a n t i g e n s ) .
that B c e lls
60
o r CBA/N B c e l l s
( ! - i n d e p e n d e n t t yp e I
a r e a b sen t
in t h e CBA/N s t r a i n
Because p r e v i o u s e x p e r i m e n t s
r e s p o n s i v e t o PVP and D N P - F i c o l l ,
have an a l t e r e d
Co i r r a d i a t i o n ,
ab ility
e x p e r i m e n t s were d esi gn ed t o
i ndep en den t
type 2 a n tigens.
wh ol e body.
M ic e t hen recei ved, o p t i m a l
type I a n t i g e n ) ,
(!-independent
have shown
both ! - i n d e p e n d e n t
investigate p o s t-ir r a ­
t y p e I a n t i g e n s and T -
Mi ce r e c e i v e d 550rads ^ C o
irrad iatio n
doses o f e i t h e r LPS ( T - i n d e - .
PVP ( ! - i n d e p e n d e n t
type 2 a n t ig e n ,
t y p e 2 a n t i g e n ) , o r . SE ( ! - d e p e n d e n t a n t i g e n ) .
S SS - 1 I I
PFC
post-immunization.
,( Tabl e Xl I I ) . show t h a t PFC responses t o e i t h e r S S S - 1 I I o f
PVP wer e s i g n i f i c a n t l y
than
(!-independent
(Table X l l )
responses t o t h e se a n t i g e n s were measured f i v e , d a y s
The r e s u l t s
in
to f u n c t io n a l Iy recover fo llo w in g
d i a t i o n PFC responses o f mi ce t o ! - i n d e p e n d e n t
pendent
t hymu s - I nd e p e n d en t
o r t h e i r c a p a c i t y t o s t i m u l a t e o n l y more m a t u r e B c e l l s
o n t o g e n y , whi ch c e l l s
antigens,
have shown t h a t
i n t o two m a j o r su bset s based on t h e i r a b i l i t y
to s t i m u l a t e e a r l y neonatal
antigens)
( 54)
in u n i r r a d i a t e d
(p < . 0 5 )
controls.
l ow er i n 3 0 - d a y p o s t - I r r a d i a t l o n mice
Conversely,
p o s t - I r r a d i a t l o n PFC r e ­
sponses t o LPS were, n o t found t o be s i g n i f i c a n t l y
(p < . 0 5 )
d ifferen t
T a b l e Xl I I .
D i r e c t PFC responses, o f mi ce t o S S S - I I I 3 , LPS^, PVPc , o r SEc* 30 days
f o l l o w i n g °®Co i r r a d i a t i o n 6 .
I.
60Co Z S S S - I I I
2.
Normal
3.
60CoZLPS [ T - l
4.
Normal
5.
60Co ZPVP [ T - l
6.
Normal
7.
8.
PFCZlO6 f
PFCZSpleen^
T r e a t m e n t Group
[T-l
( 2 ) Ag]
I ,604
105.18
10,374
57.86
23,487
47.40
5,459
62.40
45,436
295.70
60CoZSE [ T- D A g ] .
24,000
164.30
Normal
59,875
174.80
(I)
Ag]
controlZLPS
.
( 2) Ag]
c o n t r o l ZPVP
controlZSE
i.p .
a M i ce were
immunized w i t h 0 . 5 yg o f sss-111
6 M i ce we re
immunized w i t h
C M i c e we re
immunized w i t h 0 . 2 5 yg o f PVP i . v .
1 . 0 yg o f LPS I . v .
control
15.6 %
16.45.
17,625
c o n t r o l / S S S - 1I I
% Normal
122.0 %
'
21 .1 %
9 4 . 0 %.
in s a l i n e .
in s a l i n e .
in. s a l i n e ,
^ M i c e wer e immunized w i t h 0 . 2 5 ml o f a . 10% s u sp e n s ion o f SE in s a l i n e i . v .
e M i c e r e c e i v e d 550 rads ^ C o
i r r a d i a t i o n 30 days p r i o r t o
immunization.
^ A r i t h m e t i c mean o f s i x m i ce d e t e r m i n e d f i v e days f o l l o w i n g
immunization.
88
from LPS-PFC responses o f u n i r r a d i a t e d , c o n t r o l s .
irrad iatio n
S E - s p e c i f i c PFC responses
Furthermore,
in mi ce d i d n ot d i f f e r
c a n t l y f rom S E - s p e c i f i c PFC responses o f u n f r r a d i a t e d
H o s i e r e t a I . proposed t h a t T - I nd ependent
dent
post­
s ig n ifi­
controls.
t y p e I and f - indepen-,
t y p e 2 a p t igens ad dr essed d i f f e r e n t s u b p o p u l a t i o n s o f B c e l l s .
P o s t-irrad iatio n
experiments
dence t h a t t h e B c e l l s
antigens f a i l s
(Table X I I )
presented here provide e v i ­
s u b p o p u l a t i o n addr essed by T - i n dep en den t
t o f u n c t i o n a l I y r e c o v e r 30 days f o l l o w i n g
whereas t h e B c e l l
type 2
irrad iatio n ,
p o p u l a t i o n addr essed by T - i ndependent .
type I a n t i ­
gens r e c o v e r s f u n c t i o n a l I y by day 30 p o s t - i r r a d i a t i o n .
PVP Optima I - D ay T i t r a t i o n In 3 0 - d a y P o s t - I r r a d I a t i o n
M ic e and Normal Mi ce
Because one o f t h e ways in which
body s y n t h e s i s
Is t o
l e n g t h e n t he
ionizing
radiation
affects
lag phase o f a n t i b o d y p r o d u c t i o n
(.5), e x p e r i m e n t s w e re d esi gn ed t o e x c l u d e t h i s p o s s i b i l i t y
anism o f
cell
t h e obser ved p o s t - i r r a d i a t i o n
subpopulations
dent
t yp e
r espondi ng
tion.
mal
S im ilarly
controls.
r e c o v e r y di chot omy between B
type 2 an tig e n s .
i r r a d i a t i o n , wh ol e body.
ed a n . o p t i m a l , dose o f PVP i . v .
as a mech­
t o T - d e p e n d e n t a n t i g e n s o r T - indepen­
I a n t i g e n s and T - i nd ependent
c e i v e d 550 rads ^ C o
an ti- .
on days 3 0 ,
3),
Mi ce r e ­
Groups o f mi ce r e c e i v ­
3 2 , o r 33 p o s t - I r r a d i a ­
immunized groups o f u n l r r a d i a t e d mi ce ser ve d as n o r ­
89
The r e s u l t s
( T a b l e X l V)
show t h a t o p t i m a l
wer e o b t a i n e d on day f i v e p o s t - i m m u n i z a t i o n
g ro u ps . F u r t h e r m o r e , o p t i m a l
were d r a m a t i c a l l y
P V P - s p e c i f i c responses
in both p o s t - i r r a d i a t i o n
PVP-PFC responses o f p o s t ? I r r a d i a t ion mi ce
l ower t han o p t i m a l
PVP-PFC responses b f u n i r r a d i a t e d
controls.
These d a t a
i mpl y t h a t peak P V P - s p e c i f i c PFC responses o f mice were,
not. o bser ved a f t e r day f i v e
Therefore,
lations
in p o s t - 1 r r a d i a t ion m i c e .
t h e p o s t - I r r a d i a t ion r e c o v e r y di chotomy between B c e l l
r espondi ng t o e i t h e r T - d e p e n d e n t and T - i ndep en den t
t i g e n s o r T - 1ndependent
cr e as e d
post-immunization
type 2 antig ens
popu­
t y p e I an­
is n ot s i mply due t o an i n ­
lag phase in a n t i b o d y p r o d u c t i o n ,
P V P - s p e c i f | c PFC Responses in I r r a d i a t e d
and S e l e c t i v e l y R e c o n s t i t u t e d Mice
E x pe ri m en t s wer e d esi gn ed t o d e t e r m i n e what c e l l
optively
t r a n s f e r r e d , c ou ld r e s t o r e P V P - s p e c i f i.c PFC responses
i r r a d i a t i o n mice.
Mi ce r e c e i v e d 550r ad s
p o s t - I r r a d i a t i o n mi ce r e c e i v e d e i t h e r
o r bone marrow c e l l s
tive
p o p u l a t i o n s , ad­
i.v .
groups or
uni r r a d i a t e d
controls .that
•
Co, wh ol e body.
Inclu ded.as
controls.
irra d ia te d -s e le c tiv e ly
immunized w i t h an o p t i m a l
On day 7
8
1x10° s p l ee n c e l l s , t h y m u s
U n i r r a d i a t e d mi ce r e c e i v i n g
t r a n s f e r s o f c el Is wer e
irrad iatio n ,
60
in p o s t - .
s i m i l a r adop­
On day 30 p o s t -
r e c o n s t i t u t e d mi ce and
received s i m i l a r c e l l
dose o f PVP i . v .
cells,
p o p u l a t i o n s were
Day 30 p o s t - i r r a d i a t ion
90
Table XIV.
PVP o p t i m a l day t i t r a t i o n
and normal m i c e . 3
T r e a t m e n t Group
in 3 0 - d a y p o s t - i r r a d i a t i o n mi ce
■ D i r e c t P V P - s p e c i f i c PFCZlO6b
^Normal
control
I.
Normal
2.
Day 30 p o s t - i r r a d i a t i o n
day 5 PVP response
58,80
Day 30 p o s t - i r r a d i a t i o n
day 5 PVP response
.41,50
Day 30 p o s t - i r r a d i a t i o n
day 7 PVP response
0.89
0.20 %
Day 30 p o s t - i r r a d i a t i o n
day 8 PVP response
3,12
2.06 %
3-
4.
5.
control
151,00
39.9 %
.
27.48 %
a M ic e r e c e i v e d 550r ad $
Co i r r a d i a t i o n on day 0,
M i c e were immunized
on e i t h e r day 3 0 , 3 1 , 3 2 , o r 33 w | t h 0.25 ug o f PVP i . v ,
^ A r i t h m e t i c mean f o r s i x mi ce d e t e r m i n e d on day 38 p o s t - i r r a d i a t i o n .
91
m i ce t h a t wer e n ot s e l e c t i v e l y
r e c e i v e d an o p t i m a l
The r e s u l t s
r e c o n s t i t u t e d and normal mi ce a l s o
dose o f PVP ? , v .
( T a b l e XV)
and wer e
show I r r a d i a t e d mice, t h a t w e re r e c o n s t i t u ­
ted w i t h e i t h e r spleen c e l l s ,
thymus c e l l s o r bone marrow c e l l s
much h i g h e r P V P - s p e c I f i c PFC responses t han
c e i v e d no c e l l s ,
i r r a d i a t e d mi ce t h a t r e ­
populations.
t o n o t e t h a t a d o p t i v e t r a n s f e r o f thymus c e l l s
responses o f normal mi ce
(51% o f normal
appear ed t o be t h e most e f f i c i e n t c e l l
i r r a d i a t i o n RFP-RFC responses o f mi ce
c ells,
m ic e .•
I t was
co n tro l).
population
Bone marrow c e l l s
in r e p a i r i n g
(155% o f normal
repairing
o r bone marrow c e l l s
into
interesting
i mp a i r e d t h e PVP-PFC
i mpl y t h g t a d o p t i v e t r a n s f e r o f e i t h e r
thymus c e l l s ,
capable o f
had
b u t n ot d i f f e r e n t f rom normal mi ce t h a t r e c e i v e d
adoptive tr a n s fe r of s im ila r c e ll
These d a t a
i n c l u d e d as c o n t r o l s .
post­
c o n t r o l %.
IxlO
8
s p l een
i r r a d i a t e d mi ce
is
p o s t - i r r a d i a t i o n P V P - s p e c i f i c RFC responses o f
92
T a b l e XV.
D i r e c t PVP-PFC responses o f a d o p t i v e l y r e p a i r e d mice
30 days f o l l o w i n g °^Co i r r a d i a t i o n , 3
T r e a t m e n t Group
P V P - s p e c i f i c PFC/1
control
I.
Irradiation
2.
Normal
3.
Day 30 p o s t - i r r a d i a t i o n
Thymus c e l I r e p a i red
1 4 9 , 00
4.
Normal
131.00
50.97 %
5.
Day 30 p o s t - i r r a d i a t i o n
Bone marrow r e p a i r e d
397.00
154.47 %
6.
Normal
187.00
72:76 %
7.
Day 30 p o s t - i r r a d i a t i o n
Spl een c e l l r e p a i r e d
187.14
7 2. 81 %
Normal
281.95
8.
control
Normal
control
control/Thymus c e l l s
c o n t r o l / B o n e marrow
c o n t r o l / S p l een c e l l s
12.02 %
30.90.
257.00
.
57.97 %
109.70%
.
a Mi ce r e c e i v e d 550r ad s ^ C o i - r a d i a t i o n on day 0 .
M i ce r e c e i v e d
1x10° thymus c e l l , s p l e e n c e l l s , o r bone marrow c e l l s i . v . on day
7 post-irradiation.
Mi ce wer e immunized w i t h 0 . 2 5 yg o f PVP i . v .
on day 30 p o s t - i r r a d i a t i o n .
.
,
A r i t h m e t i c mean o f f i v e mi ce d e t e r m i n e d f i v e days a f t e r
i m mu n i z a t i o n .
DISCUSSION
Results presented
in f i g u r e s
I a n d .2 provide d i r e c t evidence f o r
t h e thymus dependency o f _T. m u s c u i ? e l i m i n a t i o n
because d i v i d i n g
dividing
Furthermore,
forms wer e obser ved t h r o u g h o u t nude i n f e c t i o n s w h i l e
forms were d r a m a t i c a l l y
and NLM m i c e ,
from m i c e .
l i m i t e d a f t e r day 12 Pl
in both Nu-TG
t h e s e d a t a su ggest t h e thymus-dependency o f
the a b la s t i c
phenomenon.
,
A l t h o u g h nude mi ce wer e
temia,
i n cap ab le, o f c l e a r i n g T.' mu scu li
t h e y w e re obser ved t o e x e r t some c o n t r o l
compared t o mi ce t h a t had r e c e i v e d s u b l e t h a l
]\
muscuii
infection
mouse model
early
innate
I).
led t o t h e p r op osal
Early
demonstrate e a r l y
o v e r p a r a s i t e m i a when
irrad iatio n
(37*38).
participate
( F i g u r e 3)
parasitemia
o v e r p a r a s i t e m i a was
role
cle arance o f T\
Macrophage p a r t i c i p a t i o n
(Figure
and
I),
t he
investigated.
(9,
30-34,46)
i m mu n i t y , b ut may a l s o
m u s c u i i from t h e p e r i p h e r a l
in
immune e l i m i n a t i o n o f T_.
m u s c u i j has a l s o been proposed by V l en s e t a l .
ported
( 13)
t h a t macrophages may not o n l y
in e a r l y n o n s p e c i f i c
in t h e f i n a l
blood o f m i c e .
reveale d
in
Because nude mi ce have
S t u d i e s u si ng t r y p a n b l u e t o a l t e r macrophage a c t i v i t y
jn nude and NLM mi ce
to
in v e s t ig a t io n s o f the T. muscuir
o v e r T . mu scu li
th a t e a rly control
p l a y an i m p o r t a n t
prior
l e v e l s o f a c t i v a t e d macrophage a c t i v i t y
control
.
parasi­
t h a t macrophages may be i n v o l v e d
immunity t o _T. muscuI i
p a r a d o x i c a l l y high
po ssib ility
(Figure
.
( 6 1 , 7 1 ) , who have r e ­
t h a t trypanosomes a r e f r e q u e n t l y seen a d h e r i n g t o and being
94
rapidly
killed
by bl ood monocytes d u r i n g
mu scu Ii
infection.
Paradoxically,
the terminal
t h e se d a t a
s t a g e s of. T^.
imply t h a t
the
in v i v o
a d m i n i s t r a t i o n o f t r y p a n b l u e , wh ich has been r e p o r t e d t o have t r y p a n o ­
cidal
ac tiv ity
muscuI i
(75),
can under c e r t a i n
c o n d i t i o n s a c t u a l l y enhance T \
i n f e c t i o n s o f m i ce .
The thymus dependency o f _T. muscu11 e l i m i n a t i o n does not d i s t i n ­
g u i s h between t h e r o l e o f c e l l - m e d i a t e d o r humoral
of
immunity t o T_, muscu! j .
acquired
body,
Final
is due t o t he combined a c t i o n o f a
muscul I
f i r m and e x t en d t h e s e t h e o r i e s .
immune o r normal
X * muscul i
ten tial
fer
from t he p e r i p h e r a l
an ti­
bl ood o f mice
t o be dependent on a c e l l - m e d i a t e d mechanism
r a t h e r t han a d i r e c t a n t i b o d y e f f e c t
fers of
in itia l
and a t h ym u s - i n d e p e n d e n t t r y p a n o c i d a l
clearance of T\
has been h y p o t h e s i z e d
in mechanisms
P r e v i o u s work has suggest ed t h a t
immunity t o T \ mu scu Ii
thymus- dependent a b l a s t l n
factors
(72).
The d a t a
Nude mi ce t h a t
(Figures 4-9)
con­
received adoptive trans
p o p u l a t i o n s o f s p l e e n o r thymus c e l l
prior
to
i n f e c t i o n wer e a b l e t o g e n e r a t e %. m u s c u l i e l i m i n a t i o n po­
(Figure 4 ).
Conversely,
regimens o f e i t h e r Abl
or
nude mi ce t h a t
received passive t r a n s ­
IRS serum d e m o nst r at ed a marked reduc­
tion
in p a r a s i t e m i a but we re n ot a b l e t o g e n e r a t e T\ m u s c u l ? e l i m i n a ­
tion
potential
t r a n s f e r o f Abl
parasitemia
(Figures 6 - 9 ) .
I t was
interesting
to note th a t passive
in nudes d i d g e n e r a t e a r e m a r k a b l e r e d u c t i o n
( F i g u r e s 6 and 7) W h i l e p a s s i v e t r a n s f e r o f
in %DF and
IRS had l i t t l e
95
e f f e c t on %DF y e t
These d a t a a r e
investigators
in agreement w i t h
( 69 )
and t r y p a n o c i d a l
trypanocidal
reduced p a r a s i t e m i a d r a m a t i c a l l y
a n t i b o d i e s w h i l e day 28 Pl mouse serum c o n t a i n s o n l y
antibodies.
description of ab la s tin
that controls
and
Experimental
immunosuppressant s t u d i e s
an i mmu no gl ob ul in , . p o s s i b l y
ablastin a c t iv it y
(66),
t h e concept o f a
reproductio n without, k i l l i n g
i n t r i g u e d many i n v e s t i g a t o r s .
chemi cal
t h e h y p o t h e s i s proposed by e a r l y
t h a t day 18 Pl mouse serum c o n t a i n s bot h a b l a s t i n
Si nce, t h e o r i g i n a l
serum f a c t o r
( F i g u r e s 8 and 9 ) .
IgGp
( 23)
However,
p a r a s i t e s has
e v i d e n c e ac cr ue d
indicates
in p h y s i c o ­
that ablastin
t he n o n a b s o r b a b i l i t y o f
■
from a n t i s e r u m w i t h homologous trypanosomes and t h e
in ab ility
to
to rically
perplexing
iso late
its
e lic itin g
antigen(s)
pr obl em in u n d e r s t a n d i n g
(23)
has p r e s e n t e d a h i s ­
t he n a t u r e o f a b l a s t i n
and t h e mechanisms o f
its action.
a b l a s t i n has been t h e
l a c k o f dep en dab le In v i v o assays f o r a b l a s t i c
ac tiv ity ,
is
One o f t h e problems
in t h e st udy o f
P r e v i o u s d a t a have shown t h a t nud e. mi ce a r e n ot c a p a b l e o f
p r od uc in g a b l a s t i n
(Figure
I and 2)
has a pro fo un d e f f e c t on T_. mu scul j
observations
suggest t h e u t i l i t y
ob a b l a s t i n a c t i v i t y .
but t h a t p a s s i v e l y t r a n s f e r r e d A b l .
parasitemia
in nude m i c e .
These
o f t h e nude mouse as an in v i v o assay
Dat a p r e s e n t e d
in f i g u r e s 7 and 8 c o n f i r m t h i s
p r op o sa l
and d e m o n s t r a t e t he r e l i a b i l i t y
ablastin
activity.
of this
in v i v o assay o f
96.
Because a b l a s t i n
I.
mu scul i
(23),
it
is supposedl y
seemed t h a t d i v i s i o n a l
s o u rce o f a n t i g e n ( s )
that e l i c i t s
s u p p o s i t i o n were t r u e a d i v i d i n g
shoul d r e s u l t
forms m i g h t be a p r i m a r y
t he p r o d u c t i o n o f a b l a s t i n .
(35%-50%DF)
To t e s t
population of I ,
(Figures
10 and 11)
enriched population o f T\
this
hypothesis,a
dividing
clearly
antiserum.
m u s c u l i can absor b a b l a s t i c a c t i v i t y
the antibody nature o f a b l a s t i n ,
f or m-
I n d i c a t e t h a t a . d i v i d i n g f or m-
These d a t a p r o v i d e d t h e c r u c i a l
that ablastin
this
muscul? was o b t a i n e d from i r r a ­
mouse a n t i s e r u m , w h i l e a n o n d i v i d i n g p o p u l a t i o n , o f T \ m u s c u l I
can n o t .
If
form -enriched population of I . muscul?
d i a t e d , T_. muscul ? - j n f e c t e d mi ce and was used t o absorb Abl
The d a t a
forms o f
in a more a p p r o p r i a t e a n t i g e n p r e p a r a t i o n f o r . t h e a bso rp ­
tio n of a b l a s t i c antiserum.
enriched
in te ra c tin g with dividing
from
(<5%DF) '
e v i d e n c e needed t o e s t a b l i s h
Furthermore*
t hese d a t a suggest
i s a " n o r m a l " a n t i b o d y t h a t can be absorbed f ro m a n t i ­
serum w i t h homologous t rypanosomes.
Previous attempts
t o absorb ab­
lastin a c tiv ity
from a n t i s e r u m w i t h homologous trypanosomes have
probably f a i l e d
due t o a low %DF in t h e trypanosome p r e p a r a t i o n s used..
The r e s u l t s o f t h e s e e x p e r i m e n t s a l s o c o n f i r m e d t h e h y p o t h e s i s
antigens e l i c i t i n g
amounts
production o f a b l a s t i n are present
in a d i v i d i n g
that
jn a p p r e c i a b l e
p a ra s ite population,
K e n d a l l , . , in h is o r i g i n a l
d e s c r i p t i o n o f I T , muscul j . ( 4 4 ) ,
a n o t h e r s u r p r i s i n g a s p e c t o f T\ muscuI i
serology;
the
noted
lack of a s p e c ific
:
97
d e t e c t a b l e a g g l u t i n a t i n g a n t i b o d y response t o t he p a r a s i t e .
q uen t
investigations
report th e ir f a i l u r e
i*
mu scul i
have f u r t h e r a b f u s c a t e d t h e
to d e te c t a s p e c i f i c a g g lu tin a tin g
( 7 2 ) , w h i l e Chang and Du san ic
g IutInating
issue.
response t o JT. m u s c u l I .
( 12)
V i en s e t a l .
response t o
r e p o r t a d e t e c t a b l e ag-
The a b i l i t y
o f T\
musculI ' antigens
t o p a s s i v e l y absor b o n t o t h e s u r f a c e o f mouse e r y t h r o c y t e s
well
documented,
It
seemed t o me t h a t
c y t e s m i g h t p r o v i d e an i d e a l
due t o t h e r a p i d
had been
these a n tig e n -c o a te d e r y t h r o ­
i n d i c a t o r system f o r
m uscul!-s p e c ific agglu tin atin g
Subse­
antibodies
t he d e t e c t i o n o f T_.
in mouse serum.
r a t e o f e r y t h r o p h a g o c y t b s is observed
However,
in T , : muscul.i-
I n f e c t e d mi ce most a n t i g e n - c o a t e d E r y t h r o c y t e s are. r a p i d l y c l e a r e d
from t h e c i r c u l a t i o n ,
I proposed t h a t
i r r a d i a t e d mi ce t h a t had been
a l l o w e d t o d e v e l o p a f u l m i n a n t ' J \ mu s c u I i
more a p p r o p r i a t e s o u rce f o r
The d a t a p r e s e n t e d
in t a b l e
. d e m o n s t r a t e t h a t 7 \ mu scu lj
p a r a s i t e m i a m i g h t be a
musculj a n tig e n -c o a te d e r y t h r o c y t e s .
111 c o n f i r m t h i s
h y p o t h e s i s and c l e a r l y
'
a n t i g e n - c o a t e d e r y t h r o c y t e s o b t a i n e d from
i r r a d i a t e d mi ce a r e c a p a b l e of. b ei ng a g g l u t i n a t e d w i t h T \ m u s c u l i - spec ific
antisera.
These o b s e r v a t i o n s su gg est t h a t a g g l u t i n a t i n g a n t i ­
bod ies a r e produced by mi ce
thermore,
this
in response t o T . m u s c u l i
a p p r o p r i a t e e r y t h r o c y t e and serum c o n t r o l s
system t o be a s e n s i t i v e and s p e c i f i c assay f o r
anti-T.
muscul i a g g l u t i n a t i n g
antibodies.
Therefore
infection.
(Table
III)
Fur­
show
the d etectio n of
I co n cl u d e t h a t
..
98
this
h e m a g g l u t i n a t i o n t e c h n i q u e p r o v i d e s a new arid u s e f u l
f o r the q u a n t i t a t i o n o f T\ musculj - s p e c i f ic a n tib o d ie s
The a b i l i t y o f p a r a s i t i c
infections
(2,3,16,2 6,52).
whi ch
However,
little
(2 ,3 ).
tria l,
It
is c e r t a i n l y
e r r o r and a d j u s t m e n t ,
method f o r
( 26)
and s u p p r e s s i v e
r e a s o n a b l e t o assume t h a t ,
parasites
t h e e v a s i o n o f host
documented
Recent s p e c u l a t i o n has cen­
t e r e d on p a r a s i t e a c t i v a t i o n o f s u p p r e s s o r T c e l l s
(3).
immune r e ­
is known about t he mechanisms by
immune responses a r e a f f e c t e d
macrophages
in mouse serum.
to. suppress h o st
sponses t o v a r i o u s a n t i g e n s and mi togens has been w e l l
technique
t hrough
have e v o l v e d more t han one
immune r esp on ses,
T_, muscul i
Infection
s t r o n g l y d e p r esses t h e a b i l i t y
o f mi ce t o respond t o a n t i g e n s o r m i t o ­
gens
Furthermore,
in v i v o o r
in v i t r o
mi ce s e v e r e l y suppress t h e
(3).
I n v j tro
s p l ee n c e l l s
from i n f e c t e d
responses o f s p l e en c e l l s
from non-
i n f e c t e d mi ce when t h e two p o p u l a t i o n s o f c e l l s a r e c o - c u l t u r e d .
g a n t work r e c e n t l y
r e p o r t e d by A l b r i g h t e t a I .
m u s c u l ? - induced h y p o r e s p o n s i veness
in m i ce
(3)
Ele­
sug gest s t h a t T_,
is n ot due t o s u p p r es so r T
c e l l s o r s u p p r e s s i ve macr ophages, but r a t h e r t o s o l u b l e p a r a s i t i c com­
ponents a c t i n g
studies
d i r e c t l y on B c e l l s o r t h e i r p r e c u r s o r s .
However,
t o d a t e have n o t ad dr essed t he e f f e c t o f T_. muscul ? induced
h yp or es po ns iv en es s on a n t i g e n p r i m i n g , devel opment and f u n c t i o n . o f
memory c e l l s ,
o r on secondar y
.
immune r e s p o n s e s .
I have approached
t h e s e q u e s t i o n s by exami ni ng d i r e c t and i n d i r e c t RFC responses t o SE
I
99
and d i r e c t PFC responses t o PVP o f mi ce a t v a r i o u s
muscui? p a r a s it e m ia .
The d a t a
e a r l i e r observations
nude mi ce
(3).
(Table V I I I )
required f o r the
r es p o n s i v e n e s s
co ncept t h a t
(tables
I V - V I I I ) c o n f i r m and ext en d
S t u d i e s conducted
the
These o b s e r v a t i o n s a r e
immune d e f e c t o bser ved
prim arily.
immunogens
IV)
a r e not
(Table
Furthermore,
(Table V ) .
t he
s t u d i e s u si ng SE and PVP as
su ggest t h a t T . m u s c u i I p a r a s i t e m i a
The r e s u l t s
if
in agreement w i t h
in Tz m u s c u i ? i n f e c t e d mice
p r i m a r y PFC responses t o e i t h e r T - d e p e n d e n t
sitemia
muscui? - in fe c te d
i n d u c t i o n o r e x p r e s s i o n o f Tz mus£i j n_-i nduced hypo-
in m i ce .
SE a r e a f f e c t e d
in T \
p rovide d i r e c t evidence th a t T c e l l s
involves B c e l l s ,
antigens.
t imes d u r i n g T_.
inhibits
(SE) o r T - i nd ependent
a l s o suggest t h a t secondary
(PVP)
immune responses t o
a n t i g e n p r i m i n g was a t t e m p t e d d u r i n g Tz m u s c u i ? p a r a ­
However,
the data
( T a b l e s Vl
and Vl I.) . c l e a r l y
c a t e t h a t once a n t i g e n p r i m i n g has o c c u r r e d , memory c e l l
and subsequent seco nd ar y
immune responses a r e n ot a l t e r e d
in d i­
devel opment .
by T . m u s c u i i
infection.
■ Havi ng p r e v i o u s l y shown t h a t
their a b ility
I),
to control
parasitem ia.
i r r a d i a t i o n on s e v e r a l
Dat a p r e s e n t e d
l e a s t one component o f t h e h ost
elim ination
in
Tz m u s c u i i p a r a s i t e m i a than nude mi ce ( F i g u r e
I examined t h e e f f e c t s o f ^ C o
Tz mu scuI i
i r r a d i a t e d mi ce a r e more i mp ar i ed
in f i g u r e
p a r am et e r s o f
12 su gg est t h a t a t
immune response r e q u i s i t e f o r Tz m u s c u i i
in mi ce has a r a d j o s e n s i t i v i t y
that
lies
between 350 and 550
100
rads
Co.
C onversely,
tant
i n d ic a t e t h a t although
immunity t o J_. muscui ? is
ment o f a c q u i r e d
once d e v e l o p e d ,
these data
acquired
immunity t o t h i s
the develop­
radiosensitive
parasite
(Figure
is q u i t e
12),
radioresis­
(Table X ) .
Recent
investigations
muscui? can p e r s i s t
have shown t h a t m u l t i p l i c a t i v e forms o f T\
in t h e vasa r e c t a o f t h e ki dn eys o f
12 m o n t h - r e c o ­
v e re d mi ce t h a t a r e r e f r a c t ! I e t o c h a l l e n g e s o f homologous trypanosomes
(69).
As a r e s u l t o f
these observations
it
has been h y p o t h e s i z e d
that
t h e vasa r e c t a o f t h e k i d n e y r e p r e s e n t s an immunol ogi cal I y p r i v e l e d g e d
s i t e and t h a t
to th is
immune mechanisms a r e r e s p o n s i b l e f o r
anatomical
location
(65,70).
t h e s e q u e s t r a t i o n o f T \ muscuI i
I studied
(Table X ) .
b al an c e d h o s t - p a r a s i t e
r e l a t i o n s h i p maintained
m u s c u i ! - r ec o v e r e d mi ce
i s not
for
tant
if
the sequestration event,
parasites
t he e f f e c t s o f ^®Co on
in t h e vasa r e c t a o f ki d n ey s o f 'Ti.
m u s c u i ! - r e c o v e r e d mi ce
o b s e r v a t i o n s suggest t h a t
lim iting
The d a t a c l e a r l y show t h a t
the f i n e l y
in t h e vasa r e c t a o f T .
i n t e r u p t e d by ^®Co i r r a d i a t i o n .
an immune mechanism is
These
indeed r e s p o n s i b l e
t hen t h i s mechanism is q u i t e
radioresis­
(as one m i g h t e x p e c t ) .
My a t t e n t i o n was s u b s e q u e n t l y d i r e c t e d
t o t h e i n v e s t i g a t i o n o f what
p o p u l a t i o n s o f s yn g e n e i c c e l l s m i g h t be a b l e t o r e p a i r IT. m u s c u i i e l i m ­
ination p o te n tia l
only
in
i r r a d i a t e d mice.
immune s p l e e n c e l l s
The d a t a
r e s t o r e T , mu s c u i j
(Figure
elim ination
13)
show t h a t
potential
to
101
mi ce
if
adoptive tra n s fe r
T h e r e was e v i d e n c e t h a t
e a rly control
is p er f or med f i v e
immune s p l e e n c e l l s were not a b l e t o r e p a i r
o f parasitem ia,
but
mu scu li was acco mp li sh ed w i t h i n
(Figure
that
13).
received
mally
e l i m i n a t i o n o f jT.
t h e same t i me p e r i o d as normal
immune s p l e e n c e l l s
parasitemia
in
If
irrad iatio n
shoul d enhance T \
before adoptive tr a n s f e r o f
muscul? e l i m i n a t i o n
a b l e t o c o l l a b o r a t e w i t h donor
suppor t , t h i s
in t h a t
immune s p l e e n c e l l s
t h e proposed m i n i m a l l y
s houl d be f u n c t i o n a l I y
immune s p l e e n c e l l s .
h y p o t h e s i s ; mi ce t h a t
^Co
transferred
received
r ec o v e r e d and
The d a t a
that
i r r a d i a t i o n wer e g r e a t l y enhanced
t he a c t i o n o f t r a n s f e r r e d
demonstrable only a f t e r
h o st components
in
that
controls.
immune s p l e e n c e l l s was
s i x days p o s t - t r a n s f e r ex cl u d e d t h e p o s s i b i l i t y
transferred
immune s p l e e n c e l l s
in t h e r e s t o r a t i o n o f T_. muscul i
i r r a d i a t e d mice.
25
in t h e i r . a b i l i t y
o f any d i r e c t c y t o t o x i c e f f e c t on t h e p a r a s i t e and f u r t h e r
the hypothesis
(Figure
immune s p l e e n c e l l s
t o e l i m i n a t e T_. muscul i even when compared t o u n i r r a d i a t e d
The o b s e r v a t i o n
( F i g u r e .13)
t h i s were t h e c a s e , w a i t i n g a l o n g e r p e r i o d o f
r a d i o s e n s i t i v e ho st component(s)
days f o l l o w i n g
i r r a d i a t e d mice
f i v e days p o s t - i r r a d i a t i o n
r a d i o s e n s i t i v e host component and t h e a d o p t i v e l y
time a f t e r
controls
t h e r e m i g h t be a c o l l a b o r a t i o n o c c u r r i n g between a m i n i ­
immune s p l e e n c e l l s .
14)
in s p i t e o f t h i s ,
The k i n e t i c s o f jT. mu scu li
suggested t h a t
days p o s t - i r r a d i a t i o n .
A p articu larly
intriguing
reinforced
collaborate w ith ,
elim in atio n potential
a s p ec t o f t h e s e d a t a was
.
the o b s e rv a tio n
spleen c e i l s
d iffe re n t
t h a t the a c t io n o f a d o p t iv e ly
in 30 day p o s t - i r r a d i a t i o n mi ce
from t h e a c t i o n o f a d o p t i v e l y
in normal m i c e .
transfer of
result
V i en s e t a l .
( 71)
immune s p l e e n c e l l s
transferred
(Figure
transferred
have shown t h a t
in h ib itio n o f parasitem ia.
i n d ic a t e the e x q u i s i t e
reg u latio n o f the
immune r esponse ( 5 , 2 0 , 4 3 )
t h a t h ost n e g a t i v e r e g u l a t i o n o f donor
( F i g u r e 5)
show t h a t
T\ mu scu Ii
elim ination
60
Co.
However,
in NLM m i c e .
it
tion
14),
is t e m p t i n g t o propose
is
i m p ai r ed
data presented e a r l i e r
are capable o f a c c e le r a t in g
Another p o s s i b i l i t y
is t h a t once
in t h e day 25
immune s p l e e n c e l l s
a re able to func­
( p o s s i b l y macro­
and b r i n g about an a c c e l e r a t e d e l i m i n a t i o n o f t h e p a r a s i t e m i a .
In subsequent a t t e m p t s
to c h a r a c t e r i z e the
i mmunol ogi cal
p o s t - i r r a d i a t i o n mi ce an i n t e r e s t i n g o b s e r v a t i o n was made.
(Figure
that
th at provide negative
i n c o o p e r a t i o n w i t h h o st e a r l y c o n t r o l mechanisms
phages)
reports
immune s p l e en c e l l s
immune s p l e e n c e l l s
(Figure
t h e s im u l t a n e o u s
in a s l i g h t and t r a n s ­
e a r l y c o n t r o l mechanisms have been r e e s t a b l i s h e d as
p o s t - i r r a d i a t i o n mi ce
immune s p l een c e l l s
In v i e w o f
ra d io s e n s itiv ity of cells
in mi ce 25 days f o l l o w i n g
14) was s t r i k i n g l y
and i n o c u l a t i o n o f p a r a s i t e s d i d not
in e l i m i n a t i o n o f t h e p a r a s i t e but m e r e l y
ient q u a n tita tiv e
immune
15)
i n d i c a t e a conspi cuous di chot omy
The d a t a
in t h e r e c o v e r y o f RFC
responses o f mice t o SE o r RVP f o l l o w i n g wh ol e body
SE-PFC responses were t o t a l l y
status of
60
uCo i r r a d i a t i o n .
r e c o ve r e d by day 30 p o s t - i r r a d i a t i o n ,
103
whereas PVP-PFC responses o f mi ce were s t i l l
This d i f f e r e n c i a l
gens was s t i l l
(Figure
15)-
sequelae
dram atically
impaired.
r e c o v e r y o f PFC responses o f mice t o d i f f e r e n t a n t i -
a p p a r e n t as
Ionizing
long as 60 days f o l l o w i n g
radiation
in mammals ( 5 ) ;
shown t o be e x q u i s i t e l y
Co i r r a d i a t i o n
i nduces a v a s t and complex a r r a y o f
components o f t h e
radiosensitive
(5)•
immune response have been
Because o f t h e m u l t i p l e
effects of
irrad iatio n
to e x p la in
t h e di chot omy o f p o s t - I r r a d i a t i o n PFC responses o f mice t o
SE and PFP ( F i g u r e
(59)
in v i v o , numerous mechanisms c o u l d be p o s t u l a t e d
15).
Recent
investigations
by P l a y f a i r and Purves
have suggest ed two s u b p o p u l a t i o n s o f B c e l l s
n ot r e q u i r e T c e l l
Bg, d i d .
Jenni ngs and R i t t e n b e r g
evidence f o r separate subpopulations o f B c e l l s
pendent and T - dependent
servations
: antigens.
Other
investigators
(2 0 ,28,29 ,36,40,42,50 ,55,62).
(20,41,59),
Consequently,
If
that w ill
the data
not
In l i g h t o f t h e se ob­
n ot o n l y
but a l s o show a d i f f e r e n t i a l
in f i g u r e
respond t o
radio-
I conducted a s e r i e s o f e x p e r i m e n t s t o
f u r t h e r d e f i n e t h e n a t u r e o f t h e PFC response di chotomy
d i a t i o n mice.
present
have a l s o des­
one engagi ng e x p l a n a t i o n f o r t h e d a t a
classes o f an tig e n s ,
sen sitivity.
did
r esp on di ng t o ! - i n d e ­
15 m i g h t be t h a t s u b p o p u l a t i o n s o f m a t u r e B c e l l s
d ifferen t
( 41)
h e t e r o g e n e i t y u si ng a v a r i e t y o f t e c h n i q u e s
be d i s cu s s e d h e r e
Bp
h e l p t o g e n e r a t e an immune r esp on se, whereas t he
second s u b p o p u l a t i o n ,
cribed B c e ll
one o f w h i c h ,
in f i g u r e
in p o s t - i r r a ­
15 wer e r e v e a l i n g a t r u e d i f f e r e n ­
104
tia l
rad io sen sitivity
6o
low doses o f
populations.
cantly
Co i r r a d i a t i o n
Mi ce t h a t
( p < . 05)
following
in B c e l l
impaired
s u b p o p u l a t i o n s one m i g h t e x p e c t even
to
inhibit
r e c e i v e d as l i t t l e
in t h e i r a b i l i t y
(Table X I ) .
t o respond t o PVP 30 days
that
these data
(PVP) a n t i g e n s c h o s e n ,
15;Table XI)
!-dependent
carrier
The r e s u l t s
( T a b l e Xl I )
( o v al b u mi n )
(DNP)
linked
wer e not s i g n i f i c a n t l y a l t e r e d .
B cell
that
d e n t a n t i g e n s can be d i v i d e d
to s t i m u l a t e e a r l y
I antigens)
or the capacity
im- ,
(Figure
I S= Ta bl es
radiosensitivity
respond t o ! - d e p e n d e n t a n t i g e n s and
respond t o ! - i n d e p e n d e n t a n t i g e n s .
H o s i e r , Mond, and Gol d in gs
a b ility
is a d i f f e r e n t i a l
subpopulations t h a t
( p < . 05)
PFC responses t o DNP-Ovalbumin
From t h e s e r e s u l t s
I co nc lu d e t h a t t h e r e
subpopulations
(F ico IT ).
c l e a r l y d e m o n s t r a t e t h a t 30 day p o s t - i r r a d i a ­
p a i r e d , whereas 30 day p o s t - i r r a d i a t i o n
between B c e l l
and
to e i t h e r a
or a !-independent c a r r i e r
responses o f mice t o D NP - Fi co l I wer e s i g n i f i c a n t l y
and Xl I )
(SE)
i t was d eci ded t o exami ne t h e p o s t ­
PFC responses t o a s i n g l e hapt en
Xl
(Figure
i n f l u e n c e d by t h e n a t u r e and c o m p l e x i t y o f t h e ! - d e p e n d e n t
irrad iatio n
tion
t o respond t o SE was a c ­
.
S i n c e t h e r e was a p o s s i b i l i t y
T - i nd ependent
sub-
as I OOrads ^ C o were s i g n i f i ­
i r r a d i a t i o n , whereas t h e i r a b i l i t y
t u a l l y augmented
wer e
the r a d i o s e n s i t i v e B c e l l
( 54)
have r e p o r t e d
that
thymus
indepen­
i n t o two m a j o r subset s based on t h e i r
neonatal
o r CBA/N c e l l s
(!-independent
t o s t i m u l a t e o n l y m at ur e B c e l l s
Type
In o n t o - .
105
g e n y , whi ch c e l l s a r e a b se n t
in t h e CBA/N s t r a i n
(!-in d e p e n d e n t - type 2
antigens).
Because e x p e r i m e n t s
t o t h i s p o i n t had o n l y addr essed p o s t -
irrad iatio n
PFC responses o f mi ce t o I - independent
type 2 antigens*
it
was d e c i de d t o exami ne p o s t i r r a d i a t i o n PFC responses o f m i ce t o T i n d e p e n d en t ;
type
I a n t i g e n s as w e l l .
-
t y p e 2)
type I)
and PVP ( ! - i n d e p e n d e n t
t y p e 2) wer e s e v e r e l y
and SE ( ! - d e p e n d e n t ) w e re n ot d r a m a t i c a l l y a l t e r e d .
f u r t h e r dissected
in m i c e ,
Because o f
subpopulations
(B^ c e l l s )
as w e l l
as t h e i r d i f f e r e n t i a l
type I or ! - in d e p e n d e n t
th e.ab ility
of
ionizing
k i n e t i c s o f antibody production
( 5)
a b ility
t o g e n e r a t e a peak PFC r esp on se.
radiation
..
t o s e v e r e l y slow t he
in the
type 2 a n t i ­
length o f time required
The d a t a p r e s e n t e d
in t a b l e . XIV r u l e
p o s s i b i l i t y and s t r e n g t h e n t h e argument t h a t B c e l l
to !-in d ep en d en t
to r e -
i t was p o s s i b l e t h a t p o s t - i r r a d i a ­
gens was p r i m a r i l y due t o an a l t e r a t i o n
r espondi ng
can be
type 2 a n t ig e n s .
i n h i b i t i o n o f PFC responses o f mi ce t o ! - i n d e p e n d e n t
out th is
I
i n t o s m a l l e r su bset s based on t h e i r p o s t - i r r a d i a t i o n
spond t o ! - i n d e p e n d e n t
tion
(T-
responses o f mi ce t o LPS ( ! - i n d e p e n ­
c on cl ud e t h a t ! - i n d e p e n d e n t B c e l l
PFC responses
.
PFC responses o f mi ce t o S S S - 11 I
i m p a i r e d , whereas p o s t - i r r a d i a t i o n
dent
( T a b l e Xl I I ) c l e a r l y .
■
.
indicate that p o s t-ir r a d ia tio n
i n d ependent
The d a t a
type 2 an tig en are e x q u i s i t e l y
subset s
radio­
sensitive.
If
these data
(Figure
15; ..Tables X l - X l I I )
do d i s t i n g u i s h a p o s t ­
106
ir r a d ia tio n defect
in a B c e l l
subset one m i g h t e x p e c t t h e a d o p t i v e
t r a n s f e r o f e i t h e r s y n g e n e i c bone marrow c e l l s o r s p l e e n c e l l s
more e f f i c i e n t
in r e p a i r i n g p o s t - i r r a d i a t i o n PFC responses o f mi ce t o
a I - i n d ependent
type 2 antigen
s yn g e n e i c thymus c e l l s .
position,
s ib ility
cell
(PtfP)
t han t h e a d o p t i v e t r a n s f e r Of
. Dat a p r e s e n t e d
in t a b l e XV c o n f i r m t h i s sup­
a l t h o u g h n o t . a s d r a m a t i c a l l y as
o f parathymic
lymph node c e l l s
p r e p a r a t i o n s may i n p a r t e x p l a i n
p a rtia lly
I had e x p e c t e d .
contaminating
the a b i l i t y
C o llectively,
these data
(Figure
15; T a b l e s X l - XV)
b e f o r e measur i ng PFC responses t o v a r i o u s
is a new and u s e f u l
of this
B cell
(Bj
c e ll)
o f T - i nd ep en den t
in t h e
id en tificatio n
.
suggest t he
550r ad s
60
Co
subsets.
Use
o f two d i f f e r e n t
su bset s t h a t a r e r e s p o n s i v e t o T - i n de p e n d e n t
type 2 a n tig en s.
to
classes of a n t i r
technique f o r d is s e c tin g B c e l l
t e c h n i q u e has r e s u l t e d
thymus
o f thymus c e l l s
t e c h n i q u e o f a l l o w i n g mi ce t o ' r e c o v e r 30 days f o l l o w i n g
gens
The pos­
injected
r e p a i r p o s t - i r r a d i a t i o n PFC responses t o PVP.
irrad iatio n
t o be
t ype
I
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Blackwell
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