The immunology of spontaneous cure in the Nippostrongylus brasiliensis-mouse system
by William Hiram Benjamin
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Microbiology
Montana State University
© Copyright by William Hiram Benjamin (1980)
Abstract:
Either cells or serum from immune animals has been found to be able to abrogate the patency of a
larval N. bra-siliensis infection when given to naive mice. All pools of cells that could cause abrogation
of a larval patency were mesenteric lymph node cells collected from mice on days 9-12 after a primary
N. brasiliensis infection. This correlated with a lymphadenopathy that was shown to occur at about
days 6-12 after larval infection. No cell pools collected from secondary infections demonstrated the
ability to protect a naive mouse to this extent nor did cells collected before or after this period. Pools of
serum from mice 13 days after a second infection were commonly found to be able to transfer as high a
level of protection as protective cells. A lot of variability in the degree of protectivity was found in both
the serum and cell pools, even in pools that were apparently prepared in the same manner.
The serum factor was found to precipitate in 50% saturated NH4(SO4)2, was resistant to 56 C for 30
minutes, was dialyzable, was freeze-thaw resistant and apparently didn’t bind to larvae in a way that
adaptively transferred immune cells could act on the larvae. Protective serum was more effective when
given on day 3 of a larval infection than when given on day 0 only. Protective serum injected
intraduodenally on day 4 of an infection had no effect on the patency of the worm infection.
Expulsion of a N. brasiliensis larval infection by mice is very radio-resistant as shown by normal
expulsion after 350 rads on day -1 only slightly delayed expulsion after 450. rads, The lesion created by
450-550 rads given before a larval infection can be repaired by 1 x 10^8 immune mesenteric lymph
node cells and worms are expelled nearly at the normal time in mice given 550 rads and normal
thymocytes on day -1.
Attempts were made to transfer immunity to nude mice with protective immune serum or immune
mesenteric lymph node cells or a combination of the two, none of which transferred any apparent
protection.
Evidence is presented that suggests that the expulsion of N. brasiliensis from mice isn’t as
prostaglandin dependent as the N. brasiliensis rat system. Neither aspirin nor in-domethacin delayed
expulsion times. Experiments in which prostaglandins were injected intraduodenally also had little
effect on an ongoing infection which is different than had been shown in the rat system.
The role of macrophages in the expulsion of a N. brasiliensis infection in mice was approached by
using a macrophage lysozyme inhibitor, trypan blue. Larval brasiliensis infections in mice pretreated
with trypan blue, then maintained on a chronic treatment schedule maintained their worms longer than
controls. Mice given normal adult worms intra-duodenally after pretreatment and continued chronic
treatment with trypan blue expelled their worm burden about the same time as non-trypan blue treated
mice. These results suggest that trypan blue interfered in an afferent vs. effector immune function. STATEMENT OF PERMISSION TO COPY
In p r e s e n tin g t h i s t h e s i s in p a r t i a l f u l f i l l m e n t of
t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t M ontana S t a t e
.
U n i v e r s i t y , - I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y
a v a ila b le f o r in sp e c tio n .
I f u r t h e r a g re e t h a t p e rm iss io n
f o r e x t e n s i v e c o p y in g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s
may be g r a n t e d by my m a j o r p r o f e s s o r ,
by t h e D i r e c t o r o f L i b r a r i e s .
or,
in h is absence,
I t i s u n d e r s t o o d t h a t any
c o p y in g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n
s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .
S ig n a tu re
D a te
& St"
/ / 77 <? ________
THE IMMUNOLOGY OF SPONTANEOUS' CURE IN THE NIPPOSTRONGYLUS
BRASIL IE N SIS-MOUSE SYSTEM
ty
WILLIAM HIRAM BENJAMIN, JR.
A t h e s i s s u b m itte d , i n p a r t i a l f u l f i l l m e n t
o f th e re q u ire m e n ts f o r th e d eg ree
of
MASTER OF SCIENCE
in
M i c r o b io l o g y
A pproved:
C hairnfan 6 G r a d u a te ConmjLttee
MONTANA STATE UNIVERSITY
Bozeman, M ontana
J u n e, 1980
iii
ACKNOWLEDGMENTS.
I g r a t e f u l l y a ck n o w le d g e t h e s u p p o r t and g u i d a n c e
g i v e n by Dr, N. D. Reed, my a d v i s o r .
Thanks i s g i v e n f o r
h i s t a k i n g tim e when t h e r e was 'no tim e i n h i s b u s y s c h e d u l e
and e s p e c i a l l y f o r h i s c o n f i d e n c e i n me.
S p e c i a l t h a n k s go t o f e l l o w g r a d u a t e s t u d e n t s , D ennis
B i e r and B rad B r o o k s , f o r many h o u r s o f d i s c u s s i o n , t e c h n i ­
c a l h e l p and f r i e n d s h i p .
I w ould a l s o l i k e t o t h a n k my w i f e , S a r a h , f o r h e r
e n c o u r a g e m e n t, l o v e , p a t i e n c e and t y p i n g a b i l i t y d i s p l a y e d
d u r i n g t h i s w ork.
I a ls o a p p r e c ia te th e t r a i n i n g in th e a r t
o f mouse c a t c h i n g r e c e i v e d from my d a u g h t e r , ,H e a t h e r .
TABLE OF CONTENTS
Page
V I T A ................... .... ............................................................................ ....
ACKNOWLEDGMENTS
. . . . . . . . . .
TABIjE OF CONTENTS
. . . .
..................................................................................
LIST OF TABLES................... ....
LIST OF FIGURES
...................
. .
ii
ill
iv
. . ...............................................
v ii
.................................................................................
x
ABSTRACT ..................................................... ................................. ....
INTRODUCTION . . . . . .
MATERIALS AND METHODS
........................ '.
. . . . .
. . .
xi
..................................
I
.......................
14
P a r a s i t e s ............................
l4
F e c a l E x a m in a t io n s
15
A n im als
.........................................................
.................................
'15
N e c ro p sy and Worm R e c o v e ry ..........................................................
16
EP$
17
. . . . . . . . . . . . . . . . . . . . . . . .
C e ll T ra n s fe r
T ry p an B lu e
. . . . . . . .
................................................
17
. . . . . . . . .
...............................................
17
A s p i r i n .......................... . . . . ' ....................................................
18
I n d o m e t h a c i n ............................................................................................
18
P e r Os T r e a tm e n t P r o c e d u r e ..........................................................
19
Ammonium S u l f a t e P r e c i p i t a t i o n o f Serum
19
I r r a d i a t i o n o f Mice
SRBC P l a q u e A s sa y
. . . . . .
..............................................................
...................................................................................
20
20
V
L ap o ro to m y f o r I n s e r t i n g A d u l t Worms
In tra d u o d e n a lly
. . . . ..........................................................
21
In tra d u o d e n a l I n je c tio n s
21
Serum
. . . . . . . . .
.........................
.................................. .... ..................................................................
F r e e z i n g , Thawing a n d H e a t i n g S e r u m ........................ ....
C y c lo p h o sp h am id e ...................
PCA
P ro s ta g la n d in s
. . . . . . . . . .
S t a t i s t i c a l T r e a tm e n t
RESULTS
...................
.
22
.............................
22
...........................................
22
......................................
23
. . . . . .
. . . . . . . . . . . . . .
. . . .
. . . . . . .
21
...................
......................
. . . . . . .
. . . . .
23
25
C h ro n ic T ry p a n B lu e T r e a tm e n t o f Mice and t h e
E l i m i n a t i o n o f JNL_ b r a s i l i e n s i s ...........................................
25
P r o s t a g l a n d i n s a n d t h e R esponse o f Mice t o a
L a r v a l I n f e c t i o n o f Ni b r a s i l i e n s i s
.......................
.
35
w i t h IMLNC t o Normal Mice . . . . .
43
Immune Serum G iven
a)
. b)
c)
C l a s s i f i c a t i o n o f Immune Serums a s Mouse
P r o t e c t i v e o r Worm P r o t e c t i v e ...................
43
Ammonium S u l f a t e F r a c t i o n a t i o n o f Mouse
P r o t e c t i v e Serum . . . . . .
.................................
45
Mouse P r o t e c t i v e Serum A d s o rb e d on IL 3
an d A b s o rb e d w i t h IL 3 ................................................
48
A d o p tiv e T r a n s f e r w i t h IMLNC t o Mice o f Im m unity
. t o Ni b r a s i l i e n s i s L a r v a l I n f e c t i o n W ith o u t
Immune Serum . . . . . . .
................................................
.
52
• M e s e n t e r i c L ym phadenopathy O b s e rv e d D u rin g
N. b r a s i l i e n s i s L a r v a l I n f e c t i o n . ..................................
60
vi
V a r io u s D oses o f Y - I r r a d i a t i o n o f Mice on
S u b s e q u e n t IL 3 C h a ll e n g e a n d t h e R e s t o r a t i o n
o f Normal E x p u l s i o n w i t h IMLNC i n I r r a d i a t e d
MlC e
e
e
o
e
e
e
o
o
e
e
e
o
o
e
o
s
e
o
e
e
e
e
e
'
62
Impiune Serum T e s t e d F o r I t s M o u s e - P r o t e c t i v e
A c t i v i t y When G iven A lo n e . . . . . . . . . . . . . .
69
T r a n s f e r r i n g P a s s i v e Im m unity I n t o Nudes U s in g
IMLNC and P r o t e c t i v e Serum
. . . . . . . . . .
SG
DISCUSSION
. . . . . . . . . . . . . . . . . . . . . .
LITERATURE CITED
.............................................................................
8?
101.
LIST OF TABLES
T able
I.
II.
III.
IV.
V.
VT.
V II.
V III.
IX.
X.
X I.
. '
Page
S p e c ific
b r a s i l i e n s i s PCA t i t e r o f serum
o f n o rm a l and t r y p a n b l u e - t r e a t e d m ice
. . . .
28
E f f e c t s o f t r y p a n b l u e t r e a t m e n t o f m ice on
a N. b r a s i l i e n s i s l a r v a l i n f e c t i o n
. . . . . .
29
Worms r e c o v e r e d a f t e r t r a n s f e r o f n o rm a l
r a t worms i n t o n o r m a l, t r y p a n b l u e - t r e a t e d
and nude m ic e ........................................... .................................
3^
EPG an d worms r e c o v e r e d fro m
b ra s ilie n s is
i n f e c t e d m ic e t r e a t e d w i t h a s p i r i n
. . . . . .
37
EPG an d worms r e c o v e r e d fro m N1, b r a s i l i e n s i s
i n f e c t e d m ice t r e a t e d w i t h i n d o m e t h a c in . , . .
39
The e f f e c t o f p r o s t a g l a n d i n (PGE^, PGEg,
PGFgoli) i n j e c t e d i n t r a d u o d e n a l l y on day 5 o f
a l a r v a l i n f e c t i o n i n n o r m a l m ice . . . . . . ’.
,
The e f f e c t o f p r o s t a g l a n d i n (PGE^, PGEg
PGFgoO i n j e c t e d i n t r a d u o d e n a l l y on day 5 o f
a l a r v a l i n f e c t i o n i n nude m i c e .............................. .
42
A c t i v i t y o f ammonium s u l f a t e f r a c t i o n s o f a
mouse p r o t e c t i v e serum
. . . . . .
........................
49
The e f f e c t o f • i n v i t r o i n c u b a t i o n i n
p r o t e c t i v e serum on IL 3 a n d t h e a b i l i t y
o f i n v i t r o i n c u b a t i o n o f IL 3 t o d e p l e t e
serum o f p r o t e c t i v e f a c t o r ...........................................
51
The a b i l i t y o f IMLNC c o l l e c t e d a f t e r a
p r i m a r y o r s e c o n d a r y i n f e c t i o n o f N.
' b r a s i l i e n s i s g iv en w ith o r w ith o u t p r o te c tiv e
serum t o e f f e c t a d o p t i v e t r a n s f e r o f p r o t e c t i o n .
from a 300 IL 3 c h a l l e n g e t o n a i v e m ic e
. . . . .
E x p e r im e n t A.
The a b i l i t y o f MLNC c o l l e c t e d
on d i f f e r e n t d ays a f t e r a p r i m a r y i n f e c t i o n
o f m ice t o a d o p t i v e l y p r o t e c t n a i v e m ice
from a c h a l l e n g e w i t h 300 IL 3 . . . . . . . . .
54
56
v iii
• X II.
X III.
XIV.
Xy.
XVI.
XVII.
X V III.
XIX.
jQC,
E x p e r im e n t B.
The a b i l i t y o f MLNG
c o l l e c t e d on d i f f e r e n t days a f t e r a
p r i m a r y i n f e c t i o n o f m ic e t o a d o p t i v e l y
p r o t e c t , n a i v e m ice from a c h a l l e n g e w i t h
IL 3 ...................................... ......................................................... ....
57
The a b i l i t y o f MLNC c o l l e c t e d fro m d o n o r
m ic e on d i f f e r e n t d ays a f t e r a s e c o n d a r y
i n f e c t i o n w i t h 300 IL 3 t o p r o t e c t n a i v e
r e c i p i e n t m ic e from a c h a l l e n g e w i t h
la rv a e
....................................... . . . . . . . . . . .
58
The num ber o f n u c l e a t e d c e l l s r e c o v e r e d from
t h e MLN o f m ic e a t v a r i o u s t i m e s a f t e r a .
p r i m a r y o r s e c o n d a r y N^_ b r a s i l i e n s i s
in fe c tio n . . . . . . .
........................ . ...................
6l
The e f f e c t on t h e e x p u l s i o n o f worms o f
d i f f e r e n t d o s e s o f Jf*- i r r a d i a t i o n g iv e n b e f o r e a N, b r a s i l i e n s i s l a r v a l i n f e c t i o n
64
. . .
The a b i l i t y o f day 10 IMLNC t o r e p a i r t h e
e x p u l s i v e m echanism o f m ic e g i v e n 450 o r
550 r a d s ' / - i r r a d i a t i o n b e f o r e a l a r v a l
c h a l l e n g e ........................................... ............................ ....
? •
65
The a b i l i t y o f day 9 IMLNC o r n o rm a l
th y m o c y te s t o r e c o n s t i t u t e t h e a b i l i t y o f
m ice g i v e n 5 5° r a d s o f ^ - i r r a d i a t i o n b e f o r e
a l a r v a l c h a lle n g e to ex p el th e i n f e c ti o n . . .
67
D i r e c t p l a q u e - f o r m i n g c e l l s (PFC) a g a i n s t
SRBC on day 4 a f t e r a n t i g e n c h a l l e n g e i n
m ice g i v e n 55° r a d s and I x 10° th y m o c y te s
18 d ays b e f o r e
. ........................................... .......................
68
The e f f e c t o f mouse p r o t e c t i v e serum on a
300 ILo i n f e c t i o n when g i v e n a l o n e o r w i t h
IMLNC ............................. ..................................................................
70
The e f f e c t on EPG and worm r e c o v e r y o f
mouse p r o t e c t i v e serum a l o n e
. . . . . . . . .
72
ix
XXI.
The e f f e c t o f one o r two i n j e c t i o n s o f
mouse p r o t e c t i v e serum on a l a r v a l
in fe c tio n
. . . . . . . . . . . . . . . . . .
XXII.
'!
E f f e c t o f mouse p r o t e c t i v e serum g i v e n on '
day 0 o r day 3 o f a l a r v a l i n f e c t i o n ...................
75
X X III.
E f f e c t o f h e a t o r f r e e z e - t h a w on t h e a c t i v i t y .
o f mouse p r o t e c t i v e serum . ............................. ....
77
The e f f e c t on a l a r v a l i n f e c t i o n o f m ice
o f p r o t e c t i v e serum i n j e c t e d i n t o t h e •
duodenum . . . . . . . . . . . . . . . . . . .
79
E f f e c t o f c y c lo p h o s p h a m id e o r c y c lo p h o s p h a m id e
a n d p r o t e c t i v e serum on a l a r v a l i n f e c t i o n
o f m ice . . . . . . . . . . . . . . .
...................
81
The e f f e c t o f p r o t e c t i v e serum o r p r o t e c t i v e
serum and IMLNC on a l a r v a l i n f e c t i o n . o f .
nude m ice
..............................................................
.
83
The e f f e c t o f v a r i o u s c o m b i n a t i o n s o f
p r o t e c t i v e se ru m , IMLNC and th y m o c y te s on
a l a r v a l i n f e c t i o n o f . n u d e m ice . . . . . . . .
85
XXIV.
XXV.
XXVI.
XXVII.
LIST OF FIGURES
F ig u re
1.
2.
3.
4-
5.
Page
The e f f e c t o f c h r o n i c t r y p a n b l u e t r e a t m e n t
on t h e EPG p r o d u c t i o n o f a Nj_ b r a s i l i e n s i s
l a r v a l i n f e c t i o n .........................................................
26
The e f f e c t o f t r y p a n b l u e t r e a t m e n t o f m ice
on t h e e l i m i n a t i o n o f n o r m a l r a t worms
g i v e n by l a p o r o t o m y t o m ice
...........................................
31
The e f f e c t o f t r y p a n b l u e t r e a t m e n t o f m ic e
on t h e e l i m i n a t i o n o f n o r m a l r a t worms
g i v e n by l a p o r o t o m y t o n o r m a l and nude m ic e
. .
33
EPG f e c e s from m ice g i v e n IMLNC and p o o l s o f
immune serum w h ic h w ere f o u n d t o be "worm
p ro te c tiv e "
.............................................................................
46
EPG f e c e s from m ice g i v e n IMLNC and p o o l s o f
immune serum w h ic h w ere f o u n d t o be "mouse
p r o t e c t i v e " .............................................................. ....
4?
ABSTRACT
E i t h e r c e l l s o r serum fro m immune a n im a l s h a s b e e n
f o u n d t o be a b l e t o a b r o g a t e t h e p a t e n c y of a l a r v a l
bras i l i e n s i s i n f e c t i o n when g i v e n t o n a i v e m ic e . A l l p o o l s o f
c e l l s t h a t c o u l d c a u s e a b r o g a t i o n o f a l a r v a l p a t e n c y were
m e s e n t e r i c lymph node c e l l s c o l l e c t e d from m ice on d ays 9 -1 2
a f t e r a p r i m a r y N1. b r a s i l i e n s i s i n f e c t i o n .
T h is c o r r e l a t e d
w i t h a ly m p h a d e n o p a th y t h a t was shown t o o c c u r a t a b o u t days
6 -1 2 a f t e r l a r v a l i n f e c t i o n .
No c e l l p o o l s c o l l e c t e d from
seco n d a ry i n f e c t i o n s d e m o n stra te d th e a b i l i t y to p r o t e c t a
n a i v e mouse t o t h i s e x t e n t n o r d i d c e l l s c o l l e c t e d b e f o r e o r
a f t e r th is p e rio d .
P o o l s o f se ru m from m ice 13 d a y s a f t e r a
s e c o n d i n f e c t i o n w ere commonly f o u n d t o be a b l e t o t r a n s f e r
as h ig h a l e v e l o f p r o t e c t i o n as p r o t e c t i v e c e l l s .
A lo t o f.
v a r i a b i l i t y i n t h e d e g r e e o f p r o t e c t i v i t y was f o u n d i n b o t h
t h e serum and c e l l p o o l s , ev en i n p o o l s t h a t w ere a p p a r e n t l y
p r e p a r e d i n t h e same m an n e r.
The serum f a c t o r was f o u n d t o p r e c i p i t a t e i n 50$
s a t u r a t e d NH^C8 0 4 ) 2 » was r e s i s t a n t t o 56 C f o r 30 m i n u t e s , '
was d i a l y z a b l e , was f r e e z e - t h a w r e s i s t a n t and a p p a r e n t l y ■
d i d n ' t b i n d t o l a r v a e i n a way t h a t a d a p t i v e l y t r a n s f e r r e d
immune c e l l s c o u l d a c t on t h e l a r v a e .
P r o t e c t i v e serum was
more e f f e c t i v e when g i v e n on day 3 o f a l a r v a l i n f e c t i o n
t h a n when g i v e n on day 0 o n l y .
P r o t e c t i v e serum i n j e c t e d
i n t r a d u o d e n a l l y on day 4 o f an i n f e c t i o n had no e f f e c t on
t h e p a t e n c y o f t h e worm i n f e c t i o n .
E x p u l s i o n o f a N1. b r a s i l i e n s i s l a r v a l i n f e c t i o n by m ice
i s v e r y r a d i o - r e s i s t a n t a s shown by n o rm a l e x p u l s i o n a f t e r
350 r a d s on day - I
o n l y s l i g h t l y d e l a y e d e x p u l s i o n a f t e r 450.
rads,
The l e s i o n c r e a t e d by. 4 5 0 -5 5 0 r a d s g i v e n b e f o r e a
l a r v a l i n f e c t i o n c a n be r e p a i r e d by I x 10° immune m e s e n t e r i c
lymph node c e l l s an d worms a r e e x p e l l e d n e a r l y a t t h e n o rm a l
tim e i n m ice g i v e n 550 r a d s and n o r m a l th y m o c y te s on day - I .
A t t e m p t s w ere made t o t r a n s f e r im m unity t o nude m ice
w i t h p r o t e c t i v e immune serum o r immune m e s e n t e r i c lymph node
c e l l s o r a c o m b i n a t i o n o f t h e tw o , none o f w h ic h t r a n s f e r r e d
any a p p a r e n t , p r o t e c t i o n .
E v id e n c e i s p r e s e n t e d t h a t s u g g e s t s t h a t t h e e x p u l s i o n
o f N. b r a s i l i e n s i s from m ice i s n ' t a s p r o s t a g l a n d i n d e p e n d e n t
a s t h e NU_ b r a s i l i e n s i s r a t s y s te m .
N e ith e r a s p i r i n n o r in d o m e'th a cin d e l a y e d e x p u l s i o n t i m e s .
E x p e r im e n ts i n w hich
p r o s t a g l a n d i n s w ere i n j e c t e d i n t r a d u o d e n a l l y a l s o h a d l i t t l e
e f f e c t on an o n g o in g i n f e c t i o n w h ic h i s d i f f e r e n t t h a n had
b e e n shown i n t h e r a t s y s te m .
The r o l e o f m a c ro p h a g e s i n t h e e x p u l s i o n o f a Nl b r a s i l ­
i e n s i s i n f e c t i o n i n m ice was a p p r o a c h e d by u s i n g a m acrophage
x ii
ly so z y m e i n h i b i t o r , t r y p a n b l u e . L a r v a l
b ra s ilie n s is
i n f e c t i o n s i n m ic e p r e t r e a t e d w i t h t r y p a n b l u e , t h e n m a in ­
t a i n e d on a c h r o n i c t r e a t m e n t s c h e d u l e m a i n t a i n e d t h e i r worms
lo n g e r th a n c o n tr o ls .
Mice g i v e n n o rm a l a d u l t worms i n t r a d u o d e n a l l y a f t e r p r e t r e a t m e n t and c o n t i n u e d c h r o n i c t r e a t m e n t
w i t h t r y p a n b l u e e x p e l l e d t h e i r worm b u r d e n a b o u t t h e same
tim e a s n o n - t r y p a n b l u e t r e a t e d m ic e .
These r e s u l t s s u g g e s t
t h a t t r y p a n b l u e i n t e r f e r e d in. a n a f f e r e n t v s . e f f e c t o r . i m m u n e
fu n c tio n .
INTRODUCTION
T here a r e s e v e r a l r e a s o n s t o s t u d y t h e immune r e s p o n s e
o f mammalian h o s t s t o i n t e s t i n a l n e m a to d e s .
The m o st ob­
v i o u s i s t h e l a r g e num ber o f humans an d t h e i r e c o n o m i c a l l y
i m p o r t a n t d o m e s tic a n i m a l s • t h a t a r e i n f e c t e d w i t h n e m a to d e s
(1 8 ,3 2 ).
are :
Some o f t h e more common human i n t e s t i n a l n e m a to d e s
A s c a r i s , w h ich i n f e c t s a p p r o x i m a t e l y one i n e v e r y f o u r
p e o p l e i n t h e w o r ld ; hookworms, w h ic h i n f e c t a p p r o x i m a t e l y
400 m i l l i o n p e o p l e ; and T r i c h u r i s , w hich i n f e c t s a p p r o x i ­
m a t e l y 350 m i l l i o n p e o p l e w o rld w id e ( 5 3 ) •
I n t e s t i n a l nema­
t o d e s a r e n ' t j u s t a t h i r d w o r ld p r o b le m a s shown by W a r r e n 's
e s t i m a t e s o f human i n f e c t i o n i n t h e U n i t e d S t a t e s ( 6 4 ) .
O t h e r s t u d i e s show h a l f o r more o f c h i l d r e n i n some lo w incom e r u r a l c o m m u n itie s i n t h e S o u t h e a s t U .S . a r e i n f e c t e d
w ith A s c a ris o r T r ic h u r is ( I ),
A s u r p r i s i n g am ount o f
d i s e a s e - - b o t h c h r o n i c d i s e a s e , s u c h a s an em ia a n d m a l n u t r i ­
t i o n , and t h e more a c u t e m e d i c a l e m e r g e n c ie s , w h ic h a r e
u s u a l l y c a u s e d by A s c a r i s — o c c u r s i n t h e U .S . a s a r e s u l t
o f i n t e s t i n a l n em ato d e i n f e c t i o n .(I ).
The im m u n o lo g ic a l r e s p o n s e o f humans t o t h e s e n e m a to d e s
i s n ? t w e ll s tu d ie d .
The a p p a r e n t l a c k o f e x p u l s i o n o f t h e
a d u l t worms and e v e n t h e a p p a r e n t l a c k o f d e v e l o p e d r e s i s r t a u c e t o s u b s e q u e n t l a r v a l i n f e c t i o n i s w e l l known ( 4 7 )
( a l t h o u g h n o rm a l a d u l t s i n A s c a r i s endem ic a r e a s seem t o be
2
more r e s i s t a n t t o c h a l l e n g e t h a n t h e i r c o u n t e r p a r t s from
non en d em ic a r e a s ( 3 7 ) ) «
The l a c k o f p r o t e c t i o n i s e v i d e n t
ev en i n a p p a r e n t l y n o rm a l a d u l t i n d i v i d u a l s , e v e n th o u g h
t h e i n d i v i d u a l s h a v e a n t i b o d i e s o f s e v e r a l c l a s s e s to
S e v e r a l a n t i g e n s r e l e a s e d by o r p r e s e n t
(3 7 ,3 8 ).
on t h e p a r a s i t e s
Some r e s e a r c h e r s have d e m o n s t r a t e d c e l l - m e d i a t e d
im m unity t o be a c t i v e i n i n t e s t i n a l nem atode i n f e c t i o n s i n
humans w i t h o u t e v i d e n c e o f p r o t e c t i v e im m unity ( 59) >
C u r r e n t l y , t h e b e s t way t o c o n t r o l m o st o f t h e s e i n t e s ­
tin a l in fe c tio n s,
a t l e a s t i n h u m an s, i s e d u c a t i o n and h i g h ­
e r liv in g sta n d a rd s.
How ever, c o n s i d e r i n g t h e p o o r r e s p o n s e
t o t h i s a p p r o a c h , v a c c i n e s seem t o be a u s e f u l , p o s s i b l e a d ­
j u n c t .• B e c a u se o f t h e p a r a s i t e - r e l a t e d p r o b le m s i n c u r r e d i n
l i v e s t o c k m an a g em en t, n e m a to d e v a c c i n e s w i l l a lw a y s b e . i n
demand (47)»
Much e f f o r t h a s b e e n made i n t r y i n g t o p r e p a r e
v a c c i n e s i n n e m a t o d e - h o s t s y s te m s w i t h v e r y few u s a b l e r e ­
su lts
( 5 )The f i r s t v a c c i n e s t e s t e d f o r nem atode d i s e a s e s were
p a tte r n e d a f t e r th e b a c t e r i a l v a c c in e s .
A tte n u a te d ( i r r a d i ­
a t e d ) worms and v a r i o u s p r e p a r a t i o n s o f k i l l e d worms haVe
been t r i e d .
T hese have m u l t i p l e d e f i c i e n c i e s s u c h a s :
lac k
o f e f f e c t i v e p r o t e c t i o n , b e in g h a rd to p re p a re and s t o r e ,
and r e q u i r i n g a d j u v a n t s and l a r g e v o lu m e s.
3
The n e x t s t e p i n p r e p a r i n g v a c c i n e s a g a i n s t . i n t e s t i n a l
n e m a to d e s seems t o be f i n d i n g o u t how. t h e s e p a r a s i t e s can
be a c t e d upon by t h e h o s t ,
p o n sib le f o r p r o te c tio n ,
t h e h o s t r e s p o n s e w h ic h i s r e s ­
an d t h e a n t i g e n t h a t s t i m u l a t e s
t h i s p r o t e c t i v e r e s p o n s e (5)■
The b e s t s y s te m i n w hich to
s t u d y t h i s p r o b le m w ould be a p a r a s i t e t h a t s t i m u l a t e s
l o n g - l a s t i n g a n d c o m p le te im m unity i n i t s h o s t .
S ev eral
su c h s y s te m s w i t h l a b o r a t o r y a n im a l s a s t h e h o s t a r e known
( 6 2 ) , b u t e v e n i n t h e b e s t c h a r a c t e r i z e d o f t h e s e s y s te m s ,
n e i t h e r t h e m echanism o f e x p u l s i o n n o r t h e p r o t e c t i v e a n t i g en (s)
is
known.
The N i p p o s t r o n g y l u s b r a s i l i e n s i s r a t s y s te m i s p r o b a b l y
t h e m o st c o m p l e t e l y s t u d i e d o f a l l o f t h e i n t e s t i n a l nema­
to d e -h o s t sy ste m s.
I w i l l now c o n f i n e m ost o f my re m a rk s
t o t h i s s y s te m b e c a u s e t h e s y s te m I am u s i n g i s t h e a n a l o ­
gous s y s te m o f N^_ b r a s i l i e n s i s i n m i c e .
The r a t s y s te m h a s
b e e n e x t e n s i v e l y r e v i e w e d ( 1 0 , 1 1 , 1 8 , 2 7 » 4 2 ,4 3 » 4 4 ) i n t h e
f a i r l y r e c e n t l i t e r a t u r e a n d ' I w on’ t a t t e m p t t o d u p l i c a t e
th is .
The b i o l o g y , h o s t , g e o g r a p h i c a l d i s t r i b u t i o n ,
t e m a t i c s and l i f e
H a ley ( 1 0 , 1 1 ) .
sys-
c y c l e have b e e n a d e q u a t e l y r e v i e w e d by
I w i l l b r i e f l y g iv e th e l i f e
c y cle h e r e .
N. b r a s i l i e n s i s was f i r s t d e s c r i b e d i n 1914 by T r a v a s s o s and h a s b e e n r e c o v e r e d i n t h e w i l d from R a t t u s n o r v e g i -
4.
c u s , R a t t u s r a t t u s and Mus m u s c u l a r i s .
T y p ic a l o f a l l
n e m a t o d e s 9 t h e r e a r e f o u r m o u lts d u r i n g t h e l i f e
■
c y c le o f
b ra s ilie n s is.
The im m ature e g g s a r e p a s s e d i n t h e f e c e s
o f th e h o s t and,
i n p r o p e r c o n d i t i o n s , e m b ry o n a te and h a t c h
in a b o u t I day.
The f i r s t m o u lt o c c u r s on a b o u t day I ,
w ith th e second s h o r t l y t h e r e a f t e r .
These f i r s t two l a r v a l
s t a g e s , c a l l e d r h a b d i t o i d l a r v a e , f e e d on l i v e b a c t e r i a i n
s o il- f e c e s o r fe c e s-c h a rc o a l m ix tu re s.
The t h i r d l a r v a l
s t a g e , w h ich i s t h e f i l a r i f o r m o r i n f e c t i v e s t a g e , c r a w l up
t o a h i g h p o i n t w here t h e y w a it, u n t i l a p r o s p e c t i v e h o s t
p a sse s by.
The l a r v a e , w hich a r e a c t i v e l y t h e r m o t a c t i c ,
p e n e t r a t e n o rm a l r a t s k i n and f e e d t h e i r way t o t h e lu n g s
v i a t h e b l o o d s t r e a m a n d / o r l y m p h a t i c c h a n n e ls i n 11-1 5
h oi^rs.
The l a r v a e f e e d on b lo o d i n t h e l u n g s and m o u lt a t
a b o u t 36 h o u r s a f t e r i n f e c t i o n .
They a r e t h e n c oughed up ■
and sw a llo w e d and t h e f o u r t h s t a g e l a r v a e r e a c h t h e s m a l l
i n t e s t i n e w here t h e y t a k e up r e s i d e n c e and b e g i n t h e f o u r t h
m o u lt i n a b o u t 48 h o u r s ( 4 2 ) .
F e r t i l e eggs a r e f o u n d i n
t h e f e c e s o f t h e h o s t a t 6 d ays p o s t i n f e c t i o n .
The a d u l t
v worms l i v e i n t h e duodenum and je ju n u m w here t h e y f e e d on
h o s t t i s s u e b u t cause l i t t l e
i n t h e way o f s p e c i f i c p a t h o ­
lo g ic le s io n s in l ig h t in f e c tio n s .
The i n f e c t i o n i n r a t s i s e s s e n t i a l l y t e r m i n a t e d a f t e r
5
12 d ays ( 4 2 ) .
The worms a r e e l i m i n a t e d by s p o n t a n e o u s c u re
w h ic h may l e a v e a s m a l l r e s i d u a l worm b u r d e n b u t w hich c o n ­
f e r s c o m p le te im m unity t o s u b s e q u e n t i n f e c t i o n s .
T h is
phenomenon i s p r o b a b l y t h e b e s t s t u d i e d e x p r e s s i o n o f h o s t
im m unity t o a g u t nem atode and h a s b e e n r e v i e w e d e x t e n s i v e ­
ly (1 8 ,4 2 ,4 3 ,4 4 ).
Most o f t h e work c o n d u c t e d t o d e te r m i n e t h e a c t i v e com­
p o n e n t s and m echanism s o f a c t i o n i n t h e e x p u l s i o n o f t h e
w oras d u r i n g s p o n t a n e o u s c u r e h a s b e e n done i n r a t s .
S t u d i e s have shown t h e immune e x p u l s i o n o f l a r v a l i n f e c t i o n
i n m ice c l o s e l y p a r a l l e l s o r i s i d e n t i c a l t o t h a t s e e n i n
ra ts,
ex cep t i t occu rs a l i t t l e
10) t h a n i n r a t s
e a r l i e r i n m ice (d a y 9 o r
( day 1 2 ) ( 2 8 , 2 9 ) .
a ra t stra in of
b ra s ilie n s is.
T h is work was done w i t h
I t was f o u n d t h a t m ice
r e j e c t b o t h t h e r a t s t r a i n and t h e mouse a d a p t e d s t r a i n o f
N. b r a s i l i e n s i s on t h e same day b u t a g r e a t e r p e r c e n t a g e o f
t h e mouse a d a p t e d s t r a i n r e a c h t h e a d u l t s t a g e ( 6 6 ) .
One a p p r o a c h t o u n d e r s t a n d i n g t h e m echanism s o f ' e x ­
p u l s i o n and t o a f f i r m t h e im m u n o lo g ic a l n a t u r e o f s p o n t a n ­
e o u s c u r e i s t o t r y t o i n h i b i t e x p u l s i o n o f worm b u r d e n s by
im m u n o s u p p r e s s io n .
Some t o o l s t h a t d e l a y e x p u l s i o n a r e
n e o n a t a l thym ectom y, c o r t i s o n e d e r i v a t i v e t r e a t m e n t , l e t h a l
x -irra d ia tio n ,
t h o r a c i c d u c t d r a i n a g e , and a n t i t h y m o c y t e
6
serum ( 2 7 ) .
R e c e n tly ,
i t h a s b e e n shown t h a t a th y m ic m ice
( n u d e ) d o n ' t e x p e l a worm b u r d e n u n l e s s t h e y a r e thymus
g r a f t e d o r i n j e c t e d w i t h th y m o c y te s ( 1 5 » 1 6 ,3 4 ) .
g e th e r,
Taken t o ­
t h e s e r e s u l t s s u g g e s t a thymus d e p e n d e n t s t e p i s
n e c e s s a r y i n t h e d e v e lo p m e n t o f immune e x p u l s i o n o f N.
b ra s ilie n s is.
R a ts w ere o b s e r v e d t o r e t a i n t h e i r worm b u r d e n l o n g e r
t h a n n o r m a l when i n f e c t e d d u r i n g two s p e c i a l p e r i o d s o f t h e
n o rm a l r a t l i f e
c y c le .
One was d u r i n g t h e n e o n a t a l p e r i o d
b e f o r e 7 - 9 weeks o f a g e , d e p e n d i n g - o n t h e s t r a i n
t h e o t h e r was d u r i n g l a c t a t i o n ( 4 ) .
i n g a f t e r day
c a re fu lly ,
( 4 0 ) , and
When t h e worms r e m a in ­
from e i t h e r o f t h e s e c a s e s a r e exam ined
i t i s e v i d e n t t h a t t h e y a r e a b n o rm a l; t h e s e
worms a r e g i v e n t h e l a b e l "d am a g ed ",
r a p i d l y e l i m i n a t e d from n o rm a l r a t s
i n t o t h e duodenum.
Damaged worms a r e
i f tra n sfe rre d d ire c tly
Normal w orm s, when t r a n s f e r r e d ,
are
e l i m i n a t e d i n 8 - 1 0 d a y s , b u t damaged worms a r e e l i m i n a t e d
in 5-6 days.
The damaged worms h a v e a l t e r e d g u t s t r u c t u r e ,
t h e c e l l s a r e v a c u o l a t e d and t h e g u t i s d i s t e n d e d .
T here
a r e c h a n g e s i n iso e n z y m e p a t t e r n s , r e d u c e d f e e d i n g on h o s t
t i s s u e a s m e a s u re d by
u p t a k e from t h e h o s t ,
an d t h e
f e m a le worms have a r e d u c e d num ber o f eggs i n t h e i r u t e r i .
D u rin g a n i n f e c t i o n i n a n o rm a l r a t ,
t h e worms a r e f o u n d t o
7
be damaged by day 11 a l s o , b u t a r e t h e n r a p i d l y e l i m i n a t e d
frorn t h e h o s t ( 4 4 ) .
I n m ic e , t h e damage o c c u r s e a r l i e r and
i s ,e v id e n t by day 6 ( 2 8 ).'
A s e c o n d m eth o d u s e d t o d e t e r m i n e t h e m echanism o f
s e l f - c u r e ji s t o a t t e m p t t o p a s s i v e l y immunize n a i v e a n im a ls
w i t h com ponents fro m immune a n i m a l s .
S ev eral d if f e r e n t
i n v e s t i g a t o r s h a v e f o u n d t h a t some d e g r e e o f p r o t e c t i o n can
be a c h i e v e d w i t h c e r t a i n serum p o o l s ( 4 l , 5 2 ) .
L a rg e
am ounts o f serum a r e r e q u i r e d and t h e p a s s i v e p r o t e c t i o n i s
g e n e r a l l y n o t n e a r l y a s e f f e c t i v e a s a c t i v e im m unity i n
e l i m i n a t i n g worms.
te s te d ,
I n a l a r g e g ro u p (48 p o o l s ) o f s e r a
I o u t o f 3 p o o l s was somewhat, p r o t e c t i v e
(4 l).
H y p e r im m u n iz a tio n o f d o n o rs w i t h r e p e a t e d l a r v a l i n f e c t i o n s
d i d n ’ t seem t o i n c r e a s e t h e p r o b a b i l i t y o f a p o o l b e i n g
p r o t e c t i v e o r in c r e a s e th e t i t e r o f p r o t e c t i o n (4 1 ).
A few
c e l l p r e p a r a t i o n s from immune r a t s a n d m ice h a v e b e e n f o u n d
w hich w i l l p r o t e c t nonimmune a n im a l s when t r a n s f e r r e d a lo n e
i n t o them .
However, p r o t e c t i o n a p p r o a c h i n g t h a t s e e n i n
immune a n im a l s c h a l l e n g e d w i t h a l a r v a l i n f e c t i o n h a s b e en
a c h i e v e d r e p r o d u c a b l y o n ly by g i v i n g a r a t o r mouse b o th
immune c e l l s and immune serum b e f o r e c h a l l e n g e ( 2 8 , 2 9 ).
C om bining t h e two t o o l s o f p a s s i v e and a d o p t i v e immuni­
z a t i o n an d t h e n e o n a t a l and l a c t a t i n g r a t m o d e ls w h ic h a r e
8
d e f e c t i v e i n worm e x p u l s i o n , a h y p o t h e s i s o f two s t e p r e ­
q u irem en t f o r e x p u ls io n has been p u t f o r t h (4 2 ).
B oth n e o ­
n a t a l r a t s and l a c t a t i n g r a t s c a n c a u s e t h e p h e n o t y p i c
c h a n g e s i n worms w h ic h i s c h a r a c t e r i s t i c o f dam age, b u t i n
b o t h o f t h e s e s y s te m s t h e a d u l t worms a r e e x p e l l e d v e r y
slo w ly .
Immune c e l l s t r a n s f e r r e d i n t o e i t h e r o f t h e s e s y s ­
tem s fro m a d u l t m ic e w i l l c a u s e e x p u l s i o n i n n o r m a l a n i ­
m als.
Serum t r a n s f e r r e d from n e o n a t a l and l a c t a t i n g r a t s
h a s b e e n shown t o c a u s e i n c r e a s e d p r o t e c t i o n o f r e c i p i e n t s a g a in s t l a r v a l c h a lle n g e .
From t h i s work i t i s f e l t t h a t ;
t h e r e i s a h u m o ra l f a c t o r ( a n t i b o d y ) t h a t damages t h e worms
w h ich m u st p r e c e d e t h e s t e p r e q u i r i n g ly m p h o c y te s w hich ■
causes th e ra p id e x p u ls io n (4 2 ,4 4 ).
S e v e ra l gro u p s o f r e s e a r c h e r s th e n a tte m p te d to d e fin e
t h e dam aging f a c t o r ( s ) and t h e e x p u l s i v e f a c t o r ( s ) .
stu d y th e e x p u ls iv e f a c t o r ,
To
a t r u n c a t e d i n f e c t i o n was u s e d .
Worps w ere l e f t i n one r a t u n t i l damaged, t h e n t r a n s f e r r e d
t o t h e e x p e r i m e n t a l h o s t t o d e t e r m i n e w hat c e l l s w ere r e ­
q u i r e d f o r t h e s e c o n d r a t t o e l i m i n a t e t h e worms ( 4 2 , 4 4 ) .
U s in g t h i s a p p r o a c h , damaged worms w ere t r a n s f e r r e d
in to r a t s
i r r a d i a t e d a t s u b l e t h a l (440 r a d s ) o r . l e t h a l (750
radg) l e v e l s .
T hese r a t s w ere t h e n r e c o n s t i t u t e d t o f i n d
o u t th e c e l l u l a r req u irm en t f o r e x p u ls io n .
I t was fo u n d
9
t h a t t h e l e t h a l l y - i r r a d i a t e d a n i m a l s r e q u i r e d b o t h an im­
mune lymph node c e l l , p r e s u m a b ly l y m p h o c y te s , and a bone
m arrow com ponent t o e f f e c t e x p u l s i o n w i t h i n 4 d a y s .
The
s u b l e t h a l l y i r r a d i a t e d a n im a l n e e d e d o n ly t h e immune .lymph
node c e l l ( 6 , 2 5 ) .
S e v e r a l c e l l t y p e s w ere p o s t u l a t e d a s
b e i n g t h e one s u p p l i e d by t h e bone m arrow .
One o f t h e more
f a v o r e d p o s s i b i l i t i e s was t h e m onocyte o r m ac ro p h ag e ( 4 4 ) .
I t r e c e n t l y h a s b e e n shown t h a t r a t s l e t h a l l y i r r a d i a t ­
ed w i t h 6 5 0 - 7 5 0 r a d s ,
d e n a lly ,
t h e n g i v e n damaged worms i n t r a d u o - ' '
c an be r e c o n s t i t u t e d t o e x p e l t h e worms i n 3 days
i f g iv en Ig
t h o r a c i c d u c t ly m p h o c y te s c o l l e c t e d from r a t s
i n f e c t e d 7 - 8 days o r 13 days b e f o r e c o l l e c t i o n .
One o f two
p o o l s o f MLNC a l s o c a u s e d e x p u l s i o n from l e t h a l l y i r r a d i ­
a t e d a n im a l s ( 4 5 ) .
' A n o t h e r i n t e r e s t i n g t h i n g shown by t h i s work i s t h a t
e v e n th o u g h t h e r e a r e i n t h o r a c i c d u c t lymph on d a y s 7 and
8 c e l l s c a p a b le o f c a u s in g e x p u ls io n ,
t h e worms n o r m a l l y
w o u l d n 't e v e n be damaged u n t i l day 10 o r 1 1 .
T hese . r e s u l t s
s u g g e s t t h a t i n a p r i m a r y i n f e c t i o n w i t h Ni . b r a s i l i e n s i s
t h e dam aging s t e p a c t u a l l y o c c u r s a f t e r t h e r e a r e c e l l s
c a p a b le o f e f f e c t i n g th e e x p u ls io n i n th e t h o r a c i c d u ct
lymph, m aking t h e dam aging s t e p t h e r a t e l i m i t i n g s t e p .
It
i s w e l l d ocum ented t h a t p o o l s o f c e l l s from l i k e i n f e c t i o n s
10
have d i f f e r e n t a c t i v i t y when t r a n s f e r r e d ( 2 3 , 4 l ) .
The m echanism o f a c t i o n o f t h e c e l l c a u s i n g t h e e x p u l ­
s i o n i s unknown.
I t i s f e l t t h a t t h e worms a r e n ' t k i l l e d
i n t h e s m a l l i n t e s t i n e b u t d i e i n t h e a d v e r s e e n v ir o n m e n t
o f th e c o lo n (4 2 ) .
T h e re fo re , th e c e l l t h a t causes e x p u l­
s i o n w ould o n l y h a v e t o c a u s e t h e . worm t o l o s e i t s , h o l d f o r
a w h ile .
B e c a u se no c e l l h a s b e e n s e e n t o a t t a c h t o t h e
worm i n t h e lum en o f t h e i n t e s t i n e ,
i t i s t h o u g h t t o be a
s u b s t a n c e r e l e a s e d by c e l l s o r an e n v i r o n m e n t a l c h an g e i n
th e g u t t h a t cau ses e x p u ls io n (4 6 ).
W orkers i n t h i s s y s te m have assum ed t h a t t h e h u m o ral
dam aging f a c t o r i s a n t i b o d y and h a v e t r i e d t o f i n d t h e
c l a s s t h a t was p r o d u c e d a t t h e r i g h t : tim e i n an i n f e c t i o n
to. c a u s e damage.
One i n t e r e s t i n g t h i n g a b o u t p a r a s i t i c i n ­
f e c t i o n s h a s b e e n t h e h i g h IgE l e v e l s , b o t h s p e c i f i c f o r
worm a n t i g e n and s p e c i f i c f o r u n r e l a t e d a n t i g e n s t h e a n im a l
comes i n c o n t a c t w i t h ( 4 3 ) .
The r o l e o f IgE i n p a s s i v e
p r o t e c t i o n and i t s a c t i v e a m in e - r e le a s in g r o l e in a c t i v e
i n f e c t i o n s h a s l a r g e l y b e e n d i s c o u n t e d I n damage o r e x p u l ­
s i o n o f Njl b r a s i l i e n s i s
(4 3 ,4 4 ).
R e c e n tly , w o rm -sp e c ific
IgA h a s b e e n f o u n d i n t h e g u t o f i n f e c t e d r a t s a t t h e tim e
t h a t damage i s o c c u r i n g .
The im p o r t a n c e o f t h i s c l a s s i n
th e h o s t - p a r a s i t e r e l a t i o n s h i p h a s n 't been d e fin e d ( 4 9 ,5 4 ) .
11
One s t u d y (2 0 ) d e m o n s t r a t e d t h a t t h e i n v iv o p r o t e c t i v e
f r a c t i o n o f p r o t e c t i v e s e r a f r a c t i o n a t e d on one t y p e o f an .
i o n e x c h a n g e colum n c o n t a i n e d m o s t l y a n t i b o d y o f t h e IgQ^
s u b c l a s s and p r o t e c t i v e f r a c t i o n s w ere f o u n d t h a t c o n t a i n e d
little
IgE , IgM o r IgA.. .
One. r e a l p r o b le m w i t h s t u d y i n g t h e dam aging a n t i b o d y i s
t h a t i t can o n l y be d e m o n s t r a t e d t o damage worms i n v i v o .
Worms l i v e j u s t a s l o n g i n immune p r o t e c t i v e serum a s t h e y
do i n n o rm a l r a t serum i n v i t r o
(2 1 ,3 1 ).
A n o th e r re a so n
t h a t t h e e f f e c t o f a n t i b o d y c a n ' t be m o n i t o r e d i n v i t r o
is
b e c a u s e , ev en i n t h e optimum c u l t u r a l , c o n d i t i o n s , a f t e r t h e
worrn i s k e p t i n v i t r o f o r 3 d a y s , i t l o o k s e x a c t l y l i k e i n
v i v o damaged worms i n a l l r e s p e c t s ( 3 1 ) .
R e c e n t l y , n o n - s t e r o i d a n t i - i n f ! a m a t o r y a g e n t s su c h a s
a s p i r i n and i n d o m e t h a c in h a v e b e e n d e m o n s t r a t e d t o d e l a y
e x p u l s i o n o f worms i n a r a t l a r v a l i n f e c t i o n .
B e ca u se a
known a c t i v i t y o f t h e s e a g e n t s i s t o i n h i b i t p r o s t a g l a n d i n
s y n t h e s i s , w orkers in A u s t r a l i a (9 ) i n j e c t e d p u r i f i e d p r o s ­
t a g l a n d i n s d i r e c t l y i n t o t h e g u t on day 6 o f a n i n f e c t i o n
and f o u n d . t h a t t h i s i n c r e a s e d e x p u l s i o n .
T h is work i n t e r ­
p r e t e d w i t h some e v e n more r e c e n t work s u g g e s t s t h a t p r o s ­
t a g l a n d i n s by t h e m s e l v e s c a u s e damage t o t h e worm ( 5 0 ).
T h is i n c o n c l u s i v e e v i d e n c e ,
c o u p le d w i t h t h e f a c t t h a t
12
a serum p r o t e c t i v e c a p a c i t y d o e s n ' t c o r r e l a t e w i t h any
known a n t i b o d y a c t i v i t y and t h e f a c t t h a t s e r a from m u l t i ­
p l y i n f e c t e d r a t s d o n ’ t p r o t e c t more o f t e n n o r a t h i g h e r
d i l u t i o n s t h a n s e r a from a n im a l s i n f e c t e d o n l y o n c e , s u g ­
g e s t s a f a c t o r o t h e r t h a n a n t i b o d y b e i n g t h e "dam aging
a n t i b o d y ",
T h is i s a l s o s u g g e s t e d by t h e i n c o n s i s t e n c y o f
an alo g o u s p o o ls o f a n tis e ru m to t r a n s f e r p a s s iv e p r o t e c t i o n
( 4 l )..
Some work done i n t h e mouse m odel a l s o s u g g e s t s t h a t
th p dam aging f a c t o r may be o t h e r t h a n im m u n o g lo b u lin .
Mice
p a n s p e c i f i c a l l y 'im m u n o su p p ressed w i t h a n t i - p . e x p e l l e d t h e i r
worms a t t h e same tim e a s t h e n o rm a l c o n t r o l s u g g e s t i n g
;
th k t s p e c i f i c a n tib o d y i s n ’t n e c e s s a ry f o r e x p u ls io n ( 1 7 ) . .
T h is t h e s i s w i l l a d d r e s s t h e f o l l o w i n g a r e a s o f . t h e
N. b r a s i l i e n s i s s y s te m :
1)
The m ac ro p h ag e h a s b e e n s u g g e s t e d a s a p o s s i b l e
a c t i v e p a r t i c i p a n t i n t h e e x p u l s i o n o f N. b r a s i l i e n s i s from
ra ts.
I w i l l i n v e s t i g a t e t h e r o l e o f t h e m ac ro p h ag e i n t h e
s e l f - c u r e phenomenon i n m ice by u s i n g t h e m ac ro p h ag e I y s o some i n h i b i t o r ,
2)
try p a n b lu e .
The r o l e o f p r o s t a g l a n d i n s i n t h e e x p u l s i o n o f N.
b r a s i l i e n s i s w i l l be i n v e s t i g a t e d u s i n g p r o s t a g l a n d i n i n ­
h i b i t o r s and s y n t h e t i c p r o s t a g l a n d i n s .
3)
, ..
I b e l i e v e t h a t t h e h u m o ra l dam aging f a c t o r h a s b e en
13
in c o m p le te ly s tu d ie d .
it
O t h e r g r o u p s have j u s t assum ed t h a t
i s a n t i b o d y and t r i e d t o f i n d o u t w hat c l a s s o f immuno­
g lo b u lin i s a c tiv e o r th e a n tig e n ic s p e c i f i c i t y o f th e p ro ­
t e c t i v e a n tib o d y ,
I p r o p o s e t h a t t h e h u m o ra l p r o t e c t i v e
f a c t o r may n o t be im m u n o g lo b u lin an d w i l l a t t e m p t t o f i n d
,
p r o t e c t i v e s e r a p o o l s from immune m ice and c h a r a c t e r i z e t h e
fa c to r.
MATERIALS AND METHODS
P a ra s ite s
A m o u s e - a d a p te d s t r a i n o f N_i_ b r a s i l i e n s i s was u s e d i n
a l l e x p e r i m e n t s . ■ T h is s t r a i n was d e r i v e d from a r a t
s t r a i n by D r. R. B. W e s c o t t , ' W a sh in g to n S t a t e U n i v e r s i t y ,
P u llm a n , WA. ( 5 6 , 6 6 ) a n d h a s b e e n p a s s a g e d s e r i a l l y
t h r o u g h a t l e a s t 400 g e n e r a t i o n s i n m ic e .
N. b r a s i l i e n s i s was m a i n t a i n e d i n o u r l a b o r a t o r y by
i n j e c t i n g 900 IL^ i n 0 ,1 ml s a l i n e s u b c u t a n e o u s l y on t h e
d o r s a l a s p e c t o f t h e m ouse.
F e c e s w ere t h e n c o l l e c t e d on
d a y s 6, 7 and 8 p o s t - i n f e c t i o n by k e e p i n g m ice i n c a g e s
w i t h 0 .8 cm inesh w i r e b o tto m s o v e r m o i s t paper, t o w e l s .
The f e c e s w ere t h e n com m inuted w i t h w a t e r and m ixed w i t h
m o i s t g r a n u l a r a n im a l bone c h a r c o a l ( VWR S c i e n t i f i c , San
F r a n c i s c o ) i n a f e c e s t o c h a r c o a l r a t i o o f 1 : 4 V:V,
The
m i x t u r e was d i v i d e d b e tw e e n p e t r i d i s h e s and i n c u b a t e d a t
room t e m p e r a t u r e .
in c u b a tio n p e rio d .
M o i s t u r e was m a i n t a i n e d t h r o u g h o u t t h e
L a r v a e w ere u s e d b e tw e e n d a y s 5 and 20
a f t e r t h e c u l t u r e was i n i t i a t e d .
The l a r v a e w ere h a r v e s t e d from c h a r c o a l c u l t u r e s by
p l a c i n g 40 C 0,85% NaCl i n a s m a l l f u n n e l W ith a s c r e e n
(50 m esh/cm ) w h ic h was c o v e r e d by 2 l a y e r s o f Kimwipes
( K im b e rly C l a r k C o ., N e e n a n , W i s c o n s i n ) .
The l a r v a e
15
m i g r a t e d t h r o u g h t h e Kimwipes and w ere r e c o v e r e d a f t e r I
h o u r by o p e n in g a clam p on t h e end o f t h e f u n n e l .
The
l a r v a e w ere t h e n w ashed t w i c e i n 0.85% NaCl by c e n t r i f u g ­
i n g a t 200 g f o r I .5 m i n u t e s .
A su s p e n s io n o f 3,000 o r
9 , 0 0 0 IL ^ /m l was p r e p a r e d by c o u n t i n g worms i n 0 . 1 ml o f
su s p e n s io n u n d er a s te re o s c o p e ,
c o r r e c t volum e o f s a l i n e .
t h e n r e s u s p e n d e d in ' t h e
Mice w ere t h e n i n f e c t e d w i t h
300 o r 900 IL ^ a s a b o v e ,
F e c a l E x a m in a t io n s
The m o d i f i e d M cM aster t e c h n i q u e o f W h itlo c k ( 6 7 ) was
f u r t h e r a l t e r e d f o r u s e i n e s t i m a t i n g t h e num ber o f worm
eggs p e r gram o f f e c e s (E P G ).
F e c a l m a t e r i a l was commi­
n u t e d w i t h s a t u r a t e d NaCl ( s p . g r .
gm f e c e s t o 30 ml s a l i n e .
u s e d w i t h 7 .5 ml s a l i n e ,
1 .2 0 ) in a r a t i o o f I
Commonly, 0 .2 5 gm f e c e s was
A p o r t i o n o f t h i s s u s p e n s i o n was
r a p i d l y t r a n s f e r r e d t o a f e c a l egg c o u n t i n g cham ber
( C u t l e r - H a v e r - L o c k h a r t L a b o r a t o r i e s , Shawnee M i s s i o n ,
K S .) .
C ounts o f eggs from b o t h g r i d s o f t h e cham ber w ere
a v e r a g e d and c o r r e c t e d by a d i l u t i o n f a c t o r o f 200 t o
a r r i v e a t t h e EPG c o u n t .
A n im als
A l l m ice u s e d i n t h i s work a r e e i t h e r BALB/c r a i s e d in'
16
t h i s i n s t i t u t i o n o r a BALB/c Bom. l i n e c a r r y i n g t h e nude
( nu) gene.
The l a t t e r l i n e was b o u g h t from B o m h o ltg o a r d ,
L t d . , Ry, Denmark a n d m a i n t a i n e d i n t h i s l a b o r a t o r y by
m a t i n g h e t e r o z y g o t e s ( + / n u ) t o o b t a i n nude ( n u / n u ) c o n ­
g e n i t a l l y th u m u s- d e f i c i e n t m ice and t h e i r l i t t e r m a t e s
( h e r e a f t e r d e s i g n a t e d LM) w h ic h a r e o n e - t h i r d w ild , ty p e
( + / + ) and t w o - t h i r d s h e t e r o z y g o t e s ( + / n u ) .
A l l m ic e were
m a i n t a i n e d on Wayne Lab b l o x and a c i d i f i e d w a t e r a d l i b i ­
tu m .
A l l a n im a l s w ere b e tw e e n 8 - an d 12-w eeks o l d a t t h e .
b e g i n n i n g o f an e x p e r i m e n t u n l e s s o t h e r w i s e n o t e d .
The
rg .ts u s e d t o d o n a te worms w ere Lew is s t r a i n f e m a l e s .
N e c ro p s y and Worm R e co v e ry
Mice w ere k i l l e d by c e r v i c a l d i s l o c a t i o n an d t h e e n t i r e
s m a l l i n t e s t i n e was q u i c k l y rem oved.
The e n t i r e l e n g t h o f
t h e s m a l l i n t e s t i n e was o p e n ed w i t h s c i s s o r s and p l a c e d i n
37 C 0 . 8 # NaCl on I t h i c k n e s s o f g a u z e s u s p e n d e d i n a
p l a s t i c 250 ml s p e c im e n c u p .
The t e m p e r a t u r e was m a in ­
t a i n e d i n a w a t e r b a t h f o r 1 -2 h o u r s .
I n t e r m i t t e n t m ix in g
was u s e d t o a l l o w t h e a d u l t worms' t o m i g r a t e t o t h e b o tto m
o f th e cup.
The g u t was t h e n ex am in ed f o r r e s i d u a l worms.
The worms t h a t h a d m i g r a t e d t o t h e b o tto m o f t h e cup were
t h e n c o u n te d u n d e r a s t e r e o s c o p e a t a b o u t IOX a s t h e y were
17
p i c k e d up w i t h a P a s t e u r p i p e t t e on a 10 ml s y r i n g e .
EP$
The num ber o f eggs i n i n d i v i d u a l f e m a le worms (EP$) was
c o u n te d u n d e r a m ic r o s c o p e a t 63X on l i n e d m ic r o s c o p e
s l i d e s . EP9 from 20 t o 50 f e m a le worms w ere c o u n t e d from
e a c h mouse a n d t h e mean was c a l c u l a t e d .
EP$ fro m m ice
w h ic h h a d l e s s t h a n 20 f e m a le worms w e r e n ' t r e c o r d e d .
C e ll T ra n s fe r
C e l l s f o r t r a n s f e r w ere p r e p a r e d by d i s s e c t i n g o u t t h e
o r g a n o f i n t e r e s t - - e i t h e r t h e m e s e n t e r i c lymph node o r
th y m u s - - a n d g e n t l y s c r e e n i n g t h r o u g h a 100 mesh s t a i n l e s s
s t e e l s c re e n to p re p a re s in g le c e l l s u s p e n s io n s .
The
c e l l s w ere t h e n c o u n te d i n a hemocyt o m e t e r , w ashed once i n
b a l a n c e d s a l t s o l u t i o n and r e s u s p e n d e d t o t h e c o r r e c t co n ­
c e n t r a t i o n and, u n le s s o th e rw is e in d ic a te d ,
I x 10® c e l l s ■
w ere i n j e c t e d IV i n a 0 . 5 ml v o lu m e .
T rypan B lue
T ry p an b l u e was p u r c h a s e d from K a l l e s t a d L a b s , I n c . ,
M i n n e a p o l i s , M in n e s o ta and p r e p a r e d a c c o r d i n g to , H ibbs
(13).
The t r y p a n b l u e was d i s s o l v e d i n d o u b l e - d i s t i l l e d
w a t e r and d i a l i z e d a g a i n s t w a t e r f o r 72 h o u r s a t 4 C.
18
The d i a l y z e d m a t e r i a l was t h e n l y o p h i l i z e d and s t o r e d a t room
te m p era tu re u n t i l u sed .
T ry p an b l u e was r e s u s p e n d e d i n 0-.85$
NaCl t o a c o n c e n t r a t i o n o f 10 m g/m l.
Mice i n t h e t r y p a n b l u e
t r e a t e d g r o u p s r e c e i v e d 3 mg IP 24 h o u r s o r 48 h o u r s b e f o r e
t h e b e g i n n i n g o f t h e e x p e r i m e n t and 0 . 5 mg o r I mg IP 3 h o u r s
o r I day b e f o r e t h e e x p e r i m e n t .
Mice i n a l l t r e a t e d g ro u p s
w e re t h e n g i v e n 0 . 5 , I o r 2 mg e v e r y t h i r d day s u b c u t a n e o u s l y
u n t i l t h e e x p e r i m e n t was t e r m i n a t e d .
Dosage p r o t o c o l s a r e
.
i n d i c a t e d in f o o t n o te s to t a b l e s and f i g u r e le g e n d s .
.
A s p irin
A s p i r i n was a c q u i r e d i n t a b l e t form from B a y e r Company,
New Y ork, N.Y.
The p e r c e n t a g e a c t i v e i n g r e d i e n t ( a c e t y l -
s a l i c i l i c a c i d ) was d e t e r m i n e d .
Two h u n d r e d mg o f t h e a c t i v e
i n g r e d i e n t w ere d i s s o l v e d i n 2 ml o f 9 5 $ e t h y l a l c o h o l . t h e n
d i l u t e d t o 5° ml w i t h a 0 . 5 $ Tween 80 i n w a t e r s o l u t i o n .
T h u s , t h e f i n a l c o n c e n t r a t i o n o f a c e t y l s a l i c i l i c a c i d was
4 mg/ml a n d 20 gm m ic e w ere g i v e n 0 . 2 5 m l, w h ic h i s a p p r o x i ­
m a t e l y 50 m g/kg, t w i c e d a i l y .
C o n t r o l g r o u p s w ere g i v e n t h e -
d ilu e n t w ith o u t a s p i r i n ( 3 ,2 4 ).
In d o m eth ac in
C a p s u l e s o f I n d o c i n w ere o b t a i n e d from M erck, S h a rp &
..
19
Dohme.
I t was f o u n d t h a t t h e a c t i v e i n g r e d i e n t ( i n d o m e th a -
c i n ) was IO^ o f t h e c a p s u l e c o n t e n t ' s w e i g h t .
The d r u g was
d i s s o l v e d i n 0.125% Tween 80 t o a n a c t i v e i n g r e d i e n t c o n c e n ­
t r a t i o n o f 0 . 0 4 m g /0 , 2 ml w h ic h was g i v e n by sto m a c h tu b e
t w i c e d a i l y b e g i n n i n g on day 0 ( 2 ).
P e r Os T r e a tm e n t P r o c e d u r e
.
Mice w ere g i v e n d r u g s p e r os u s i n g i n t r a m e d i c p o l y ­
e t h y l e n e t u b i n g ( C la y Adams, P a r s i p p a n y , N. J . ) w i t h o u t s i d e
d i a m e t e r o f 0 .0 4 8 i n c h e s .
A I ml t u b e r c u l i n s y r i n g e w i t h a
21 G n e e d l e i n s e r t e d i n t o t h e t u b i n g was u s e d t o i n j e c t t h e ’
drug.
A to n g u e d e p r e s s o r was c u t t o 7 mm i n w i d t h on one
end w h ich w ould f i t c r o s s w i s e i n t h e m o u s e 's m o u th .
A h o le
was d r i l l e d i n t h e to n g u e d e p r e s s o r j u s t l a r g e enough to
a llo w th e tu b e to p a ss th ro u g h .
I t was p o s i t i o n e d so t h e "
t u b e was g u i d e d down t h e e s o p h a g u s when t h e awake mouse was
h e ld to m a in ta in a s t r a i g h t neck.
The t u b i n g was i n s e r t e d
w i t h a g e n t l e t w i s t i n g m o tio n u n t i l i t r e a c h e d t h e sto m ac h
and t h e c o n t e n t s o f t h e s y r i n g e w ere t h e n e m p tie d i n t o t h e
m o u s e 's s to m a c h .
Ammonium S u l f a t e P r e c i p i t a t i o n o f Serum
S a t u r a t e d ammonium s u l f a t e was a d d e d d r o p w is e t o serum
t h a t had b e e n d i l u t e d 1 :1 w i t h 0.85% NaCl u n t i l a volume
20
e q u a l t o t h e d i l u t e d serum h a d b e e n a d d e d .
P re c ip ita tio n
was t h e n a l l o w e d t o p r o c e e d a t 4 C f o r 30 m i n u t e s .
Then,
t h e p r e c i p i t a t e was s e p a r a t e d by c e n t r i f u g i n g 800 g f o r 30
m in u t e s a t 4 C.
The p r e c i p i t a t e was r e s u s p e n d e d i n p h o s ­
p h a t e b u f f e r e d s a l i n e pH 7 .2 (PBS) and b o t h t h e p r e c i p i t a t e
( g l o b u l i n - r i c h ) and t h e s u p e r n a t e n t ( a l b u m i n - r i c h ) f r a c t i o n s
w ere d i a l i z e d a g a i n s t 200 v olum es PBS f o r 12 h o u r s .
Each
f r a c t i o n was r e c o n s t i t u t e d t o t h e o r i g i n a l serum volum e and
g i v e n t o t e s t m ic e , 0 . 5 ml IV and 0 . 5 ml IP ( 6 5 ) .
I r r a d i a t i o n o f Mice
Mice w ere i r r a d i a t e d u s i n g a ^ 0CO s o u r c e w h i l e i n d i v i d ­
u a l l y i m m o b iliz e d i n a s l o w l y - r e v o l v i n g p l a s t i c c o n t a i n e r .
S o u rc e t o s k i n d i s t a n c e was a lw a y s 70 cm; t h e f i n a l dose
re c e iv e d is l i s t e d
in th e a p p r o p r ia te p la c e s in th e t e x t .
SRBC P la q u e A ssay
The a n t i b o d y r e s p o n s e o f m ice was q u a n t i t a t e d by a
s l i d e m o d if ic a tio n (33) o f J e r n e 's
in - g e l te c h n iq u e .
(1 9 ) l o c a l i z e d h e m o ly sis-
Mice were p l a q u e d on day 4 a f t e r b e i n g :
imm unized IV w i t h 0 . 2 5 ml o f a 10$ SRBC s u s p e n s i o n .
21
L ap o ro to m y f o r I n s e r t i n g A d u l t
Worms I n t r a d u o d e n a l l y
Normal r a t worms ( day 7 p o s t - i n f e c t i o n ) w ere t r a n s ­
f e r r e d t o t h e duodenum o f m ice u s i n g t h e m ethod o f J a c o b s o n
& Reed ( l 6 ) .
B rie fly ,
t h e m o u s e 's sto m a c h a n d p r o x i m a l duo­
denum w ere e x t e r i o r i z e d t h r o u g h a 3 - 5 mm v e n t r a l i n c i s i o n .
A p u r s e s t r i n g s u t u r e was p l a c e d i n t h e g r e a t e r c u r v a t u r e o f
t h e sto m a c h a n d a P a s t e u r p i p e t t e c o n t a i n i n g 150 worms was
i n s e r t e d i n t o a p u n c t u r e wound i n t h e a r e a c i r c u m s c r i b e d by
th e p u rse s t r i n g .
The 150 worms w ere d e p o s i t e d i n t h e duo­
denum a p p r o x i m a t e l y 1 -2 cm from t h e p y l o r i c s p h i n c t e r .
The
p i p e t t e was rem oved and t h e p u r s e s t r i n g s u t u r e was t i g h t e n ­
ed.
.
In tra d u o d e n a l I n je c tio n s
The duodenum was e x t e r i o r i z e d a s i n worm t r a n s f e r and
t h e d r u g o r serum was t h e n i n j e c t e d i n t r a l u m e n a l l y i n t o t h e
duodenum w i t h i n I cm o f t h e p y l o r i c s p h i n c t e r w i t h a 30 G
n e e d le .
Serum
Mice w ere b l e d r e t r o o r b i t a l l y w h i l e u n d e r e t h e r a n e s ­
th e s ia .
The b l o o d was t h e n a l l o w e d t o c l o t a t room t e m p e r ­
a t u r e f o r 2 h o u r s , t h e n i t was r e f r i g e r a t e d f o r 2 4 , h o u r s .
22
The serum was t h e n s e p a r a t e d from t h e c l o t and c e n t r i f u g e d
t o s e p a r a t e c e l l s from t h e se ru m .
The serum was t h e n p a s s e d
t h r o u g h a 0 .4 5 p.m m i l l i p o r e f i l t e r and s t o r e d a t - 2 0 C u n t i l
i t was u s e d , when i t was thaw ed a t 37 C.
F r e e z i n g , Thawing an d H e a t i n g Serum
Serum was f r o z e n i n a m e t h a n o l h a t h a t -5 0 C and thaw ed
on i c e .
T h is was r e p e a t e d 5 t i m e s .
th e a p p ro p r ia te e x p e rim e n ts .
fro zen ,
Serum was t h e n u s e d i n
A s e c o n d sam ple o f. serum was
th aw ed and t h e n m a i n t a i n e d i n a 56 C w a t e r h a t h f o r
60 m i n u t e s , t h e n u s e d i n t h e a p p r o p r i a t e e x p e r i m e n t s .
C y c lo p h o sp h am id e
C y c lo p h o sp h am id e (Mead, J o h n s o n & C o ., E v a n s v i l l e ,
I n d i a n a ) was d i s s o l v e d i n PBS t o a c o n c e n t r a t i o n o f 4 mg/ml.
O n e - h a l f ml was g i v e n t o m ice on day 2 o f a l a r v a l i n f e c t i o n
(34).
PCA
IgE r e s p o n s e s w ere m e a s u re d i n Lew is fe m a le r a t s u s i n g
h e t e r o l o g u s p a s s i v e c u ta n e o u s a n a p h y l a x i s (PCA) a s s a y (48).
Mouse serum s p e c im e n s w ere d i l u t e d s e r i a l l y 2 - f o l d and 0 ,1
ml o f t h e s e d i l u t i o n s w ere i n j e c t e d i n t r a d e r m a l l y i n t o th e
shaved hack o f a r a t .
F o u r serum s p e c im e n s c o u l d he t i t r a t -
23
e d t o 2® o r 2*^ i n one r a t .
F o r t y - e ig h t hours l a t e r th e
r a t s w ere g i v e n I ml IV o f a s o l u t i o n c o n t a i n i n g 2 ,0 0 0 worm
e q u i v a l e n t s o f Njl h r a s i l i e n s i s a n t i g e n and 5 mg o f E vans
b lu e .
The' r a t s w ere k i l l e d a f t e r 30 m in u t e s and t h e r e s u l t s
w ere r e a d a s p o s i t i v e w here a b l u e c i r c l e c o u l d be s e e n on
th e u n d e rs id e o f th e s k in .
The a n t i g e n u s e d i n t h i s t e s t
was p r e p a r e d by c o l l e c t i n g worms from r a t s i n f e c t e d w i t h
4 ,5 0 0 IL^ 8 d ays b e f o r e c o l l e c t i o n .
These worms w ere c o u n t ­
ed and m a c e r a t e d t h o r o u g h l y i n a g l a s s t i s s u e h o m o g in i z e r .
The p r e p a r a t i o n was t h e n c e n t r i f u g e d a t a b o u t 700 g f o r 10
m in u t e s and t h e s u p e r n a t e n t was f r o z e n and u s e d a s a n t i g e n
i n t h e PGA t e s t .
P ro sta g la n d in s
S y n t h e t i c p r o s t a g l a n d i n s u s e d i n t h i s s t u d y w ere k i n d l y
d o n a te d by t h e U pjohn Company, K alam azoo, M ic h ig a n .
The
p r o s t a g l a n d i n s PGE1 , PGEg and PGFgo c w ere r e c e i v e d a s c r y s ­
t a l l i n e s o l i d s an d w ere d i s s o l v e d i n
mg i n I m l.
e t h y l a l c o h o l , 10
T h is was f u r t h e r d i l u t e d 1 :1 0 w i t h 0 .2 M sodium
p h o s p h a t e b u f f e r , pH 7 . 4 .
O n e - t e n t h ml o f t h i s was i n j e c t e d
i n t r a d u o d e n a l l y f o r a d o sa g e o f 100 pg/m ouse ( 2 4 ) .
S t a t i s t i c a l T r e a tm e n t
Worm r e c o v e r y d a t a a r e u s u a l l y g i v e n a s a r i t h m a t i c mean
24
± s ta n d a r d d e v ia ti o n (S D ).
B e c a u se o f t h e v a r i a n c e s e e n i n
t h i s s y s te m , t h e num ber o f worms r e c o v e r e d from i n d i v i d u a l
m ice i s a l s o l i s t e d m o st o f t h e t i m e .
The m ethod u s e d t o
g e t EPG d a t a p r e c l u d e d any s t a t i s t i c a l t r e a t m e n t a s f e c e s
from a l l m ice i n a g r o u p were c o l l e c t e d on t h e same t r a y .
The S t u d e n t T t e s t was u s e d t o a s s e s s t h e s i g n i f i c a n c e o f
d i f f e r e n c e s n o t e d b e tw e e n means and a p r o b a b i l i t y
v a l u e o f l e s s t h a n 0 .0 $ i n d i c a t e d s i g n i f i c a n c e .
(p) •
R
E
S
U
L
T
S
C h r o n ic T ry p an B lue T r e a t m e n t o f Mice and t h e
E lim in a tio n o f
h ra s ilie n s is
The r o l e o f m a c ro p h a g e s i n e l i m i n a t i o n o f a n i n f e c t i o n
of
h r a s i l i e n s i s was a p p r o a c h e d by t h e m ethod o f i n h i b i t ­
i n g m acro p h ag e l y s o s o m a l enzymes w i t h t r y p a n b l u e (TB).
T h is c h e m ic a l i n h i b i t i o n was c h o s e n b e c a u s e o f i t s
apparent
l a c k o f m arked e f f e c t on t h e a f f e r e n t and e f f e r e n t lim b s o f
t h e a c q u i r e d immune r e s p o n s e , e x c e p t f o r t h e k i l l i n g by
m a c ro p h a g e s o f tu m o r c e l l s
(1 3 ,2 6 ).
The e x p e r i m e n t a l d e s i g n o f t h e f i r s t TB e x p e r i m e n t i s
shown b e lo w :
Day -3
Day - I
Day 0
Day 3 and E v e ry
3 r d Day
T h e re a fte r
Group I
-
-
300IL .
-■
Group 2
3mg TB IP
0.5mg TB IP
300IL .
0 . 5mg TB SQ
F o u r o f t h e m ice i n t h e e x p e r i m e n t a l g ro u p d i e d b e f o r e .
m o n i t o r i n g o f egg c o u n t b e g a n .
Egg c o u n ts p e r gram (EPG)
f e c e s w ere m o n i t o r e d d a i l y s t a r t i n g on day 6 ( F i g u r e I ) .
The egg c o u n t s i n t h e TB g ro u p w ere s u s t a i n e d a t a h i g h
l e v e l f o r a b o u t 6 days l o n g e r t h a n t h o s e i n t h e c o n t r o l
group.
T h ere was a d e f i n i t e h i g h p l a t e a u i n t h e EPG c o u n ts
w h ich s u g g e s t s t h a t t h e worm damage p h a s e was d e l a y e d .
0 — 0 Group I
X
5 m ice g i v e n l a r v a e o n l y .
X Group 2
4 m ice g i v e n IP 3 mg day
- 2 , 0 . 5 mg day - I and SQ 0 . 5 mg
e v e r y t h i r d day t h e r e a f t e r .
DAYS POST INFECTION
F ig u re I .
The e f f e c t o f c h r o n i c t r y p a n b l u e t r e a t m e n t on t h e EPG p r o d u c ­
t i o n o f a N. b r a s i l i e n s i s l a r v a l i n f e c t i o n .
2?
E x p u l s i o n o c c u r e d , b u t may have b e e n d e l a y e d a l i t t l e .
The
worm s p e c i f i c IgE p r o d u c t i o n i n t h e s e m ice was m e a su re d by
p a s s i v e c u ta n e o u s a n a p h y l a x i s (PCA) an d was f o u n d t o be
r i s i n g from day 18 t o d a y ' 25 i n t h e T B - t r e a t e d g r o u p , w hich
m ig h t be e x p e c t e d i f t h e worms w ere m a i n t a i n e d l o n g e r .
The
PCA t i t e r s a c h i e v e d ,in t h e T B - t r e a t e d m ice shows t h a t t h e
IgE r e s p o n s e was f a i r l y n o rm a l ( T a b le I ) .
B ecau se t h e f i r s t TB e x p e r i m e n t ( F i g u r e I ) s u g g e s t e d
t h a t TB p r o l o n g s t h e tim e o f egg p r o d u c t i o n i n a l a r v a l i n ­
fe c tio n ,
t h e f o l l o w i n g e x p e r i m e n t was d e s i g n e d t o d e te r m i n e
i f TB i n h i b i t s t h e dam aging s t e p o r t h e e x p u l s i o n s t e p .
Day -2
Day I
Day 0
__________
__________
_______
Group I
-
-
3 0 0 IL 3
Group 2
3mg T B ,IP
Img TB,SQ
30011^
Day 2 and E v ery
3 r d Day
T h e re a fte r
Img TB1SQ
Some m ice from e a c h g ro u p w ere k i l l e d on days 6, 8, 10
and 12 and t h e num ber o f worms r e c o v e r e d was r e c o r d e d .
As
a m e a su re o f damage, t h e num ber o f eggs p e r f e m a le (EPS) was
a ls o d e te rm in e d (T ab le I l) ,.
The T B - t r e a t e d g r o u p a p p a r e n t l y
a l l o w e d more worms t o r e a c h m a t u r i t y by day 6 ( P c . 05) and
a l s o r e t a i n e d more worms on day 10 ( P c . 0 5 ) t h a n d i d t h e
c o n tr o l group.
The EPS d a t a w ere i n c o n c l u s i v e .
By day 12,
28
T a b le I .
S p e c i f i c N1. b r a s i l i e n s i s PCA t i t e r o f serum o f
n o rm a l a n d t r y p a n b l u e - t r e a t e d m ic e .
Group I
C o n t r o l m ice
Group 2
T rypan b l u e - t r e a t e d mice*3
5* Day p o s t i n f e c t i o n .
D See F i g u r e I , t r e a t m e n t o f g r o u p 2.
Day 1 8 a
Day 25^
■128
64
64
256
T a b le I I .
E f f e c t s o f t r y p a n b l u e t r e a t m e n t o f m ic e on a Njl b r a s i l i e n s i s
..la rv a l in fe c tio n ..
C o n tro l
No. Mice Wormsb
Rec.
EPGc K i l l e d
Day
6
9 ,0 0 0
Day
7
4 ,9 0 0
Day
8
900
Day
9
400
Day 10
0
4
73
(± 3 2 7 )
~
EPSd
EPGc
8 .3
3 1 ,2 0 0
• T ry p an b l u e a
No. Mice
Worms"
K ille d
Rec.
5
1 3 5 .4
( ± 3 3 -6 )
1 2 .5
4
157
( ± 4 7 .7 )
1 9 .3
5
145'
( ± 5 3 .2 )
1 3 -4
4
15
(0 -6 l)
-
44,000
4
98
(± 1 1 2 )
1 3 .7
1 8 ,3 0 0
1 6 ,3 0 0
-
4
6 ,6 0 0
(± 1 )
Day 11
0
Day 12
0
EPSd
4 ,6 0 0
3
(± 1 )
1 ,4 0 0
a E ach mouse g i v e n 3 mg TB IP day - 2 , I mg SQ day - I and I mg TB SQ e v e r y
t h i r d day t h e r e a f t e r .
D Worms R e c . = a v e r a g e num ber o f worms r e c o v e r e d / i n f e c t e d m ouse..
" EPG = a v e r a g e worm e g g s /g r a m o f i n f e c t e d mouse f e c e s .
"■ EPS = a v e r a g e e g g s / f e m a l e worm.
Worms r e c o v e r e d
Day 6 C o n t r o l v s . T e s t P< =05
Day 8 C o n t r o l v s . T e s t NS
Day 10 C o n t r o l v s . T e s t P < .0 5 0
30
t h e worms h a d v i r t u a l l y a l l b e e n e x p e l l e d from a l l b u t one
mouse' i n t h e e x p e r i m e n t a l g r o u p .
The EPG were, h i g h e r i n t h e
T B - t r e a t e d g r o u p an d r e m a in e d h i g h e r a b o u t 5 days ( T a b le
II).
I b e l i e v e d t h a t i f a d u l t worms w ere i n o c u l a t e d i n t o t h e
g u t o f T B - t r e a t e d m ic e , t h e r e w ould be l e s s s t i m u l a t i o n o f
t h e immune r e s p o n s e a n d a g r e a t e r e f f e c t o f TB m ig h t be
re a liz e d .
T h e r e f o r e , worms w ere r e c o v e r e d from 7- d a y i n f e c ­
t i o n s o f L ew is r a t s an d 150 worms w ere p l a c e d by la p o r o to m y
i n t h e duodenum o f m ice o f 2 g r o u p s .
The c o n t r o l g ro u p was
made up o f n o rm a l 1 0 - w e e k - o ld l i t t e r m a t e s .
The t e s t g ro u p
was 1 0 - w e e k - o ld l i t t e r m a t e s w h ic h h a d b e e n g i v e n 2 p r i m a r y
d o s e s (3 mg day - 2 ,
I mg day - I ,
I P ) o f TB, and t h e n were
m a i n t a i n e d on I mg TB s u b c u t a n e o u s l y e v e r y t h i r d d a y .
EPG
f e c e s c o u n t s , m o n i t o r e d b e g i n n i n g on t h e day a f t e r l a p o r o ­
tomy, w ere s i m i l a r i n a l l g r o u p s ( F i g u r e 2 ) .
I t was p o s s i b l e t h a t t h e worms w ere damaged d u r i n g t h e
t r a n s f e r from r a t s t o i m m u n o l o g ic a l l y c o m p e te n t m ic e and
t h a t f y t r a n s f e r r i n g worms i n t o nude m ic e , t h e s t a t u s o f t h e
t r a n s f e r r e d worms c o u l d . b e d e d u c e d .
o f m ice w ere u s e d .
litte rm a te s .
To t e s t t h i s ,
3 g ro u p s
Two g ro u p s c o n s i s t e d o f 1 0 - w e e k - o l d .
The m ic e i n Group 2 w ere p r e t r e a t e d w i t h TB
t h e n m a i n t a i n e d on 2 mg e v e r y t h i r d d a y , w h e r e a s , t h e m ice
I
O— -O
Group I
Normal littermates .
X— *
Group 2 TB t r e a t e d LKi 3mg
IP* day 2, I mg IP day - I ,
e v e r y t h i r d day.
UJ 25 -
DAYS POST LAPOROTOMY
Figure 2.
The effect of trypan blue treatment of mice on the elimination
of normal rat worms given by laporotomy to mice.
32
i n Group I w ere n o rm a l c o n t r o l s .
m ic e .
The t h i r d g r o u p was 6 nude
One h u n d r e d f i f t y n o rm a l r a t worm s, Which c o n t a i n e d
2 8 .6 EP$, w ere t r a n s f e r r e d "by la p o r o t o m y on day. O i n t o a l l
m ice and egg c o u n t s w ere m o n i t o r e d b e g i n n i n g on ddy I p o s t
t r a n s f e r ( F i g u r e 3 a n d T a b le I I I ) .
The egg c o u n t s d i d n ' t a p p e a r t o be d i f f e r e n t .
Worms
w ere r e c o v e r e d from t h e n u d e s when t h e y d i e d ( T a b le I I I ) .
The p u r g i c a l p r o c e d u r e a p p a r e n t l y was v e r y s t r e s s f u l t o t h e
n u d e p , c a u s i n g a l l b u t one t o d i e w i t h i n 9 d a y s .
The worms
r e c o v e r e d from some n u d e s a p p e a r e d t o be damaged a s m e a su re d
by EP$, th o u g h i t i s n ' t known w h e t h e r t h i s was c a u s e d by
t h e p o o r c o n d i t i o n o f t h e n u d e s o r w h e t h e r t h e worms were
damaged b e tw e e n t h e tim e t h e y w ere rem oved from t h e r a t and
t h e tim e t h e y w ere p l a c e d i n t h e n u d e s .
The h i g h and v a r i ­
a b l e num bers o f worms r e c o v e r e d from g ro u p s I and 2 a s l a t e
a s d ays 8 a n d . 9 r a i s e d t h e q u e s t i o n o f t h e s u r g i c a l p r o c e ­
d u re i n t e r f e r i n g w i t h e x p u l s i o n more t h a n t h e TB ( T a b le
III),
C o lle c tiv e ly ,
t h e s e d a t a show t h a t c h r o n i c TB t r e a t m e n t
o f m ice b e f o r e and d u r i n g a l a r v a l i n f e c t i o n w i t h Ni b r a s i l i e n s i s a l l o w s more worms to r e a c h t h e a d u l t s t a g e and t h e
worms a r e r e t a i n e d i n t h e h o s t l o n g e r ( T a b le I I ) .
T h is r e - .
s u i t s i n h i g h e r EPG c o u n t s t h a t a r e m a i n t a i n e d l o n g e r
O ----- 0
Group I
X-----
Group 2 T B - t r e a t e d l i t t e r m a t e s :
3 mg
TB IP day - 2 , I mg IP day - I , 2 mg TB
SQ day 2 and e v e r y t h i r d day t h e r e a f t e r .
EGGS/g FECES x IO3
Group 3
Normal l i t t e r m a t e s .
Nude m ic e .
A l l m ice w ere g i v e n 150 n o rm a l r a t worms
by la p o r o to m y on day 0.
DAYS POST LAPOROTOMY
F i g u r e 3-
The e f f e c t o f t r y p a n b l u e t r e a t m e n t o f m ice on t h e e l i m i n a t i o n
o f n o r m a l r a t worms g i v e n by la p o r o to m y t o m ic e .
T a b le I I I .
Worms r e c o v e r e d a f t e r t r a n s f e r o f n o r m a l r a t worms i n t o n o rm a l,
.tr y p a n b l u e - t r e a t e d , a n d nude m i c e .
Group I C o n t r o l a
Worms
Rec.
EP$
Day 5
177
4
Group 2 T ry p a n b l u e a ' ^
Worms
EP $
R ec.
Group 3 Nudesa
Worms
Rec.
EP 9
154
• 7 .7
Day 6
137
28
Day 7
l6 o
132
11.9
5 .5
115
22.5
Day 8
74
O
Day 9
53
17
9
. 0
O
6 .2 5
3-9
183
3-3
193
• 4
6 .0
-
• 170
.I
I •
O
O
O
6 .6
f ’ A l l m ic e g i v e n 150 n o r m a l r a t worms by l a p o r o t o m y on day O.
See l e g e n d F i g u r e 2 f o r TB a d m i n i s t r a t i o n s c h e d u l e .
35
(F ig u re I ,
T a b le I I ) .
TB h a s much l e s s e f f e c t on an i n f e c ­
t i o n i n m ice o b t a i n e d by t r a n s f e r r i n g a d u l t n o r m a l r a t worms
i n t o T B - t r e a t e d m ic e ( F i g u r e 2 , T a b le I I I ) .
At t h i s tim e ,
i t i s unknown w h e t h e r t h e e f f e c t s o f TB a r e c a u s e d by a
s p e c i f i c d e p r e s s i o n o f m ac ro p h ag e e f f e c t o r f u n c t i o n o r
w h e th er i t i s j u s t a d e p re s s io n o f th e g e n e r a l h e a l t h o f th e
m ouse.
P r o s t a g l a n d i n s and t h e R e sp o n se o f Mice t o a L a r v a l
I n f e c t i o n o f Ni. b r a s i l i e n s i s
E x p u l s i o n o f a Ni . b r a s i l i e n s i s i n f e c t i o n i n t h e r a t can
be d e l a y e d by p r o s t a g l a n d i n i n h i b i t o r s
(8 ,9 )•
The f o l l o w i n g
s e r i e s o f e x p e r i m e n t s was done t o exam ine t h e p o s s i b i l i t y
t h a t p r o s t a g l a n d i n s a r e i m p o r t a n t i n t h e e x p u l s i o n o f worms
from m ice a l s o .
The f i r s t e x p e r i m e n t was d e s i g n e d t o d e t e r m i n e i f
a s p ir in (a c e t y l s a l i c i l i c a c i d ) , a p ro sta g la n d in in h ib ito r ,
w ould i n h i b i t worm e x p u l s i o n .
w ere d i v i d e d i n t o 5 g r o u p s ,
o n ly l a r v a e .
d ilu e n t,
Iy .
E i g h t - w e e k - o l d B a y l o r m ice
One g r o u p , t h e c o n t r o l , g o t
Mice i n 2 o t h e r g r o u p s e a c h g o t o n l y a s p i r i n
I g ro u p o r a l l y and t h e o t h e r g ro u p i n t r a p e r i t o n e a l -
Mice i n I t e s t g ro u p e a c h r e c e i v e d 50 m g/kg a s p i r i n
tw i c e a day o r a l l y v i a g a s t r i c t u b e and m ice i n t h e se c o n d
t e s t g ro u p e a c h r e c e i v e d 50 m g/kg i n t r a p e r i t o n e a l l y .
T h is
36
'
d o se was a d o p t e d from work done i n m ice u s i n g a s p i r i n a s a
p r o s t a g l a n d i n s y n t h e s i s i n h i b i t o r ( 3 ) and was u s e d o n ly
a f t e r t h e d o sa g e (3 m g /k g , t w i c e d a i l y ) g i v e n by D i n e e n , _et
a l ( 9 ) t o r a t s was f o u n d n o t t o d e l a y e x p u l s i o n o f a l a r v a l
in fe c tio n
( d a t a n o t sh o w n ).
■•
Two m ic e i n t h e o r a l a s p i r i n and I i n t h e i n t r a p e r i t o n p a l a s p i r i n g r o u p d i e d o f i n t e s t i n a l b l e e d i n g , and s t o ­
mach u l c e r s w ere g r o s s l y v i s i b l e i n 2 o f t h e 3 m ic e .
The
egg c o u n t s i n a l l g r o u p s d r o p p e d b e tw e e n day 7 a n d 8 and a l l
g ro u p s w ere v i r t u a l l y n e g a t i v e by day 9-
B ecau se egg c o u n ts
w ere s i m i l a r i n a l l c o n t r o l g r o u p s , t h e y w ere com bined f o r
p r e s e n t a t i o n i n T a b le IV.
L ik e w ise , th e 2 d i f f e r e n t a s ­
p i r i n - t r e a t e d g r o u p s w ere com bined i n T,able IV.
As i n t h e
p r e v i o u s e x p e r i m e n t ( d a t a n o t sh o w n ), a s p i r i n d i d n o t r e p r o d u c a b ly i n c r e a s e t h e l e n g t h o f p a t e n c y o r d e l a y e x p u l s i o n o f
t h e w orm s.
A s e c o n d d ru g w h ic h i s a known p r o s t a g l a n d i n s i n h i b i ­
to r,
i n d o m e t h a c i n , was a l s o u s e d .
In d o m eth acin p r e v io u s ly
h a d b e e n u s e d i n a nem atode ( T r i c h u r i s m u r i s ) -m ouse sy ste m
and t h e i n t e r p r e t a t i o n o f t h e e x p e r i m e n t a l r e s u l t s was t h a t
i n d o m e t h a c i n d e l a y e d e x p u l s i o n by i n h i b i t i n g p r o s t a g l a n d i n
sy n th e sis (2 ).
T h ree g ro u p s o f 7 - w e e k - o ld
bALB/ c
m a le s w ere g i v e n 300
37
T a b le IV.
EPG and worms r e c o v e r e d from Njl b r a s i l i e n s i s
i n f e c t e d m ice t r e a t e d w i t h a s p i r i n ^ .
C o n tro lb
A s p i r i n '3’ c
T r e a t e d Mice
EPG Day 6
• 21,600
3 1 ,0 0 0
EPG Day 7
2 0 ,6 0 0
3 6 ,3 0 0
EPG p a y 8
3 ,0 0 0
5 ,7 0 0
EPG Day 9
200
1,300
Worms R e c o v e re d
Day 10
C o n tro l
0 ,0 ,0 ,
0 ,0 ,0
O r a l ASAcc
1 1 7 ,4 5 ,0 ,
0 ,0
IP ASAcd
3 3 ,1 2 ,1 ,
0 ,0
a A l l m ic e g i v e n JOO ILo on day O and were k i l l e d and worms
, c o u n t e d on day 10.
^
D The EPG c o u n ts f o r a l l c o n t r o l g r o u p s w ere s i m i l a r and
w ere com bined.
L i k e w i s e , t h e EPG c o u n ts o f t h e 2 d i f f e r ­
e n t a s p i r i n t r e a t m e n t g r o u p s w ere s i m i l a r and w ere com­
b in e d .
c 50 m g/kg a s p i r i n g i v e n t w i c e d a i l y from day 0, e i t h e r
o r a l l y (cc) o r i n tr a p e r i t o n e a ll y (c d ).
38
IL^ and u s e d i n t h i s e x p e r i m e n t .
t h e r tre a tm e n t.
Group I r e c e i v e d no f u r ­
Mice i n g ro u p 2 r e c e i v e d 0 .2 ml 0.125%
Tween 80 ( i n d o m e t h a c i n d i l u e n t ) p e r os t w i c e d a i l y . . Mice i n
g ro u p 3 w ere g i v e n 0 ,0 4 mg i n d o m e t h a c in t w i c e d a i l y i n 0 .2
ml volum e a s was u s e d by Bruce & W ak e lin ( 2 ) , b e g i n n i n g 6
h o u r s b e f o r e l a r v a l i n f e c t i o n and c o n t i n u i n g u n t i l t h e m ice
w ere k i l l e d .
EPG w ere d e t e r m i n e d f o r e a c h g ro u p from day 6
u n t i l day 1 0 .5 when t h e m ice w ere k i l l e d a n d t h e worms were
r e c o v e r e d ( T a b le V) .
I t a p p e a r s t h e m ice w ere k i l l e d a day
to o e a r l y t o d e t e r m i n e i f e x p u l s i o n was d e l a y e d by in d o m e th ­
a c in .
The g r e a t v a r i a b i l i t y i n t h e num ber o f worms r e c o v e r ­
ed i n e a c h g r o u p s u g g e s t s no d i f f e r e n c e b e tw e e n t h e d i l u t i o n
c o n t r o l and i n d o m e t h a c i n g ro u p s and t h a t b o t h g r o u p s were
e x p e l l i n g t h e i r worm b u r d e n .
The u l t i m a t e t e s t o f w h e t h e r p r o s t a g l a n d i n s w ould
cause e x p u ls io n i s to i n j e c t p r e p a r a tio n s o f p u r i f i e d p r o s ­
t a g l a n d i n s i n t o t h e g u t and s e e i f a worm b u r d e n i s e x p e l l e d
e a r l i e r th a n n o rm al.
T h is was t r i e d n e x t .
P ro sta g la n d in s.,
PGE^ and PGE2 , when i n j e c t e d i n t r a d u o d e n a l l y i n t o r a t s on
day 6 o f a Njl b r a s i l i e n s i s i n f e c t i o n c a u s e d t h e e x p u l s i o n o f
t h e worms by day 10 ( 2 4 ) .
T h erefo re,
two e x p e r i m e n t s were
d e s i g n e d t o t e s t t h e e f f e c t o f p r o s t a g l a n d i n s on mouse i n ­
fe c tio n s .
The f i r s t t r i a l c o n s i s t e d o f 5 g r o u p s o f l i t t e r -
T a b le V.
EPG and worms r e c o v e r e d from
b ra s ilie n s is
i n f e c t e d m ice t r e a t e d w i t h indom et h a c i n .
O
Group I
U n trea ted C o n tro l
Group 2
D ilu e n t* 3
C o n tro l
Group 3
In d o m eth ac in c
EPG Day
6
4 7 ,3 0 0
4 5 ,7 0 0
44,300
EPG Day
7
4 7 ,5 0 0
4 2 ,5 0 0
30,800
EPG Day
8
8 1 ,2 0 0
50,700
3 1 ,7 0 0
EPG Day
9
4 7 ,0 0 0
27,200
6 ,7 0 0
EPG Day 10
0
. 4oo
. 200
Worms R e c o v e re d
on Day 1 0 ;5
A verage
(P=NS)
262, 110, 68,
5 6 ,3 ,1
83+97
1 5 6 ,1 3 3 ,1 0 2 ,8 6 ,
6 7 , 12,0
92+7 2 . 2
? A l l m ic e g i v e n 300 ILo on day 0.
0 .2 0.125% Tween 80 p e r os t w i c e d a i l y from day O t o day
10.
c OcOk mg/mouse i n d o m e t h a c ih p e r os t w i c e d a i l y from day 0
t o day 10.
40
m a te s i n f e c t e d on day 0 w i t h 300 I L ^ .
f u r t h e r t r e a t m e n t (.Group I ) .
One. g ro u p r e c e i v e d no
On day 5 p o s t - i n f e c t i o n , a
s e c o n d g ro u p (G roup 2 ) was i n j e c t e d w i t h 0 .1 ml p r o s t a g l a n ­
d in d i l u e n t i n tr a d u o d e n a l l y .
Mice i n 3 g ro u p s e a c h were
g i v e n 100 pgm o f e i t h e r PGE^ ( Group 3 ) , PGE2 ( Group 4 ) o r
PGF2 ^ ( Group 5 ) •
c o u n ts d r o p p e d .
EPG f e c e s w ere m o n i t o r e d u n t i l t h e egg.
No i n c r e a s e d e x p u l s i o n was shown by t h e
p r o s t a g l a n d i n - t r e a t e d m ice a s ju d g e d by t h e egg c o u n t s a n d ,
a s t h i s e x p e r i m e n t h a d an u n u s u a l l y l o n g p a t e n c y p e r i o d ,
I
c o n c lu d e t h a t p r o s t a g l a n d i n s d i d n ' t damage t h e worms ( T a b le
V I).
.
The same e x p e r i m e n t was done w i t h n u d e s .
B e ca u se n u d e s
d o n ' t damage t h e worms ( 1 6 , 3 4 ) , p r o s t a g l a n d i n - i n d u c e d damage
s h o u l d be e a s i e r t o d e t e c t , i n n u d e s .
F iv e g r o u p s o f 6 -w e ek -
o l d n u d e s w ere i n f e c t e d on day 0 w i t h 300 I L ^ .
Group I was
u s e d a s i n f e c t i o n c o n t r o l and w a s n ' t m a n i p u l a t e d f u r t h e r .
One g ro u p ( Group 2 ) was g i v e n 0 .1 ml o f p r o s t a g l a n d i n d i l ­
u e n t i n t r a d u o d e n a l l y on day 5-
Mice i n 3 o t h e r g r o u p s e a c h
w ere g i v e n 100 pgm o f e i t h e r PGE^ ( Group 3 ) , PGE2 ( Group 4 )
o r PGF2cs^ (G roup 5 ) i n t r a d u o d e n a l l y .
As b e f o r e , e g g c o u n ts
w ere m o n i t o r e d t h r o u g h day I 6 ( T a b le V I I ) .
T a b le VI.
The e f f e c t o f p r o s t a g l a n d i n ( PGE^, PGEg, PGFg^) i n j e c t e d i n t r a d u o d e n a l l y on day 5 o f a l a r v a l i n f e c t i o n i n n o rm a l m i c e . 3Group I
- U n m a n ip u la te d
No. Mice
Group 2
■ B uffer ,
C o n tro l0
Group 3
Group 4
Group 5
" PGE1 0
PGEgd
PGFgc^ e
7 .
7
6
6
6
Day
6
32,300
4 1 ,4 0 0
16,800
33,300
36,600
Day
7
82,300
64,600
58,900
38,700
7 1 ,7 0 0
Day
8
51,600
34,000
18,000
27,600
3 4 ,7 0 0
Day
9
40,200
13,300
9,900
13,800
2 4 ,6 o o
Day 10
30,600
7,200
8,300
3,900
1 5 ,5 0 0
Day 11
5 ,4 0 0
3 ,4 0 0
5,500
7,900
8,700
Day 12
3 ,4 0 0
3 ,4 0 0
600
3,500
10,100
Day 14
1,700
100
4,4oo
4 ,3 0 0
■ 4 ,2 0 0
Day 16
200
100
500
0
300
f' 300
0.1
day
day
ILg g i v e n t o a l l m ice on day 0; a l l m ice w ere 6 - w e e k - o l d LM.
m l^ lO ^ e t h y l a l c o h o l i n p h o s p h a t e b u f f e r ' i n j e c t e d i n t r a d u o d e n a l l y on
5 . IOOpgm o f PGE1 c , PGE^d' and PGF9 ,e i n j e c t e d i n t r a d u o d e n a l l y on
5.
'
I
^
T a b le V I I .
The e f f e c t o f p r o s t a g l a n d i n (PGE^, PGEg, PGFgex,) i n j e c t e d i n t r a ­
duo d e n a l l y on day 5 o f a l a r v a l . i n f e c t i o n i n n u d e m i c e . 3Group I
U n m a n ip u la te d
Group 2
B uffer h
C o n tro l0
Group 3
Group 4
Group 5
PGEi0
PGE2 4
PGFgef
Day
6
36,700
4 1 ,4 0 0
60,500
39,000
30,600
Day
7
1 8 9 ,800'
176,600
100,300
l46,6oo
1 5 4 ,2 0 0
Day
8.
188,400
143,000
169,600.
185,400
7 8 ,8 0 0
Day
9
86,000
125,600
103,200
76,800
52,400
Day 10
1 3 7 ,8 0 0
124,000
1 0 4 ,2 0 0 •
93,800
7 6 ,0 0 0
Day 12
1 1 9 ,4 0 0
125,000
88,600
70,000
4 9 ,6 0 0
Day 16
90,400
56,4oo
7 4 ,0 0 0
38,000
29,000
■
g i v e n t o a l l m ice on day 6; a l l . m ice w ere 6 - w e e k - o l d nude m ic e .
a b le V I.
43
The d a t a d o n ’ t s u g g e s t any damage t o t h e worm i n any
g ro u p .
A n o r e x ia ,
c a u s e d by t h e s u r g e r y , d e c r e a s e s t h e
volume o f f e c e s , w h ic h i n t u r n i n c r e a s e s ' t h e EPG.
A lth o u g h
none o f t h e s e e x p e r i m e n t s ( T a b l e s IV, V, VI, V I I ) i s d e f i n i ­
tiv e ,
I b e li e v e t h a t a l l t h r e e ta k e n t o g e t h e r g iv e re a s o n to
b e l i e v e t h a t t h e mouse
b r a s i l i e n s i s s y s te m may n o t have
a s much o f a d e p e n d e n c e on p r o s t a g l a n d i n t o c a u s e e x p u l s i o n
a s t h e Njl b r a s i l i e n s i s r a t s y s te m . •
Immune Serum G iven w i t h IMLNC t o Normal Mice
a)
C l a s s i f i c a t i o n o f Immune Serum s a s Mouse P r o t e c t i v e
o r Worm P r o t e c t i v e .
A t te m p ts t o e i t h e r damage worms e a r l i e r
t h a n n o rm a l or. p r e v e n t t h e i r damage f o r an a p p r e c i a b l e
amount o f tim e i n v i v o h a v i n g f a i l e d ,
I d e cid ed to t r y to
p a s s i v e l y t r a n s f e r from immune m ic e t o n a i v e m ic e t h e
a b i l i t y t o damage t h e worm.
Immune serum and immune c e l l s
a re a p p a r e n tly n e c e s s a r y to g e t p r o t e c t i o n t h a t ap p ro x im ate s
n a t u r a l im m unity i n b o t h t h e r a t ( 2 8 ) and mouse ( 2 9 ,4 4 )
sy ste m s.
The n e x t s e t o f e x p e r i m e n t s was d e s i g n e d t o g e t a
c o n s t a n t ty p e o f immune c e l l s and f i n d p r o t e c t i v e p o o l s o f
serum by g i v i n g immune m e s e n t e r i c lymph node c e l l s
(IMLNC)
t h a t w ould s y n e r g i z e w i t h p r o t e c t i v e p o o l s o f seru m t o c a u s e
e x p u ls io n s i m i l a r to t h a t o b serv ed in a second i n f e c ti o n .
F o r t h e f i r s t e x p e r i m e n t , I c h o se IMLNC from m ice w hich
h a d b e e n g i v e n 300 IL^ on d ays - 5 6 and -32 and 900 IL^ on
8
day -13*
These IMLNC w ere g i v e n IV i n a d o se o f I x 10
'
n u c l e a t e d c e l l s on t h e same day ( 0 ) t h a t t h e r e c i p i e n t m ice
w ere i n f e c t e d w i t h 300 I L ^ .
Twelve p o o l s o f serum c o l l e c t e d
a t v a r i o u s t i m e s a f t e r one o r more N^. b r a s i l i e n s i s l a r v a l
i n f e c t i o n s w ere u s e d a s t h e immune se ru m .
E ach o f t h e 12
p o o l s was t e s t e d i n a g ro u p o f 3 m ice i n j e c t e d w i t h I ml
e a c h , 0 . 5 ml IV and 0 . 5 ml I P .
A l l m ice a ls o , r e c e i v e d IMLNC
on day 0.
Mice i n one c o n t r o l g ro u p r e c e i v e d n o rm a l m e s e n t e r i c
lymph node c e l l s
(NMLNC) o n l y , and m ice i n a s e c o n d c o n t r o l
g ro u p r e c e i v e d IMLNC o n l y .
n i n g on day 6.
EPG f e c e s w ere m o n i t o r e d b e g i n ­
The egg c o u n t c u r v e s r e s u l t i n g from t r e a t - ■
m ent w i t h t h e s e serum p o o l s d i v i d e t h e p o o l s i n t o t h r e e
d iffe re n t groups:
.
I ) S even o f 12 p o o l s t e s t e d w e r e n ' t u n ­
l i k e t h e c o n t r o l g r o u p s ( n o r m a l o r IMLNC o n l y ) .
T hese p o o l s
w ere c o l l e c t e d from m ice 13, 15» 20, 21, 2 3 , 24 an d 25 days
a f t e r a p rim a ry i n f e c t i o n .
2 ) I n 3 o f t h e p o o l s , t h e EPG
c o u n t s w ere l o w e r t h a n t h e c o n t r o l s on day 6, b u t e v e n t u a l l y
w ent v e r y h i g h and h a d a p r o l o n g e d d u r a t i o n o f p a t e n c y .
T hese w ere c a l l e d worm p r o t e c t i v e s e r a .
T hese seru m p o o l s
w ere t a k e n from m ice 19 o r 24 d ays a f t e r a p r i m a r y i n f e c t i o n
45
and from m ice i n f e c t e d on b o t h day 10 and day 32 b e f o r e
h a r v e s t i n g serum ( F i g u r e 4 ) .
3 ) Serum from 2 o f t h e 12
p o o l s was c l a s s i f i e d a s mouse p r o t e c t i v e serum b e c a u s e t h e
EPG f e c e s w ere v e r y low and d ro p p e d t o 0 b e f o r e t h e c o n t r o l s
d id ( F ig u re 5)•
in fe c tio n .
One o f t h e s e s e r a was from a day I 7 p r i m a r y
The s e c o n d was From t h e IMLNC d o n o r m ice w hich
w ere b l e d 13 days a f t e r t h e t h i r d i n f e c t i o n .
T h is l a s t p o o l
h a d n ’ t b e e n f r o z e n a n d was i n j e c t e d i n t o m ice w i t h i n 24
h o u r s a f t e r c o l l e c t i o n ( F i g u r e 5)■
The r a t i o o f mouse p r o t e c t i v e p o o l s t o n o n p r o t e c t i v e
p o o l s i s l e s s t h a n t h e one o f t h r e e r e p o r t e d i n t h e l i t e r a ­
t u r e , b u t i s c o m p a ra b le ( 4 l ) .
I d i d n ' t f i n d any c o r r e l a t i o n
b e tw e e n t h e tim e a f t e r i n f e c t i o n when a serum was h a r v e s t e d
and t h e p r o t e c t i v e t i t e r o f t h e se ru m .
may be an i m p o r t a n t f a c t o r .
M u ltip le in f e c tio n s
One o f t h e p o o l s t h a t was mouse
p r o t e c t i v e h a d n ' t b e e n f r o z e n w h ic h s u g g e s t e d t h e p o s s i b i l i ­
ty of a free ze -th aw l a b i l e f a c to r .
b)
Serum .
Ammonium S u l f a t e F r a c t i o n a t i o n o f Mouse P r o t e c t i v e
A n o t h e r s e r i e s o f e x p e r i m e n t s was d e s i g n e d t o e x ­
p l o r e some o f t h e p a r a m e t e r s o f t h i s mouse p r o t e c t i v e f a c 8
to r.
A l l t e s t a n im a l s were g i v e n I x 10 IMLNC from
3 - m o n t h - o l d BALB/ c o f e i t h e r s e x w hich had b e e n i n f e c t e d
w i t h 300 IL^ 48 and I 5 days b e f o r e t h e c e l l s w ere c o l l e c t e d .
Group I
Mice g i v e n NMLNC.
FECES x IO
180
DAYS POST INFECTION
F ig u re 4.
EPG f e c e s from m ice g i v e n IMLNC and p o o l s o f immune serum w hich
w ere f o u n d t o be "worm p r o t e c t i v e ".
Group I
a s F i g u r e 4.
Group 2
as F ig u re 4.
Group 4 IMLNC ( a s g r o u p 2) and I ml serum
g i v e n IV on day O from a p o o l c o l l e c t e d
17 days a f t e r d o n o r i n f e c t i o n .
Z
EG G S/g FECES x IO3
Group 5 IMLNC ( a s g ro u p 2 ) and I ml serum
g i v e n IV from a p o o l c o l l e c t e d from t h e
IMLNC d o n o rs when t h e y w ere k i l l e d .
-Fr
-o
5.5
F i g u r e 5.
6
6,5
7.5
8.5
DAYS POST INFECTION
9.5
EPG f e c e s from m ice g i v e n IMLNC and p o o l s o f immune serum w hich
w ere f o u n d t o he "mouse p r o t e c t i v e " .
48
A new p o o l o f mouse p r o t e c t i v e serum was u s e d .
I t was from
t h e same m ice from w h ic h t h e IMLNC w ere t a k e n .
The serum
was p r e c i p i t a t e d once w i t h 5 0 ^ ammonium s u l f a / t e and t h e
a l b u m i n - r i c h s u p e r n a t e n t and t h e g l o b u l i n - r i c h p r e c i p i t a t e
w ere d i a l i z e d a g a i n s t p h o s p h a t e b u f f e r e d s a l i n e a n d r e s u s - •
p e n d e d t o t h e o r i g i n a l volum e.
I ml o f t h e immune se ru m .
One g r o u p o f m ice was g i v e n
A s e c o n d g r o u p was g i v e n I ml o f
t h e g l o b u l i n - r i c h an d a t h i r d g r o u p o f 5 was g i v e n I ml o f
t h e a l b u m i n - r i c h s u p e r n a t e n t ( T a b le V I I I ) .
The p r o t e c t i v e a c t i v i t y was f o u n d o n l y i n t h e g l o b u l i n ric h fra c tio n .
T h is e x p e r i m e n t a l s o shows t h e mouse p r o t e c ­
t i v e f a c t o r i s d i a l i z a b l e an d i s n ' t i r r e v e r s i b l y d e n a t u r e d
by ammonium s u l f a t e p r e c i p i t a t i o n .
c)
w ith IL ^ .
Mouse P r o t e c t i v e Serum A d s o rb e d on IL^ a n d A b so rb ed
The n e x t e x p e r i m e n t s w ere d e s i g n e d t o f i n d o u t :
a ) i f t h e p r o t e c t i v e f a c t o r i n serum c o u l d be a d s o r b e d o n to
l a r v a e _in v i t r o and a l l o w immune c e l l s t o a c t on t h e l a r v a e •
when t h e y w ere i n j e c t e d s u b c u t a n e o u s l y i n t o m i c e , an d b) i f
t h e a c t i v i t y o f an immune serum c a n be a b s o r b e d o u t u s i n g a
I
l a r g e num ber o f i n f e c t i o u s l a r v a e .
The e x p e r i m e n t a l d e s i g n
w as:
1
Group I - 300 n o rm a l IL J
Group 2 - IMLNC d o n o rs i n f e c t e d w i t h 300 IL^ 28 and I 4
T a b le V I I I .
A c t i v i t y o f ammonium s u l f a t e f r a c t i o n s o f a mouse p r o t e c t i v e
seru m .
Group I
C o n tro l
Group 2
IMLNCa
Group 3
IMLNCa
Immune
Serum"b
Group 4
IMLNCa
A lb u m in - R ic h c
F ra c tio n of
Immune Serum
Group 5
IMLNCa
G lo b u lin -R ic h c
F ra c tio n of
Immune Serum
Day
6
5,100
1 9 ,2 0 0
0
8,700
100
Day
7
15,000
31,000
100
3 5 ,1 0 0
1,600
Day
8
2,500
17,600
0
15,200
1,300
Day
9
100
4 ,6 0 0
0
10,500
0
Day. 10
.0
600
'0
0
0
a Mice r e c e i v e d I x i o IMLNC fro m m ic e g i v e n 300 ILo 48 a n d 15 days b e f o r e
, t h e IMLNC w ere c o l l e c t e d .
^
I ml o f serum g i v e n IV t o e a c h mouse from a p o o l made from serum c o l l e c t ­
ed from m ic e i n f e c t e d w i t h 300 ILo 48 and 15 d a y s b e f o r e b l e e d i n g .
c The seru m ( b ) was p r e c i p i t a t e d w i t h 5 0 # ammonium s u l f a t e and b o t h t h e
p r e c i p i t a t e ( g l o b u l i n - r i c h ) and t h e s u p e r n a t e n t ( a l b u m i n - r i c h ) w ere
d i a l y z e d and r e c o n s t i t u t e d t o t h e s t a r t i n g serum volum e an d t h e e q u i v a ­
l e n t o f I ml seru m was g i v e n t o e a c h mouse i n g r o u p s 4 an d 5 fro m e i t h e r
th e a lb u m in -ric h f r a c t i o n o r th e g l o b u l i n - r i c h f r a c t i o n .
50
d a y s b e f o r e MLNC w ere c o l l e c t e d .
IL 3 .
300 n o rm al
Group 3 - IMLNC ( a s g ro u p 2 ) and I ml o f mouse p r o t e c ­
t i v e - serum g i v e n 6 h o u r s b e f o r e i n f e c t i o n
w i t h 300 n o r m a l IL 3 .
Group 4 - IMLNC ( a s g r o u p 2 ) and I ml o f t h e p o o l o f
se ru m u s e d i n g ro u p 3 a f t e r a b s o r p t i o n w i t h
4 6 ,2 0 0 IL 3 .
Group 5 - IMLNC ( a s g ro u p 2) and 300 IL 3 w h ic h h a d b e e n
a d s o r b e d i n serum from t h e same p o o l a s was
g i v e n g ro u p 3 Group 6 - IMLNC ( a s g ro u p 2 ) and 300 I L 3 w h ic h h a d b e e n
a d s o r b e d i n NMS.
^
The EPG d a t a ( T a b le IX ) s u g g e s t
i n immune serum w ere a b l e t o i n i t i a t e
t h a t la r v a e in c u b a te d
a n o rm a l i n f e c t i o n ,
ev en i n m ice g i v e n IMLNC from day I 4 a f t e r a s e c o n d i n f e c ­
tio n .
F u r t h e r m o r e , t h e serum w a s n ’ t d e p l e t e d o f i t s p r o t e c ­
t i v e f a c t o r u s i n g e v e n l a r g e num bers o f l a r v a e .
T h is i s
v e r y i n c o n c l u s i v e b e c a u s e t h e serum w a s n ’ t t i t r a t e d and t h e
I ml o f serum g i v e n may h a v e b e e n more t h a n n e c e s s a r y .
Such
a s m a l l volum e o f l a r v a e may have r e s u l t e d i n i n c o m p l e t e a b ­
s o r p t i o n and m ig h t n o t have b e e n a d e q u a t e ' t o show a d i f - .
fe re n c e in th e a ssa y used.
I b e l i e v e t h e s e d a t a ( T a b le IX) show t h a t t h e mouse
p r o t e c t i v e f a c t o r w o n 't a d s o r b o n to l a r v a e i n a way t h a t
a d o p t i v e l y t r a n s f e r r e d im m unity c an a c t on t h e l a r v a e .
The
s e c o n d p a r t o f t h e e x p e r i m e n t p r o v e d h a r d t o do c o r r e c t l y a s
T a b le IX.
The e f f e c t o f i n v i t r o i n c u b a t i o n i n p r o t e c t i v e serum on IL~
and t h e a b i l i t y o f i n v i t r o i n c u b a t i o n o f ILo t o d e p l e t e serum
of p ro te c tiv e f a c to r.
^
Group I a
Group, 2
IMLNCd
NMS
Group, 3 a
IMLNCd
IMSc
Group, 4 a
IMLNC
A b s o rb e d
IMSd
EPG Day 6
29,300
36/200
O
EPG Day 7
72,800
46,700
O
EPG Day 7 . 5
38,200
1 4 ,2 0 0
Worms
R e c o v e re d
On Day 7 .5
261,192,
139,40
216,194,
149
A v e rag e
158±93
186+34
100
■5
I
3+2.8
.
G r o u p ,5
IMLNC
I L q-Ads
IMSe
Group, 6
IMLNC
ILq-A ds
NMSf
0
35,000
30,000
0
49,500
42,800
0
11,100
1 0 ,0 0 0
0
3
-
-
I . 5±2. I
, 300 n o r m a l I I g .
d I x 108 IMLNC c o l l e c t e d from d o n o r s w h ich h a d b e e n i n f e c t e d w i t h 300 IL 3 .
c P r o t e c t i v e p o o l o f immune serum from m ice i n f e c t e d 48 and 15 days b e f o r e
, b l e e d i n g . ' O.'5 ml IV and 0 .5 IP g i v e n 6. h o u r s b e f o r e i n f e c t i o n .
E ach mouse was g i v e n I ml o f se ru m from t h e p o o l g i v e n t o g ro u p 3 w hich
■ h a d b e e n a b s o r b e d w i t h 4 6 ,2 0 0 I L y f o r 2 h o u r s .
e E ach mouse r e c e i v e d 3 0 ° IL 3 w h ic h h a d b e e n i n c u b a t e d f o r 3-0 m in u te s i n
„ serum fro m t h e p o o l g i v e n t o g r o u p 3 «
1 300 ILo w h ic h h a d b e e n i n c u b a t e d i n NMS.
Group I v s , 2 = NS ■
Group 4 v s . 5 = NS
Group 1+3 v s . 4+5 = P< . 05
52
i t i s t e c h n i c a l l y d i f f i c u l t t o g e t t h e l a r g e num ber o f
l a r v a e t h a t w ould be n e c e s s a r y t o a d e q u a t e l y a b s o r b enough
serum t o be u s e d i n t h i s a s s a y .
A d o p tiv e T r a n s f e r w i t h IMLNG t o Mice o f Im m unity t o
N. b r a s i l j e n s i s L a r v a l I n f e c t i o n W ith o u t
Immune Serum
The immune c e l l s u s e d i n t h e above e x p e r i m e n t s had b e e n
c o l l e c t e d a b o u t day I 4 a f t e r a s e c o n d l a r v a l i n f e c t i o n o f
donors.
H a v in g a p o o l o f known p r o t e c t i v e se ru m a v a i l a b l e ,
I w a n te d t o f i n d o u t i f 2 i n f e c t i o n s , w ere n e c e s s a r y t o p r o ­
duce IMLNG c a p a b l e o f c o o p e r a t i n g w i t h p r o t e c t i v e serum to
c a u s e a b r o g a t i o n o f t h e p a t e n c y o f an i n f e c t i o n .
Two p o o l s o f c e l l s w ere p r e p a r e d t o t e s t t h e a b i l i t y o f
immune c e l l s t o c o - o p e r a t e w i t h immune serum'.
One p o o l was
from m ice w h ic h h a d b e e n g i v e n a 300 IL^ p rifila ry i n f e c t i o n
9 days b e f o r e c e l l c o l l e c t i o n .
The s e c o n d p o o l was p r e p a r e d
by g i v i n g 2 i n f e c t i o n s o f 300 IL^ t o t h e d o n o r m ic e on days
32 a n d 9 b e f o r e t h e c e l l s w ere c o l l e c t e d .
B o th p o o l s o f
c e l l s w ere g i v e n t o m ic e w i t h and w i t h o u t known mouse p r o ­
t e c t i v e serum a s shown b e lo w :
Group I - C o n t r o l , 300 IL^ day 0 o n l y .
8
Group 2 - E ach mouse r e c e i v e d I x 10 IMLNC from p r i ­
m ary i n f e c t i o n d o n o r s .
53
Group 3 - E ach mouse r e c e i v e d I x 10® IMLNC a s g ro u p 2
an d I ml o f a known p r o t e c t i v e p o o l o f serum .
Group 4
Euch, mouse r e c e i v e d I x 10® IMLNC from d o n o r
m ice g i v e n 2 l a r v a l i n f e c t i o n s .
Group 5 - E ach mouse r e c e i v e d IMLNC a s g r o u p 4 and I ml
o f a known mouse p r o t e c t i v e p o o l o f serum .
A l l r e c i p i e n t g r o u p s w ere t h e n g i v e n 300 IL t h e same day t h e y w ere g i v e n IMLNC.
^
The r e s u l t s o f t h i s e x p e r i m e n t ( T a b le X) w ere s u r p r i s ­
i n g b e c a u s e t h e c e l l s from, day 9 o f t h e p r i m a r y i n f e c t i o n
seem ed t o be v e r y e f f e c t i v e by t h e m s e l v e s a t e l i m i n a t i n g
worms, w h e re a s ( a s h a d b e e n f o u n d i n p r e v i o u s work ( F i g u r e s
3 & 4 , T a b le V I I I ) ) t h e c e l l s from t h e s e c o n d a r y i n f e c t i o n
w e r e n 't.
The n e x t s t e p s u g g e s t e d by t h e s e r e s u l t s was to
t r a n s f e r c e l l s a t d i f f e r e n t d ays a f t e r an i n f e c t i o n and s e e
on w h ic h days t h e c e l l s a r e a c t i v e by t h e m s e lv e s an d on
w h ich d a y s t h e y n e e d p r o t e c t i v e serum t o e f f e c t e x p u l s i o n
o f a worm b u r d e n .
Groups o f 8 - w e e k - o l d BALB/c f e m a le m ice w ere i n f e c t e d
w i t h 300 IL^ a t 2 day i n t e r v a l s so t h a t a t day 0 c e l l s
c o u ld be c o l l e c t e d from i n f e c t i o n s o f 4 , 6 , 8 , 1 0 , 12 and
I 4 d ays d u r a t i o n .
From t h e s e d o n o r m ic e , IMLNC w ere t r a n s ­
f e r r e d i n t o r e c i p i e n t m ice and a l l r e c i p i e n t s w ere i n f e c t e d
w i t h 300 IL^ on t h a t same d a y ,
and 7 p o s t - i n f e c t i o n .
EPG w ere c o u n te d on days 6 .
The m ice w e re k i l l e d on day 8 . 5 t o
T able X.
EPG
The a b i l i t y o f IMLNC c o l l e c t e d a f t e r a prim ary o r se co n d a ry i n ­
f e c t i o n o f N. b r a s i l i e n s l s g iv e n w ith o r w ith o u t p r o t e c t i v e serum
to e f f e c t a d o p tiv e t r a n s f e r o f p r o t e c t i o n from a 3 0 0 IL ^■c h a l ­
le n g e to n a iv e m ic e .
Group I a
C o n tro l
Group 2a
P rim ary
IMLNC
Group 3
Serumb
P rim ary
IMLNC
Group 4 a S e c o n d a ry
IMLNC
Group 5
Serum " ,
S e c o n d a ry
IMLNC
Day
6
69,900
1,700
0
1 4 ,7 0 0
2,300
Day
7
86,100
3,300
0
55,600
4 ,2 0 0
Day
8
6 0 ,4 0 0
2,575
100
62,800
6,000
Day
9
25,700
900
100
30,400
2,000
Day 10
19,900
0
100
14,900
0
Worms
R e c o v e re d
On Day 10
235,212,
209,205,
171,170,
95,61
1 6 9 . 8+
61.2
A v erag e
20
10
0
0
7.5± 9.6
, A l l m ic e r e c e i v e d 300 IL^ on day
D I ml IY o f known p r o t e c t i v e p o o l
^ l x 10° IMLNC fro m m ice i n f e c t e d
11 I x 108 IMLNC fro m m ice i n f e c t e d
tra n sfe r.
Group I v s . 4 =■NS
Group I v s . . 2 = P< . 05
.
I
0
.
• 5±1
-
205
149
57
10
5
4
1 3 6 .7 ± 7 4
6 . 313.2
0.
o f serum on day 0.
w i t h 300 I L 3 9 days b e f o r e t r a n s f e r ,
w i t h 300 IL 3 on days 32 and 9 .b e f o r e
Group 2 v s . 3 = NS
Group 4 v s . 5 = P < - 05
55
r e c o v e r worms ( T a b le X I ) .
The egg c o u n t an d worms r e c o v e r e d d a t a s u g g e s t t h a t i f
t h e m ice h a d b e e n k i l l e d a day e a r l i e r t h e r e w o u ld have b e e n
a more n o t i c a b l e d i f f e r e n c e i n t h e worm c o u n t s .
The worm
r e c o v e r y d a t a a r e c o n s i s t e n t w i t h a l l immune c e l l s i n c r e a s ­
i n g e x p u l s i o n by day 8 . 5 b u t t h e EPG f e c e s s u g g e s t t h a t day
8 and 10 c e l l s w ere a c t i v e e a r l i e r t h a n any o f t h e o t h e r
c e ll p re p a ra tio n s.
The p r e v i o u s e x p e r i m e n t was r e p e a t e d b u t t h i s tim e day
10 c e l l s w ere t i t r a t e d
( T a b le X II & T a b le X I I I ) .
The worms
r e c o v e r e d i n t h i s e x p e r i m e n t have b e t t e r s e p a r a t i o n among
g r o u p s , p o s s i b l y b e c a u s e t h e m ice w ere k i l l e d a day e a r l i e r .
The day 8 c e l l s show v e r y l i t t l e
a c t iv i t y in th is e x p e ri­
m en t, w h ic h d e m o n s t r a t e s t h e v a r i a b i l i t y o f t e n o b s e r v e d i n
t h i s s y s te m .
The day 10 IMLNC g ro u p i s t h e o n l y g ro u p i n '
w h ic h t h e worms r e c o v e r e d d a t a i s s i g n i f i c a n t l y d i f f e r e n t
fro m t h e c o n t r o l a t t h e P C .05 l e v e l , b u t t h e day 12 c e l l
g ro u p i s s i g n i f i c a n t l y d i f f e r e n t a t t h e P < .0 6 l e v e l ,
sug­
g e s t i n g t h a t t h e tim e t h a t t h e c e l l s from t h e MLN w ere a b l e
t o c a u s e e x p u l s i o n s h i f t e d from b e f o r e day 10 t o a f t e r day
10 i n t h i s e x p e r i m e n t .
I b e l i e v e t h a t a c e l l p o p u l a t i o n was e i t h e r m i g r a t i n g
t h r p u g h t h e MLN on d ays 8 -1 2 o r was b e i n g p r o d u c e d i n t h e
T able XI.
E xp erim en t A. The a b i l i t y o f MLNC c o l l e c t e d on d i f f e r e n t days
a f t e r . a p rim ary i n f e c t i o n o f m ice to a d o p t iv e ly p r o t e c t n a iv e
m ice from a c h a lle n g e w ith 3 0 0 I L ^ .a
Grp I
Grp 2^
Day 4
MLNC
Grp 3°
Day 6
MLNC
Grp 4^
Day 8
MLNC
Grp 5
Day 10
MLNC
Grp 6f
Day 12
MLNC
Grp 7^
Day 14
MLNC
EPG Day 6.
22,200
11,300
11,900
600
1,100
9,500
1 5 ,3 0 0
EPG Day 7
2 8 ,4 0 0
6,100
3 ,4 o o
200
500
8,900
7,400
6,900
1,300
1 , 100.
0
0
200
1 ,6 0 0
EPG Day 8 '
Worms
R e c o v e re d
A v e rag e
220,163,
128,89,
73,57
59,19,
30,0
10,126,
4 6 ,0
38,0,
0 ,0 ,0
1 ,0 ,0 , • 121, 50,
0 , 0,0
31 ,1 ,0
1 2 1 . 6±
6 1 .7
27±24.7
4 8 ± 5 5 .5
7 . 6+17
.2 ± .4 l
4 0 . 6±
57.3
1 1 6 ,4 3
23,1,0
3 6 . 6±
. 47.8
. A l l r e c i p i e n t m ic e w ere g i v e n 300 ILo on day 0.
,
,
A l l m ic e r e c e i v e d I x 10° MLNC from d o n o r m ice g i v e n 300 IL 3 4 , 6C, 8 ,
IOe , 12^ o r
d a y s b e f o r e t h e c e l l s w ere t r a n s f e r r e d .
T ab le X II.
E xp erim ent B. The a b i l i t y o f MLNC c o l l e c t e d on d if f e r e n t , days
a f t e r a prim ary i n f e c t i o n o f m ice to a d o p t i v e l y -p n o te c t n a iv e
m ice from a c h a lle n g e w ith 3 0 0 IL ^. a
Grp 4 d
Day 8
MLNC
Grp I
Grp Zb
Day 4
MLNC
EPG Day 6
29,400
25,300
2,900.
5,700 .
EPG Day 7 .
2 3 ,4 0 0
31,300
9,700
Worms
R e c o v e re d
On Day 7
,
A v e ra g e
253
207
181
150
139
64
1 6 5 . 7+
6 4 .6
167
165
148
144
100
63
1 3 1 . 2±
4 0 .4
Grp 6 ^
Day 12
MLNC
Grp 7^
Day 14
MLNC
0
1 4 ,1 0 0
1 7 ,4 0 0
7,400
0
7,200
1 2 ,1 0 0
157 '
143
111
106
21
223
142
123
118
■ 4l
9
I
I
I
I
0
78
150
144
112
88
49
' 4
226
170
157
112
82
IO7 . 5&
52.9
109+
76.1
91±
56.7
148±
55.3
Grp 3 °
Day 6
MLNC
Grp 5
Day 10
MLNC
1 3 . 7±
3 1 .6
A l l r e c i p i e n t m ic e w ere g i v e n 300 I L - on day 0.
,
,
A l l m ice r e c e i v e d I x 10° MLNC from d o n o r m ice g i v e n 300 IL 3 4 , 6° , 0 ,
IOe , 12^ o r l 4 § d a y s b e f o r e t h e c e l l s w ere t r a n s f e r r e d .
Worms r e c o v e r e d I v s . 2 , 3 , 4 , 7 = NS'
I y s . 5 = P<.03
I v s . 6 = P < , 06
T able X I I I .
The a b i l i t y o f MLNC c o l l e c t e d from donor m ice on d i f f e r e n t
days after* a se c o n d a r y i n f e c t i o n w ith JOO IL 3 to p r o t e c t n a iv e
r e c i p i e n t m ice from a c h a lle n g e w ith l a r v a e .
Group I
Group 213
Day 3
MLNC
EPG Day 6
10,300
1 4 ,9 0 0
2,200
8,000
9,100
. 4 ,5 0 0
EPG Day 7
15,300
18,100
4 ,6 0 0
6,000
9,000
6,300
EPG Day 8
9,700
8,700
900
800
5,900
1 ,2 0 0
Worms
R e c o v e re d
A v e ra g e
Group 3°
Day 6
MLNC
Group 4
Day 9
MLNC
1 3 4 ,1 0 3 ,
95,85,78
1 1 6 ,1 0 9 ,
85,85,71
9 1 ,8 4 ,5 4
4 3 ,3 4
9 9 + 2 1 .8
9 2 . 2+
1 9 .4
6 1 . 2+2 5 . 1 7 2 .4 + 2 3 .5
109,81,65
5 8 ,4 9
Group 5 & Group 6^
Day 11
Day 13
MLNC
MLNC
108,88,78,
70,2
77,69,50
49.28
69.2+40.2
5J.6±
18.3
a A l l r e c i p i e n t m ic e w ere g i v e n 300 IL ^ on day 0.
A l l d o n o r m ice w ere i n f e c t e d on day 29 b e f o r e t r a n s f e r a n d a se c o n d i n f e c ­
t i o n was g iv e n , on d a y s 3 » 6 ° , 9 , I l e o r 13 ■ A l l m ice a l s o r e c e i v e d
I x 10° IMLNC on day 0.
Worms r e c o v e r e d
I v s . 2 , 4 , 5 = NS
I v s . 3 , 6 = P< . 0 5
59
MLN Ly d i v i s i o n and t h a t t h i s p o p u l a t i o n m ig h t be t h e c e l l
t h a t i s r e s p o n s i b l e f o r p r o d u c i n g t h e serum f a c t o r w hich i s
t h e l i m i t i n g f a c t o r i n p r i m a r y worm i n f e c t i o n r e j e c t i o n as
shown by worm t r a n s f e r e x p e r i m e n t s ( 2 8 , 2 9 , 4 4 ) .
To f i n d o u t
i f t h e r e i s an a n a l o g o u s c o l l e c t i o n o f c e l l s i n t h e MLN d u r ­
in g th e second i n f e c ti o n ,
a n e x p e r i m e n t was d e s i g n e d so t h a t
c e l l s w ould be t r a n s f e r r e d on d i f f e r e n t days a f t e r t h e
second in f e c ti o n .
Donor m ice w ere i n f e c t e d w i t h 300 IL^
29 d ays b e f o r e c e l l s were t r a n s f e r r e d .
Groups o f t h e s e m ice
w ere t h e n r e i n f e c t e d w i t h 300 IL^ on 13, 11, 9 , 6 o r 3 days
b e f o r e t h e y w ere u s e d a s c e l l d o n o r s .
Mice i n a l l e x p e r i 8
m e n t a l g r o u p s r e c e i v e d .1 x 10 IMLNC from t h e a p p r o p r i a t e
d o n o rs on day 0.
EPG f e c e s w ere m o n i t o r e d 6, ? and 8 days
a f t e r IMLNC t r a n s f e r and 300 IL^ i n f e c t i o n .
Worms w ere r e ­
c o v e r e d 8 days a f t e r t h e t r a n s f e r and i n f e c t i o n ( T a b le
X III).
Compared w i t h c e l l s . f r o m a p r i m a r y i n f e c t i o n w i t h
N. b r a s i l i e n s i s , t h e r e was a l o t l e s s p r o t e c t i o n w i t h c e l l s
from a s e c o n d i n f e c t i o n .
C e l l s t r a n s f e r r e d day 6 and day 13
a f t e r t h e s e c o n d i n f e c t i o n c a u s e d s i g n i f i c a n t ( P < .0 5 ) r e d u c e
tio-n o f worm r e c o v e r y by day 8 w h ic h i s a b o u t w h a t h a s b e e n '
s e e n by o t h e r s w i t h immune c e l l s a l o n e (2 8 ,2 9 )<
I n t h e s e 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 , I was l o o k i n g
f o r a more s i g n i f i c a n t d ro p i n worm c o u n ts on day 7 ' o r 8
6o
b e c a u s e t h i s b o r d e r l i n e l e v e l o f e x p u l s i o n i s commonly fo u n d
i n t h e r a t s y s te m when immune c e l l s a r e t r a n s f e r r e d ( 2 8 , 2 9 ) •
The i n t e r e s t i n g o b s e r v a t i o n i n t h e s e e x p e r i m e n t s was t h a t
p o o l s o f c e l l s from b o t h p r i m a r y i n f e c t i o n s c a u s e d t h e c h a l ­
l e n g e i n f e c t i o n t o be n o n p a t e n t an d t o g r e a t l y r e d u c e t h e
worm b u r d e n on d a y s 7 an d 8 ( T a b le s X I, X I I ) .
T hese a r e t h e
r e s u l t s t h a t w ould be e x p e c t e d i n a mouse im m unized by a
n o rm a l i n f e c t i o n .
None o f t h e p o o l s o f c e l l s c o l l e c t e d
a f t e r th e second i n f e c t i o n ap p ro ach ed th e s e c r i t e r i a f o r
a d o p t i v e t r a n s f e r o f im m u n ity .
T hese r e s u l t s d e m o n s t r a t e
t h a t t h e r e i s in a p rim a ry i n f e c t i o n a h ig h p e rc e n ta g e o f
c e l l s t h a t , when t r a n s f e r r e d t o n a i v e m ic e , c an c a u s e a
c h a l l e n g e i n f e c t i o n t o become n o n p a t e n t and t h a t t h e p e r ­
c e n t a g e d r o p s a f t e r day 10 o r 12 a n d a p p a r e n t l y d o e s n ' t
re a c h as h ig h a p e rc e n ta g e in a seco n d ary i n f e c t i o n .
M e s e n t e r i c L ym phadenopathy O b se rv e d D u r in g
N. b r a s i l i e n s i s L a r v a l I n f e c t i o n
An i n t e r e s t i n g o b s e r v a t i o n made i n t h e s e e x p e r i m e n t s
( T a b le s XI, X I I , X I I I ) was t h e ly m p h a d e n o p a th y t h a t o c c u r r e d
d u r i n g t h e i n f e c t i o n i n t h e IMLNC d o n o r s .
The t o t a l num ber
o f c e l l s r e c o v e r e d from t h e m e s e n t e r i c lymph n o d e s from
p r i m a r y i n f e c t i o n s o f v a r y i n g l e n g t h s i s p r e s e n t e d i n T a b le
XIV,
The num ber o f c e l l s r e c o v e r e d was l o w e s t on day 4 and
61
'No. o f C e l l s
R e c o v e re d
P rim ary ^
In fe c tio n 0
2 .3 7 x IO8
1 .5 7
X
6
3 .2 5
'X
CO
O
8
3 .1 5
X
3 .5 8
X
3 .4 5
X
CO
1 .2
x IO8
IO8
13
14
x IO8
T-4
11
12
2 .4
CO
O
X
3 .5 6
O
9
3 .7 5
4 .0 6 x IO8
O
OO
T—
I
4
O
CO
3
10
No. o f C e l l s
R e c o v e re d
S e c o n d a ry
In fe c tio n 0
Ki
Day A f t e r
L ast
In fe c tio n
The num ber o f n u c l e a t e d c e l l s r e c o v e r e d from t h e
MLN o f m ice a t v a r i o u s t i m e s a f t e r a p r i m a r y o r
s e c o n d a r y N^ b r a s i l i e n s i s i n f e c t i o n .
X
T a b le XIV.
IO^
The num ber o f c e l l s r e c o v e r e d from m e s e n t e r i c lymph n o d e s
p e r mouse on d a y s 3- a f t e r a p r i m a r y i n f e c t i o n .
c The num ber o f c e l l s r e c o v e r e d from m e s e n t e r i c lymph n odes
p e r mouse on d a y s 3- a f t e r a s e c o n d a r y i n f e c t i o n when t h e
f i r s t i n f e c t i o n was g i v e n 29 days b e f o r e .
62
i n c r e a s e d u n t i l day 10, t h e n d e c r e a s e d some b e f o r e day 14;
m o st o f t h e a c t i v i t y was. f o u n d i n t h e c e l l s c o l l e c t e d on
days 8 -1 2 ( T a b l e s XI, X I I ) .
MLN c o l l e c t e d on d i f f e r e n t d ays a f t e r a s e c o n d i n f e c ­
t i o n ( T a b l e X I I I ) a l s o showed ly m p h a d e n o p a th y w h ic h p e a k e d
e a r l i e r th a n th e p rim a ry i n f e c t i o n (day 6 v s .
day 10) ( T a b le
XIV) and by day 13 had r e t u r n e d t o n e a r n o rm a l s i z e
p o rted o b se rv a tio n ).
(u n re­
The p r o t e c t i v e c a p a c i t y o f c e l l s
t r a n s f e r r e d d u r i n g i n f e c t i o n w i t h Njl b r a s i l i e n s i s a p p e a r s t o
c o r r e l a t e somewhat w i t h t h e ly m p h a d e n o p a th y ( T a b l e s XI, X I I ,
X III).
The c e l l s r e c o v e r e d from t h e l a r g e r n o d e s a r e more
p r o t e c t i v e t h a n e q u a l num bers o f c e l l s from s m a l l e r n o d e s ,
e x c e p t day 13 a f t e r t h e s e c o n d i n f e c t i o n when t h e c e l l s were
s i g n i f i c a n t l y p r o t e c t i v e even a f t e r i n v o lu t i o n had o c c u rre d
( T a b l e s X I I I , X IV ).
V a r io u s Doses o f Y - I r r a d i a t i o n o f Mice on S u b s e q u e n t IL C h a ll e n g e an d t h e R e s t o r a t i o n o f Normal E x p u l s i o n ^
w i t h IMLNC i n I r r a d i a t e d Mice
To f u r t h e r c h a r a c t e r i z e t h e f a c t o r - p r o d u c i n g c e l l ,
r a d i o r e s i s t a n c e o f worm e x p u l s i o n was d e t e r m i n e d .
th e
T h is was
done by i r r a d i a t i n g m ice a t d i f f e r e n t d o s e s and t h e n i n f e c ­
t i n g them w i t h 300 IL^ and m o n i t o r i n g egg c o u n ts t o d e t e r ­
m ine when e x p u l s i o n o c c u r r e d .
Groups o f 5 m ice w ere g i v e n
63
e i t h e r 100, 2 5 0 , 3 5 0 , 4-50 o r 55° r a d s o f y - i r r a d i a t i o n 24h o u r s b e f o r e b e i n g i n f e c t e d w i t h 300 I L ^ .
\
'
EPG w ere m o n i- •
^
t o r e d b e g i n n i n g on day 6 ( T a b le XV).
These d a t a show t h a t 4-50 r a d s w ere r e q u i r e d t o c a u se
any d e l a y i n e x p u l s i o n o f t h e worm b u r d e n , and 550 r a d s w ere
r e q u i r e d t o g e t any a p p r e c i a b l e d e l a y o f e x p u l s i o n .
su g g e sts t h a t th e c e l l ( s )
T h is
in v o lv e d in f a c t o r p ro d u c tio n a re
v e r y r a d i o r e s i s t a n t and can f u n c t i o n n o r m a l l y a f t e r m ice
r e c e i v e 350 r a d s o f i r r a d i a t i o n .
As a s e c o n d p a r t o f t h i s e x p e r i m e n t , g ro u p s o f 3 a n i Q
m als from e a c h i r r a d i a t i o n d o se w ere g i v e n I x 10
IMLNC
from a day 10 p r i m a r y i n f e c t i o n t o a t t e m p t t o r e p a i r th e
e x p u l s i v e m echanism .
In a p re v io u s ex p erim e n t,
immune c e l l s
p r e p a r e d i n t h e same m anner t r a n s f e r r e d t h e a b i l i t y o f im­
mune e x p u l s i o n t o n o r m a l m ice by t h e m s e l v e s .
In t h is ex p er­
im e n t, a s shown i n T a b le XVI, Group 2 , t h o s e r e s u l t s w e r e n ' t
r e p r o d u c a b l e , h o w e v er t h e s e c e l l s w ere a b l e t o r e p a i r t h e
e x p u l s i v e m echanism i n t h e 4-50 r a d g ro u p and c a u s e e x p u l s i o n
t o bq e f f e c t e d 6 o r 7 d ays e a r l i e r i n t h e 550 r a d g ro u p
( T a b le XV I).
The e x p u l s i v e m echanism i n m ice f u n c t i o n e d
n o r m a l l y i n m ice r e c e i v i n g 350 r a d s o r l e s s o f ^ - i r r a d i a t i o n ,
was d e l a y e d v e r y l i t t l e
i n m ice r e c e i v i n g 4-50 r a d s and was
s e v e r e l y d e l a y e d i n 550 r a d - i r r a d i a t e d m ice ( T a b l e XVI) .
T a b le XV.
The e f f e c t on t h e e x p u l s i o n o f worms o f d i f f e r e n t d o s e s o f
Y - I r r a d i a t i o n g i v e n b e f o r e a N. b r a s i l i e n s i s l a r v a l . i n f e c t i o n .
Group I
C o n tro l
No. o f Mice
Group 2 ■ Group 3
250 r a d
100 r a d d
6
6
6
Group 4
350 r a d
'
6
Group 5
450 r a d
Group 6
550 r a d
6
6
EPG Day
6b
20,300
33,900
61,700
52,700
59,ooo
58 ,.400
EPG Day
7
27,900
50,000
82,200
72,200
9 2 ,4 0 0
1 1 8 ,8 0 0
EPG Day
8
11,700
25,300
28,800
8 0 ,0 0 0
1 0 9 ,8 0 0
EPG Day 10
0
2,500
0
16,100
1 10,0 00
.0
' 64,000
EPG Day 14
8,200 .
0
EPG Day 18
3 7 ,6 0 0
EPG Day 21
2 ,7 0 0
^ Mice w ere g i v e n i n d i c a t e d d o s e s o f i r r a d i a t i o n 24 h o u r s b e f o r e i n f e c t i o n .
Day p o s t 300 IL^ i n f e c t i o n .
T a b le XVI.
The a b i l i t y o f day 10 IMLNC t o r e p a i r t h e e x p u l s i v e m echanism
o f m ic e g i v e n 450 or- 550 r a d s / - i r r a d i a t i o n b e f o r e a l a r v a l
c h a lle n g e .
Group I
C o n tro l
No. o f Mice
6
Group 2 Gr oup 3
,
450 R adsa
IMLNC IMLNC
3
3
Group 4
450 Rads
No C e l l s
Gr oup 5
550 Rads
IMLNC
Group 6
550 Rads
No C e l l s
6
3
6
EPG Day
6C
20,300
11,800
5,4oo
52,700
■2 0 , 3 0 0
•58,400
EPG Day
7
27,900
15,000
6,300
72,200
32,300
1 1 8 ,8 0 0
EPG Day
8
11,700
3,100
8,400
28,800
15,100
1 0 9 ,8 0 0
EPG Day 10
0
0
100
0
11,600
1 1 0 ,0 0 0
3,500
64,000
EPG Day 14
a Rads ^ - i r r a d i a t i o n g i v e n t o m ic e 24 h o u r s b e f o r e i n f e c t i o n .
IMLNC— I x 108 from d o n o r m ic e g i v e n a p r i m a r y i n f e c t i o n 10 days e a r l i e r .
c Day p o s t 300 IL^ i n f e c t i o n .
66
T h is d e f e c t i n e x p u l s i o n c o u ld "be r e p a i r e d t o n o rm a l i n m ice
g i v e n 450 r a d s and p a r t i a l l y r e p a i r e d i n m ice g i v e n 550 r a d s
"by g i v i n g IMLNC fro m d o n o r m ic e g i v e n a p r i m a r y JOO I L y i n ­
f e c t i o n 10 d ays b e f o r e c e l l t r a n s f e r ( T a b le XVI).
An e x p e r i m e n t was done t o s e e i f n o r m a l th y m o c y te s
c o u ld r e p a i r t h e worm e x p u l s i v e c a p a c i t y o f 550 r a d - i r r a d i a t e d m ic e .
F iv e g r o u p s made up o f 8 - w e e k - o l d BALB/c m ice
w ere u s e d ;
Group I r e c e i v e d o n l y 300 IL^ on day 0, a s d i d
a l l o t h e r g r o u p s ; Group 2 r e c e i v e d I x 10® IMLNC from d o n o r
m ice g i v e n a p r i m a r y l a r v a l i n f e c t i o n 9 days b e f o r e t r a n s f e r
of c e lls ;
Group 3 r e c e i v e d 550 r a d s
^ - i r r a d i a t i o n ; Group 4
8
r e c e i v e d 5 5 ° r a d s ^ - i r r a d i a t i o n a n d a l s o I x 10 th y m o c y te s
from 3 - w e e k - o l d s y n g e n e i c d o n o r s ; Group 5 r e c e i v e d 550 r a d s
^ - i r r a d i a t i o n and I x 10® IMLNC a s Group 2 d i d .
m o n i t o r e d b e g i n n i n g on day 7 .
EPG were
A g a in t h e day 9 IMLNC d i d n ’ t
c a u s e e x p u l s i o n by t h e m s e lv e s i n n o rm a l m ice ( T a b l e X V II;
Group 2 ) , b u t t h e y w ere a b l e t o r e c o n s t i t u t e 550 r a d i r r a d i a t e d m ice t o g i v e a n e a r n o r m a l e x p u l s i o n tim e (Group
5).
The i n t e r e s t i n g g ro u p i n t h i s e x p e r i m e n t was Group 4
w hich was g i v e n th y m o c y te s ( T a b l e XVII, Group 4 ) .
Mice i n
t h i s g r o u p e x p e l l e d t h e i r worms by day I 4 a l t h o u g h when
p la q u e d a g a i n s t sheep re d b lo o d c e l l s
( S R B C s ) w h ic h .w e r e
g i v e n on day 1 8 , b o t h o f t h e s e m ice showed a g r e a t l y r e d u c e d
T a b le X V II.
The a b i l i t y o f day 9 IMLNC o r n o r m a l th y m o c y te s t o r e c o n s t i ­
t u t e t h e a b i l i t y o f m ice g i v e n 550 r a d s o f ( - i r r a d i a t i o n
. 'b e f o r e a l a r v a l c h a l l e n g e t o e x p e l t h e I n f e c t i o n .
Group I
C o n tro l
No. o f Mice
5 .
Group 2
IMLNCb
5
Group 3
550 R adsa
Group 4
550 R adsa
T hym ocytes0
5
5
Group 5
550 Rads'
IMLNC
5
EPG Day
?d
23,300
45,900
73,900
93,200
26,800
EPG Day
8 '
4 , 800
22,200
56,100
1 1 6 ,2 0 0
21,600
0
0
62,100
55,900
700
1 0 7 ,8 0 0
3 0 ,4 o o
600
EPG Day I 4
85,400
6oo
0
EPG Day I 6
44,300
EPG Day 18
33,500
EPG Day 20
30,800
EPG Day 22
42,200
EPG D a y .10
EPG Day 12
■
0
f- ^ - i r r a d i a t i o n g i v e n 24 h o u r s b e f o r e c e l l t r a n s f e r an d l a r v a l c h a l l e n g e ,
b I x IO8 IMLNC fro m d o n o r m ic e i n f e c t e d 9 d ays b e f o r e t r a n s f e r .
° I x IO8 th y m o c y te s from 3 - w e e k - o l d s y n g e n e i c d o n o r s .
Days' a f t e r 3 00 IL ^ i n f e c t i o n .
68
T a b le X V III.
D i r e c t p l a q u e - f o r m i n g c e l l s (PFG) a g a i n s t SRBC
on day U a f t e r a n t i g e n c h a l l e n g e i n m ice g i v e n
550 r a d s and I x 10° th y m o c y te s 18 d ays b e f o r e =
Normal C o n t r o l
550 Rads an d
a
Thymocyte R e c o n s t i t u t e d
P F C /S p le e n
PFC/106
2 1 1 ,2 5 0 1 1 7 ,6 2 8
660+8 5 . 0
7 , 688+265
a Two m ice from Group 4 i n T a b le XVII.
4 2 .0 + 2 4 .0
69
a b i l i t y t o p r o d u c e d i r e c t p l a q u e s t o t h i s th y m u s - d e p e n d e n t
a n t i g e n ( T a b le X V I I I ) .
T h is e x p e r i m e n t shows . t h a t m ice w hich a r e g i v e n thymo­
c y t e s a f t e r 550 r a d s ^ - i r r a d i a t i o n c an e x p e l a l a r v a l i n f e c ­
tio n of
b ra s ilie n s is
( T a b le X V II, Group 4 ) b e f o r e t h e i r
a n t i b o d y - p r o d u c i n g p o t e n t i a l i s r e c o v e r e d ( T a b le X V I I I ) .
IMLNC, e v e n th o u g h n o t c a u s i n g a b r o g a t i o n o f p a t e n c y i n n o r ­
m al m ice ( T a b le X V II, Group 2 ) , w ere a b l e t o r e p a i r 550 r a d i r r a d i a t e d m ice t o a n e a r n o r m a l e x p u l s i o n tim e ( T a b le XVII,
Group 5 )•
Immune Serum T e s t e d F o r I t s M o u s e - P r o t e c t i v e
A c t i v i t y When G iven A lone
The immune c e l l s I had b e e n u s i n g t o c o l l a b o r a t e w i t h
t h e immune serum d i d n ’ t c a u s e e x p u l s i o n when u s e d a lo n e
( F i g u r e s 3 , 4 & T a b le V I I I ) ,
so I d e c i d e d t o t e s t t h e p r o t e c ­
t i v e seru m f o r a c t i v i t y by i t s e l f .
T h is s h o u l d h a v e b e e n a
c o n tr o l i n e a r l i e r e x p e rim e n ts ( F ig u re s 3 ,4 , & T ab les
V II,
I X ) , b u t b e c a u s e o f t h e l i m i t e d amount o f a c t i v e serum
a v a i l a b l e , serum was g i v e n a l o n e o n l y t o s m a l l g r o u p s a f t e r
i t was known t o w ork w i t h c e l l s .
I n one o f t h e f i r s t e x p e r i m e n t s , n o rm a l l i t t e r m a t e s
(NLM) w ere u s e d an d serum g i v e n w i t h IMLNC from a day l 6 i n ­
f e c t i o n was no more e f f e c t i v e t h a n serum g i v e n a l o n e ( T a b le
T a b le XIX.
The e f f e c t o f mouse p r o t e c t i v e seru m on a 300 IL^ i n f e c t i o n
when g i v e n a l o n e o r w i t h IMLNC.
^
Group I
C o n tro l
Group 2
IMLNCa + .
Im l Serum 0
1 2 ,0 0 0
0
100
100
EPG Day ?
8,000
0
0
200
EPG Day 8
800
100
0
600
EPG Day 6 C
Worms R e c o v e re d
On Day 8
Group 3 b
Im l Serum
Group
3
b
■5ml Iserum
r\
Worms
141
96
91
72
9
I
EP$
1 3 .3
9.2
4.9
8.6
Worms
113
?4
27
0
0
EPS
2 .1
0
-
Worms
118
20
. 3
0
0
EPS
1 2 .7
— . -
Worms
74
36
EPS
1 .6
3 .7
-
a I x 10 O IMLNC fro m s y n g e n e i c d o n o r m ice g i v e n 300 ILo I 6 days b e f o r e
c e l l s w ere t r a n s f e r r e d .
^
b Serum fro m a known p r o t e c t i v e p o o l g i v e n . IV on t h e same day as a 300 ILo
in fe c tio n .
'
^
c Post in fe c tio n .
d EP$ = E g g s r e c o v e r e d p e r f e m a le worm. Eggs i n 2 0 -5 0 f e m a le worms w ere
c o u n t e d a.nd t h e mean t a k e n .
o
71
XIX).
The EPG f e c e s c o u n t s seem ed low f o r t h e num ber o f
worms r e c o v e r e d a t n e c r o p s y b u t t h e EP9 was v e r y low i n t h e
s e r u m - t r e a t e d m ic e , s u g g e s t i n g t h a t t h e worms w ere damaged
e a r l i e r t h a n n o r m a l.
A s e c o n d e x p e r i m e n t was done u s i n g 1 0 - w e e k - o ld BALB/c■
fe m a le s.
T h ree g r o u p s w ere ran d o m ly c h o s e n , t h e f i r s t o f
w h ic h c o n s i s t e d o f m ice w h ic h g o t o n l y a 300 IL^ i n f e c t i o n
on day 0.
The s e c o n d g ro u p c o n s i s t i n g o f 3 m ic e , was g i v e n
I ml o f a known p r o t e c t i v e p o o l o f serum 6 h o u r s b e f o r e t h e
larv a e.
The t h i r d g ro u p c o n s i s t i n g o f 4 m ice was g i v e n 0 . 5
■ml o f t h e same p o o l a l s o 6 h o u r s b e f o r e c h a l l e n g e .
The r e s u l t s o f t h i s e x p e r i m e n t ( T a b le XX) show a m arked
r e d u c t i o n i n t h e EPG c o u n ts o f m ice g i v e n t h e immune serum ,
b u t because o f th e g r e a t v a r i a b i l i t y ,
t h e r e i s no s t a t i s ­
t i c a l d i f f e r e n c e i n t h e worm r e c o v e r y d a t a .
The EP$ a r e
s t a t i s t i c a l l y l o w e r ( P < . 05) i n b o t h o f t h e immune se ru m tr e a te d g ro u p s.
The EPG c o u n ts a r e i n c r e a s i n g i n e a c h g ro u p
o f a n im a l s g i v e n immune serum , s u g g e s t i n g t h a t t h e serum
f a c t o r i s b e in g d e p le te d , p o s s ib ly because o f a s h o r t h a l f life ,
and t h e worms a r e r e c o v e r i n g .
The e x p e r i m e n t s u g g e s t e d by t h e above r e s u l t s was t o
g i v e immune serum on b o t h day 0 a n d day 3 t o s e e i f a h i g h
enough t i t e r o f serum f a c t o r c o u ld be m a i n t a i n e d t o c a u s e
T a b le XX.
The e f f e c t on EPG an d worm- r e c o v e r y o f mouse p r o t e c t i v e serum
a lo n e .
Group I
Group 2
Group 3
C o n t r o l _____________ Im l Serum__________ .5m l Seruma
EPG Day 6 P I b
2 1 ,1 0 0
300
700
EPG Day 7 P I
2 4 ,8 5 0
. 3,400
2,000
Worms R e c o v e re d
On Day 7 '
A v erag e
- Worms
220
208
182
179
162
142
48
163+
57
EP $
14.2
13.2
1 2 .6
1 1 .3
1 0 .2
9.8
6.2
Worms
211
158
3
1 1 . 1±
2 .7
124±
108
EP$
I
4 .4
I . 7±
2.1
5 Known p r o t e c t i v e p o o l o f serum g i v e n IV on day 0.
P o s t i n f e c t i o n - - 3 0 0 IL^ h a d b e e n g i v e n on day 0.
Worms r e c o v e r e d
I v s . 2 , 3 = NS
I v s . 3 = P < .0 5
I
Worms
204
157
137
2
EP 9
8
6
6.3
125+
86.7
5±3.5
-
73
e x p u lsio n .
T h r e e . d i f f e r e n t p r o t e c t i v e serum p o o l s w ere u s e d
in t h i s e x p erim e n t.
One ml o f e a c h p o o l w a s .g i v e n t o e a c h
mouse i n g r o u p s o f 3 o r 4 m ice on t h e day o f l a r v a l i n f e c - ■
t i o n and o t h e r g r o u p s o f 3 o r 4 m ice w ere g i v e n 0 . 5 ml from
e a c h p o o l on b o t h d ays 0 and 3•
EPG w ere d e t e r m i n e d on days
6 and 7 and a l l m ic e w ere k i l l e d on day 7 .
c o v e r e d w ere c o u n t e d .
The worms r e ­
A l l g r o u p s g i v e n t h e .s a m e t r e a t m e n t
w i t h seru m w ere p o o l e d b e c a u s e r e s u l t s o f a l l p o o l s Were
s t a t i s t i c a l l y a lik e
( T a p le XXI).
The worm c o u n t s from t h e
c o n t r o l i n f e c t i o n h a d a h i g h s t a n d a r d d e v i a t i o n , b u t were
s i g n i f i c a n t l y ( P < . 0 5 ) d i f f e r e n t from t h e g ro u p t h a t r e c e i v e d
serum on b o t h d ays 0 and 3•
T h is e x p e r i m e n t showed, t h a t I
ml o f p r o t e c t i v e serum g i v e n on day 0 o n l y w a s n ' t any more
a c t i v e t h a n I mi s p l i t b e tw e e n day 0 and day 3•
I w a n te d t o know a t w hat s t a g e i n t h e l i f e
worm t h e serum was e f f e c t i v e .
c y c le o f th e
S a rles & T a lia fe rro
( 5 2 ) .sug­
g e s t e d t h a t serum may c a u s e d e l a y e d m i g r a t i o n o r immune
p r e c i p it a te - m e d i a t e d k i l l i n g in h o s t t i s s u e , e s p e c i a l l y lu n g .
L a t e r work h a s n ' t s u p p o r t e d t h i s i d e a and s u g g e s t s t h a t
worms a r e e x p e l l e d from immune r a t s o n l y a f t e r t h e y r e a c h
th e g u t (4 2 ).
To s o l v e t h i s p r o b le m ,
I gave 0 . 5 ml o f a
p r o t e c t i v e p o o l o f serum 6 h o u r s b e f o r e t h e i n f e c t i o n t o t h e
m ice i n one g ro u p and i n a s e c o n d g r o u p , e a c h mouse g o t 0 .5
74
-
T a b le XXI.
The e f f e c t o f one o r two i n j e c t i o n s o f mouse
p r o t e c t i v e serum on a l a r v a l i n f e c t i o n .
• Group I
C o n tro l
Group 2
Immune \
Seruma ' D
Day 0
, Group 3
Immune
Seruma ' 1*
Days
0 and 3
EPG Day 6
19,500
200
0
EPG Day 7
8,800
600
0
186
127
9479
22
O
158
71
31
30
5
5
2
0
0
0
20
42
I
I
I
0
0
0
0
Worms R e c o v e re d
On Day 7
A v erag e
85±68
30+50
2 .9 ± 6
^ 300 ILo g i v e n t o a l l m ice on day 0.
I ml o f a known mouse p r o t e c t i v e p o o l o f serum g i v e n IV on
day 0.
0 . 5 ml o f serum fro m b g i v e n IV on day O and 0 . 5 ml more
g i v e n IV on day 3■
.
Worms r e c o v e r e d
I v s . 2 = NS
' I v s . 3 = P < . 05
75
T a b le XXII.
E f f e c t o f mouse p r o t e c t i v e serum g i v e n on day
0 o r day 3 o f a l a r v a l i n f e c t i o n .
C o n tro l
Day Oa
Day 3b
EPG Day 6C
13,200
0
0
EPG Day 7
13,900
0
0
50
22
8
2
2
I
I
I
0
0
0
Worms R e c o v e re d
On Day 7
A verage
242
l 6l
126
112
79
16
6
• 106+82
1 4 .2 + 1 9 .2 5
.4 + .5 5
a 0 . 5 ml o f mouse p r o t e c t i v e serum g i v e n t o e a c h mouse on
, day 0 .
.
,
0 . 5 ml serum (fro m p o o l u s e d above ) g i v e n t o e a c h mouse
on day 3 Days a f t e r a 300 IL^ c h a l l e n g e on day 0.
Worms R e c o v e re d
I v s . 2 = P < . 05
I y s . 3 = P c . 05
76
ml on day 3 o f t h e i n f e c t i o n ( T a b le X X I I ) . .
P r o t e c t i v e se ru m g i v e n IV on day 3 a p p e a r s t o be more
e f f e c t i v e i n c a u s i n g e x p u l s i o n t h a n when g i v e n on day 0 .
T h is w ould s u g g e s t t h a t t h e e l i m i n a t i o n o f a l a r v a l i n f e c ­
t i o n m ig h t be more e f f i c i e n t a f t e r t h e worms r e a c h t h e g u t
th a n w h ile th e y a re i n th e s k in o r lu n g s .
The p r o t e c t i v e f a c t o r h a s b e e n r e p o r t e d t o be h e a t
la b ile
(38).
To t e s t t h i s i n my s y s te m , t h e serum was
h e a t e d 56 C f o r 30 m i n u t e s t h e n g i v e n t o m ic e — 0 . 5 ml on day
3.
I b e l i e v e d t h a t I h a d e v i d e n c e t h a t t h e f a c t o r was
fre e z e -th a w l a b i l e ,
so one g ro u p was g i v e n on day 3 0 . 5 . ml
o f serum w h ic h h a d b e e n f r o z e n and th a w e d 5 t i m e s (T a b le
X X III).
More worms w ere f o u n d i n t h e m ice r e c e i v i n g t h e
h e a t e d se ru m , b u t t h e r e was s t i l l a l o t o f a c t i v i t y i n t h e
serum , s u g g e s t i n g t h a t e i t h e r 56 C f o r 30 m in u t e s was i n a d e ­
q u a te to i n a c t i v a t e a l l o f th e p r o t e c t i v e f a c t o r o r th e p r o ­
t e c t i v e f a c t o r i s n ' t a s h e a t l a b i l e a s was t h o u g h t .
The l e a k l e s i o n t h e o r y o f a n t i b o d y damage p r o p o s e s t h a t
t h e dam aging s t e p i s t o g e t I g E - m e d i a t e d a n a p h y l a x i s o f t h e
g u t, th u s a llo w in g p r o t e c t i v e a n tib o d ie s o f o t h e r c la s s e s to
g e t i n t o t h e g u t a n d c a u s e e x p u l s i o n o f t h e worms.
I .de­
s i g n e d an e x p e r i m e n t t o t e s t t h i s i d e a by i n j e c t i n g t h e p r o ­
t e c t i v e serum i n t o t h e g u t on day 4 when a l l o f t h e worms
77
T a b le X X III.
E f f e c t o f h e a t o r f r e e z e - t h a w on t h e a c t i v i t y
o f mouse p r o t e c t i v e se ru m .
Group I
C o n tro l
EPG Day 6d
EPG Day 7
■
Worms R e c o v e re d
On Day 7
A verage
Group 2 ■ Group 3
Immune
Immune
Serum h
Serum
H eated u
Group 4
Immune
Serum ■
F ro z e n
Sc Thawedc
13,200
0
1 ,400
.0
1 3 ,9 0 0
0
0.
0
100
83
' 11
5
0
I
0
0
0
0
.0
242
161
126
112
79
16
- 6
106+82
.0 '
0
0
I
I
.4 + . 5 5
3 9 . 8+4 7 . 7
.
.1 7 + .4 1
a Each mouse r e c e i v e d 0 . 5 ml IV o f a known mouse p r o t e c t i v e .
, p o o l o f serum on day 3 .
Serum from t h e p o o l u s e d i n Group 2 was h e a t e d 30 m in u te s
a t 56 C; e a c h mouse r e c e i v e d 0 . 5 ml o f t h e h e a t e d serum
IV on day 3»
Serum from t h e p o o l u s e d i n Group 2 was f r e e z e - t h a w e d 5'
t i m e s ; e a c h mouse r e c e i v e d 0 . 5 ml o f t h e f r e e z e - t h a w e d
j, serum IV on day 3«
A l l m ice r e c e i v e d 300 IL^ on day 0.
78
w ould be i n t h e g u t , b u t n o t a l l m o u lt e d t o t h e a d u l t s t a g e
y e t,
One h a l f ml o f t h e p r o t e c t i v e serum was i n j e c t e d i n t o
t h e duodenum v i a l a p o r o t o m y .
A n o t h e r g ro u p was g i v e n O.5
ml o f n o r m a l mouse serum i n t r a d u o d e n a l l y ( T a b le XXIV).
The
serum was p r o t e c t i v e when g i v e n IV b u t n o t when g i v e n i n t r a ­
duo d e n a l l y .
E i t h e r t h e serum w a s n ' t i n c o n t a c t w i t h t h e
worms f o r a l o n g en o u g h tim e o r serum i s n ’ t a c t i v e a g a i n s t
t h e worms i n t h e g u t lum en.
I t w ould be d i f f i c u l t t o i n -
.
j e c t serum i n t o t h e duodenum more t h a n a few t i m e s b e c a u s e
o f s u r g i c a l tr a u m a .
C y c lo p h o sp h am id e h a s b e e n r e p o r t e d t o d e l a y e x p u l s i o n
o f a worm i n f e c t i o n f o r a b o u t 6 d ays ( 3 4 ) .
In t h a t stu d y ,
c y c lo p h o s p h a m id e g i v e n on day -2 r e l a t i v e t o i n f e c t i o n d i d
n o t have an e f f e c t ,
p u lsio n .
b u t i f g i v e n on day +2 i t d e l a y e d e x ­
B ecau se I h a d t h e serum s y s te m w o r k in g ,
I b e lie v e d
I c o u ld d e t e r m i n e w h e t h e r t h i s c y c l o p h o s p h a m i d e - i n d u c e d
A
d e l a y was i n t h e h u m o ra l dam aging s t e p o r a l a t e r e x p u l s i o n
s t e p . ' I gave t h e m ic e 2 mg c y c lo p h o s p h a m id e IV, w h ic h i s
a b o u t h a l f t h e d o se g i v e n s u b c u t a n e o u s l y by M i t c h e l l ’s
g ro u p ( 3 4 ) .
The m ic e w ere d i v i d e d i n t o 6 g r o u p s and t h e
m ice i n e a c h g ro u p w ere g i v e n :
Group I - 300 IL^ on day 0, a s w ere a l l o t h e r g r o u p s .
Group 2 - Immune serum on day 0.
79
T a b le XXIV.
The e f f e c t on a l a r v a l i n f e c t i o n o f m ice o f
p r o t e c t i v e serum i n j e c t e d i n t o t h e duodenum.
Group I
C o n tro l
No. o f Mice
6
Group 2
Immune
Serum IV*3
6
■Group. 3
Immune
Serum ID c
Group 4
' NMSd
ID
6
6
EPG Day 6a
1 4 , ooo
0
21,200
9 ,4 0 0
EPG Day 7
1 4 ,8 0 0
0
85,600
9,400
EPG Day 8
1 ,0 0 0
6 ,8 0 0
4 ,8 0 0
0
7,600
2,900
EPG Day 8 .5
f' A l l m ice w ere i n f e c t e d w i t h 300 I I g on day 0.
Mice w ere g i v e n o . 5 ml IV o f a known p r o t e c t i v e p o o l o f
serum on day 0.
c On day 4 p o s t i n f e c t i o n , m ice w e r e . i n j e c t e d i n t r a d u o d e n a l , I y w i t h 0 . 5 ml o f t h e p o o l u s e d IV i n Group 2 .
On day 4 p o s t i n f e c t i o n , m ice w ere i n j e c t e d i n t r a d u o d e n a l I y w i t h 0 , 5 ml o f NMS.
80
Group 3 - Immune serum on day 3•
Group 4 - C y c lo p h o sp h am id e on day 2.
Group 5 - Immune serum day 0, c y c lo p h o s p h a m id e day 2.
Group 6 - C y c lo p h o sp h am id e day 2, immune serum day 3.
The r e s u l t s ' ( T a b l e XXV) o f t h i s e x p e r i m e n t w ere a s u r ­
p r i s e an d more e x p e r i m e n t s n e e d t o be done b e f o r e t h e y c an
be i n t e r p r e t e d .
The EPG, c o u n t s w ere d e p r e s s e d on day 6 i n
b o t h g r o u p s r e c e i v i n g serum and c y c lo p h o s p h a m id e ( Groups 5>
6) b u t h a d r e a c h e d n o r m a l l e v e l s by day 7, a f t e r w h ich t h e
worms w ere a p p a r e n t l y e x p e l l e d n o r m a l l y .
One p o s s i b l e e x ­
p l a n a t i o n o f t h e s e r e s u l t s i s t h a t a t h r e s h h o l d d o se o f b o t h
immune serum and c y c lo p h o s p h a m id e was u s e d .
T h is i s shown
i n t h e g r o u p w h ich r e c e i v e d c y c lo p h o s p h a m id e o n l y , a s worm
e x p u l s i o n was n e a r n o r m a l .
A l s o , i f t h e serum d o se was t h e
minimum n e e d e d , t h e c y c lo p h o s p h a m id e may have d e c r e a s e d t h e
p r o d u c t i o n o f t h e f a c t o r i n t h e m ice w h ic h was n e c e s s a r y t o
s u p p le m e n t t h e immune serum g i v e n t o g e t t h e f u l l b e n e f i t o f
t h e se ru m .
T r a n s f e r r i n g P a s s i v e Im m unity I n t o Nudes
U s in g IMLNC and P r o t e c t i v e Serum
Two m eth o d s o f t r a n s f e r r i n g t h e worm, dam aging s t e p t o
n a i v e n o r m a l m ice h a v e b e e n e x a m in e d ; p a s s i v e t r a n s f e r o f
p r o t e c t i o n w i t h immune serum ( T a b le X X II) and a d o p t i v e
.
T a b le XXV.
E f f e c t o f c y c lo p h o s p h a m id e o r c y c lo p h o s p h a m id e and p r o t e c t i v e
serum on a l a r v a l i n f e c t i o n o f m ic e .
Group I
C o n tro l
Group 2
Serum D
Day 0
Group 3
Serum b
Day 3 • '
EPG Day 6a
13,200
0
0
EPG Day 7
13,900
0
EPG Day 8
-
EPG Day 8 . 5
-
Group 5
C y c lo p h o s ­
p h a m id e 0
Serum 0
Day 0
Group 6
C y c lo p h o s ­
p h a m id e 0
Serum^
Day 3
2 7 ,4 0 0
1,100
7,700
0
39,500
2 4 ,0 0 0
20,000
-
-
1 0 ,4 0 0
2 ,4 0 0
500
-
-
5,800
400
200
Worms
R e c o v e re d
A v e rag e
Group 4
C y c lo p h o s p h a m id e 0
107
18
0
0
0
106±82^
l4 .2 d
Range
1 -50
.4 d
Range
0 -1
, A l l m ic e g i v e n 300 ILo on day 0.
■.Mice g i v e n 0 . 5 ml o f one p o o l o f
serum IV on days i n d i c a t e d .
25+46.5®
I
I
I
0
. 0
■ 0
.5±.55®
10
4
2
I
0
0
2 . 8±3. 8e
Mice g i v e n 2 mg c y c lo p h o sp h a m id e IV
^ on day 2.
Worms r e c o v e r e d on day 7 .
Worms r e c o v e r e d o n "d ay 8 . 5
82
t r a n s f e r o f im m unity w i t h IMLNC ( T a b le s XI, X I I ) .
N ext, t h e
r e q u i r e m e n t f o r t r a n s f e r o f some am ount o f p a s s i v e im m unity
t o nude m ic e was s t u d i e d .
When g i v e n thymus g r a f t s o r when
g i v e n th y m o c y te s 21 days b e f o r e c h a l l e n g e , n u d e s e x p e l t h e i r
worms i n a n e a r n o r m a l p a t t e r n ( 1 6 , 3 4 ) .
However, i t t a k e s
a p p ro x im a te ly I 4 days to g e t e x p u ls io n a f t e r g i v in g e i t h e r
n o rm a l s p l e e n c e l l s o r th y m o c y te s t o t h e nude a t t h e same
tim e t h e y a r e i n f e c t e d .
No one h a s r e p o r t e d g i v i n g n udes
p r o t e c t i v e serum o r c e l l s t h a t a r e p r o t e c t i v e by t h e m s e l v e s .
An e x p e r i m e n t was d e s i g n e d t o t e s t t h e a b i l i t y o f known
p r o t e c t i v e serum an d immune c e l l s t o c o l l a b o r a t e i n a nude
t o c a u s e e x p u l s i o n b e f o r e day I 4 when n u d e s g i v e n n o rm a l
s p l e n o c y t e s and th y m o c y te s w ould e x p e l a worm b u r d e n .
The .
groups u sed i n t h i s e x p e rim e n t a r e :
Group I - L M c o n t r o l , 300 ILo on day O a s a l l o t h e r
g roups.
^
I. Group 2 - LM g i v e n I x IO^ IMLNC IV from d o n o r s i n f e c ­
t e d I 6 days b e f o r e t r a n s f e r .
Group 3 - LM g i v e n I ml IV o f a known p r o t e c t i v e p o o l
o f serum on day 0.
Group 4 - Nudes g i v e n l a r v a l i n f e c t i o n o n l y .
Group 5 - Nudes g i v e n immune serum as Group 3■
Group 6 - Nudes g i v e n IMLNC a s Group 2 an d immune
se ru m as Group 3 .
Nudes g i v e n mouse p r o t e c t i v e serum a l o n e ( T a b le XXVI,
T a b le XXVI.
EPG
The e f f e c t o f p r o t e c t i v e seru m o r p r o t e c t i v e serum and IMLNC
on a l a r v a l i n f e c t i o n o f nude m ic e .
Group I
C o n tro l
NLM
Group 2
NLM ,
IMLNC
Group 3
NLM
Immunec
Serum
Group 4
Nude
Group 5
Nude
Immunec
Serum
Group 6
Nude ,
IMLNCd
I mmunec
Serum
Day
6a
1 2 ,0 0 0
13,000
0
18,300
51,800
1 ,0 0 0
Day
7
8,000
9,4oo
0
32,800
69,700
34,600
Day
8
800
1,500
0
74,500
160,200
91,400
Day 12
38,900
89,200
33,900
Day. 15
95,400
1 0 1 ,8 0 0
11,800
Day 17 '
69,500
1,700
Day 19
55,200
6 ,8 0 0
Day 21
1 1 1 ,4 0 0
Day 23
67,300
3 m ic e ,
300
2 m ic e , 5 8 ,8 0 0
, A l l m ic e g i v e n 300 I L 3 on day 0 . .
Mice g i v e n I x 10° IMLNC fro m d o n o r m ice i n f e c t e d I 6 days b e f o r e c e l l
t r a n s f e r on day 0 .
c Mice g i v e n I ml o f a known p r o t e c t i v e p o o l o f se ru m IV on day I
84
Group 5) d i d n ' t e x p e l t h e i r worms and d i e d e a r l i e r t h a n
nudps n o t g i v e n mouse p r o t e c t i v e serum (Group 4 ) .
Nudes
g i v e n mouse p r o t e c t i v e serum and day I 6 IMLNC ( Group 6)
showed a d e l a y i n e g g p r o d u c t i o n by one day.
EPG c o u n ts
seem ed t o d ro p b e tw e e n days 15 and 17, b u t i t i s d i f f i c u l t
t o t e l l b e c a u s e 3 o f t h e n u d e s w ere a p p a r e n t l y r e c o n s t i t u t e d
and e x p e l l e d t h e i r worms, w h i l e 2 o t h e r s w e r e n ’ t r e c o n s t i ­
t u t e d a n d d i e d w i t h h i g h EPG c o u n t s .
T h is shows t h a t e a c h
nude mouse n e e d s t o have EPG c o u n te d s e p a r a t e l y so t h e d a t a
from t h o s e t h a t w ere r e c o n s t i t u t e d c o u ld be s e p a r a t e d from
t h o s e w h ic h w e r e n ' t .
The l a c k o f a p o o l o f u n d i f f e r e n t i a t e d T c e l l s was f e l t
t o be a p o s s i b l e e x p l a n a t i o n f o r t h e f a i l u r e o f p r o t e c t i v e
serum t o have an e f f e c t on an i n f e c t i o n i n n u d e s , so I
s e l e c t e d 3 g ro u p s o f 2 n u d e s a n d on day 9 o f a
b ra silie n -
s i s i n f e c t i o n I g a v e them d i f f e r e n t c o m b i n a t i o n s o f c e l l s
and mouse p r o t e c t i v e serum a s f o l l o w s :
8
Group I - I x 10 th y m o c y te s I P , 0 . 5 ml immune serum
IP .
Group 2 - I x 10® th y m o c y te s I P , 0 . 5 ml p r o t e c t i v e
seru m and I x 10 IMLNC from d o n o r LM m ice
g i v e n a p r i m a r y i n f e c t i o n 9 days b e f o r e .
Group 3 - I x 10® t h y m o c y t e s , IMLNC a s G ro u p 2 .
EPG w ere m o n i t o r e d ( T a b le XXVII).
■
85
T a b l e XXVII.
The e f f e c t o f v a r i o u s c o m b i n a t i o n s o f p r o t e c ­
t i v e se ru m , IMLNC and t h y m o c y t e s on a l a r v a l
i n f e c t i o n o f nude m i c e .
Days A f t e r 8C ell T ra n sfe r
Group I
, ■
T h y m o c y te s 0
Immune Serumc
Group 2
,
Thymocytes
IMLNCd
Immune Serumc
(No. o f M ice)
(2 )
(2 )
Group 3
b
Thymocytes
IMLNCd
(2 )
-2
28,900
75,100
82,000
I
72,100
71,000
1 2 7 ,0 0 0
3
33,300
45,400
54,300
5
53,200
71,600
1 4 3 ,0 0 0
7
57,800
5 8 ,4 o o
71,500
9
44,600
35,100
46,150
11
59,500
31,400
32,000
13
3 l,8 o o
20,800
3 ,ioo
14
- I died
41,000
88,900
14 ,3 5 0
16
47,000
63,600
7,900
19
31,400
42,300
2,100
3.
, A l l m ic e were g i v e n 300 ILg 9 days b e f o r e c e l l t r a n s f e r .
A l l m ic e were g i v e n I x 10° t h y m o c y t e s IP fr o m 3 - w e e k - o l d
littem ate s.
c Mice g i v e n 0 . 5 ml o f a known p r o t e c t i v e p o o l o f serum on
d t h e day o f c e l l t r a n s f e r , I P .
Mice g i v e n I x 10° IMLNC fro m d o n o r l i t t e m a t e s g i v e n a
p r i m a r y i n f e c t i o n o f 300 IL^ 9 days b e f o r e c e l l t r a n s f e r .
86
T h i s i s a v e r y p r e l i m i n a r y e x p e r i m e n t , b u t none o f t h e
n u d e ' s c o u n t s were d e p r e s s e d u n t i l a b o u t day I 4 a f t e r t h e
c e l l s w ere g i v e n .
stitu te
The t h y m o c y t e s a p p a r e n t l y d i d n ' t r e c o n ­
t h e n u d e s a s t h e g r o u p t h a t d i d n ' t g e t IMLNC w a s t e d ,
one d y i n g on day 12 a f t e r t r a n s f e r .
The a c t i v i t y o f t h e s e
day 9 IMLNC i n a l i t t e r m a t e i n f e c t i o n i s unknown b e c a u s e t h e
l a r v a e u s e d i n t h i s e x p e r i m e n t d i d n ' t c a u s e an i n f e c t i o n i n
l i t t e r m a t e s , b u t d i d g i v e a u n i f o r m EPG d i s t r i b u t i o n i n
nudes.
L o o k i n g a t Groups 2 and 3 i n t h i s e x p e r i m e n t ,
it
a p p e a r s a s t h o u g h g i v i n g serum on t h e day IMLNC' s were g i v e n
c a u s e d t h e worms t o be r e t a i n e d l o n g e r t h a n i n t h e nu d es
t h a t r e c e i v e d IMLNC, b u t no se ru m .
The serum c o u l d have
c a u s e d the. worms t o become a d a p t e d o r i t may h a v e a c t e d a s
a b l o c k i n g serum, n o t a ll o w in g th e h o s t c e l l s to r e a c t to
the a n tig e n s .
At t h i s p o i n t ,
immunize a nude a r e unknown.
the req u irem en ts to p a s s iv e ly
■ D IS C U S S IO N
The m o st p o p u l a r t h e o r y f o r N . . b r a s i l i e n s i s e l i m i n a t i o n
i s t h e two s t e p t h e o r y p u t f o r t h by O g i l v i e an d c o -w o r k e r s '
(4 2 ,4 3 ,4 4 ).
In the f i r s t s te p ,
" a n t i b o d i e s " damage t h e
worms t h e r e b y m ak in g them i n some unknown way s u s c e p t i b l e
to the a c tio n o f a c e ll-m e d ia te d r e a c t i o n (second s te p )
w h ic h r e s u l t s i n t h e i r e l i m i n a t i o n from t h e h o s t by an u n ­
known m ech an ism .
Some o b s e r v a t i o n s w h ic h s u p p o r t t h i s p r o ­
p o s e d mechanism a r e :
worms r e c o v e r e d . i n t h e l a t e r s t a g e s o f
a normal i n f e c t i o n in r a t s
( d a y s 1 0 - 1 4 ) ( 4 2 ) o r m ic e ( day 8)
( 2 9 ) when i n o c u l a t e d i n t o n o r m a l r a t s show an a c c e l e r a t e d
e l i m i n a t i o n p a t t e r n (<day 4 v s . > day 8) and a r e c a l l e d
"damaged worms"; n e o n a t a l and l a c t a t i n g r a t s d o n ' t e x p e l
t h e i r worms e v e n t h o u g h t h e worms a p p e a r damaged n e a r l y a s
s o o n a s i n a n o r m a l i n f e c t i o n ( t h e damage h a s b e e n a s s e s s e d ,
c y t o l o g i c a l l y and b i o c h e m i c a l l y an d by e a r l i e r e x p u l s i o n
a fte r tran sfer);
e x p u l s i o n c a n be f a c i l i t a t e d i n n e o n a t a l
a nd l a c t a t i n g a n i m a l s by g i v i n g them n o r m a l l y m p h o c y t e s
( 2 2 ) ; se rum from immunoi n c o m p e t e n t n e o n a t e s c a n a c c e l e r a t e
e x p u ls io n i n normal anim als g iv e n a l a r v a l i n f e c t i o n .
these o b se rv atio n s,
From
i t has been deduced t h a t a n t i b o d i e s
f o u n d i n t h e serum o f l a c t a t i n g a n d n e o n a t a l r a t s c a u s e
damage t o t h e worm, b u t l y m p h o c y t e s from an immunocompetent
a d u l t a n i m a l a r e n e e d e d t o c a u s e t h e e x p u l s i o n o f t h e worm
88
f ro m im m u n e in c o m p e t e n t a n i m a l s .'
A ttem pts to t r a n s f e r to n a iv e anim als th e l e v e l o f
im m u n ity t o l a r v a l
b rasilien sis
in f e c ti o n t h a t is seen
i n p r e v i o u s l y i n f e c t e d a n i m a l s h a s v e r y r a r e l y b e e n accom­
p l i s h e d e i t h e r p a s s i v e l y w i t h immune se rum o r a d o p t i v e l y
w i t h immune s p l e e n o r IMLNC.
Love ( 2 8 , 2 9 ) f o u n d t h a t t o g e t
m ic e o r r a t s t o e x p e l t h e i r worms a s p r e v i o u s l y i n f e c t e d
a n i m a l s w o u ld , b o t h hyperimmune se ru m a nd immune c e l l s were
required.
T h ere a r e o n l y two r e p o r t s i n t h e l i t e r a t u r e o f
IMLNC p r e p a r a t i o n s c a u s i n g e x p u l s i o n o f a l a r v a l
b rasil­
i e n s i s i n f e c t i o n b e f o r e t h e i n f e c t i o n became p a t e n t .
Both
o f t h e s e w ere t h e e x c e p t i o n a l c a s e i n a s e r i e s o f e x p e r i ­
ments ( 2 3 , 2 9 ) .
Two o t h e r g u t - d w e l l i n g n e m a t o d e - r o d e n t
s y s t e m s ( T r i c h u r i s m u ri s -mouse ( 6 0 ) and T r i c h i n e l l a
s p i r a l i s -mouse ( 6 2 ) ) t h a t h ave b e e n e x t e n s i v e l y s t u d i e d
h a v e L een r e p o r t e d t o r e q u i r e b o t h immune c e l l s a n d immune
se ru m t o t r a n s f e r t o n a i v e h o s t s a h i g h d e g r e e o f p r o t e c t i o n
to challenge w ith in f e c tiv e l a r v a e .
T h ere h a s b e e n l i t t l e
work on t h e d a m agin g f a c t o r ,
e i t h e r i t s r e a l i d e n t i t y o r i t s o r i g i n . . The damagin g f a c t o r
f o u n d . i n serum h a s b e e n assum ed by m o st w o r k e r s i n t h i s
f i e l d t o be w o r m - s p e c i f i c a n t i b o d y .
t h o u g h t t o be a n t i b o d y , l i t t l e
B e ca u se t h e f a c t o r i s
work h a s b e e n done t o i d e n ­
89
t i f y the c e l l producing i t .
se rum on t h e worm i n v i t r o
- The l a c k o f e f f e c t o f immune
( l 4 , 5 1 ) h a s l e f t t h e mechanism o f
a c t i o n o f t h e " a n t i b o d y " unknown.
The r e c e n t work o f
■
J a c o b s o n , e t a l ( 1 7 ) s u g g e s t i n g t h a t t h e f a c t o r i n mice
i s n ' t a n t i b o d y h a s n o t b e e n f o l l o w e d up i n t h e l i t e r a t u r e
y et.
In experim ents r e p o r te d h e r e , 5 pools o f c e l l s given
0
a t a do se o f I x 10 c e l l s p e r .mouse on day 0 o f a n i n f e c ­
t i o n ( T a b l e s X, XI, X I I ) c a u s e d t o t a l a b l a t i o n o f EPG f e c e s
a nd v e r y lo w worm c o u n t s on d a y s 8 a nd 10.
A ll of these
c e l l p o o l s were p r e p a r e d from m ic e t h a t h ad b e e n i n f e c t e d
f o r 8 - 1 2 day s a f t e r a p r i m a r y i n f e c t i o n .
S e v e ra l pools of
c e l l s c o l l e c t e d fr o m m ic e 4, 6 a n d I 4 day s ( T a b l e s X, XI,
X I I ) a f t e r a p r i m a r y i n f e c t i o n o r 9 - 1 4 days ( F i g u r e s 4 , 5
an d T a b l e s V I I ,
IX ) a f t e r a seco n d i n f e c t i o n d i d n ' t cause
an y d i f f e r e n c e i n t h e i n f e c t i o n a s m o n i t o r e d by e g g c o u n t s
when t r a n s f e r r e d t o n a i v e r e c i p i e n t s .
The k i n e t i c s t u d y o f
c e l l s t a k e n a f t e r a s e c o n d i n f e c t i o n showed some d e c r e a s e i n
EPG a n d d e p r e s s e d worm c o u n t s on day 8 ( T a b l e X I I I ) , b u t d i d
n o t show t h e l e v e l o f immunity s e e n i n o t h e r c e l l p r e p a r a ­
t i o n s ( T a b l e s X, X I r X I I ) .
B e c a u s e o f t h e e f f e c t on t h e c h a l l e n g e i n f e c t i o n i n t h e
r e c i p i e n t m i c e , I b e l i e v e t h a t t h e c e l l s p r o d u c i n g t h e worm
damaging f a c t o r m u s t have b e e n t r a n s f e r r e d o r t h a t t h e c e l l s
90
t r a n s f e r r e d were c o m m i t t e d h e l p e r c e l l s w h ic h c a u s e d t h e
e f f e c t o r c e l l i n t h e r e c i p i e n t mouse t o p r o d u c e l a r g e q u a n ­
titie s
o f the f a c t o r .
I a l w a y s t r a n s f e r r e d a c o n s t a n t num­
b e r o f MLNC i n t h e e x p e r i m e n t s an d i t was a lw a y s t h e c e l l
p r e p a r a t i o n fro m d o n o r s 9 - 1 2 day s a f t e r a p r i m a r y i n f e c t i o n
t h a t caused a b l a t i o n of p atency su g g e s tin g a high percentage
o f f a c t o r - p r o d u c i n g o r h e l p e r c e l l s i n t h e c e l l s t h a t were
transferred.
The a c t i v i t y o f t h e c e l l p r e p a r a t i o n s c o r r e -
.
I a t e d w i t h t h e p e a k l y m p h a d e n o p a t h y ( T a b l e XIV) s e e n i n t h e
MLN w h ic h i m p l i e s t h a t t h e c e l l t y p e r e s p o n s i b l e f o r t h e i n ­
c r e a s e i n c e l l u l a r i t y o f t h e MLN i s t h e c e l l r e s p o n s i b l e f o r
a c c e l e r a t i o n o f worm damage i n t h e r e c i p i e n t a n i m a l .
Work p u b l i s h e d r e c e n t l y d e m o n s t r a t e d t h e same s h o r t
tim e o f h ig h a c t i v i t y a g a i n s t a l a r v a l i n f e c t i o n i n r e c i p ­
ients
( 3 4 ) o f t h o r a c i c d u c t l y m p h o c y t e s c o l l e c t e d fr o m r a t s
d u r i n g a p r i m a r y N^ b r a s i l i e n s i s i n f e c t i o n ( c e l l s from days
10 an d 15 p o s t i n f e c t i o n were a c t i v e , c e l l s from d a y s 5 and
20 were n b t ) .
The a c t i v e c e l l i n t h i s work was an I g
p o p u l a t i o n w h ic h c a u s e d t h e a u t h o r s t o p o s t u l a t e t h a t damage
and e x p u l s i o n a r e c a u s e d by t h e same c e l l p o p u l a t i o n and
t h a t t h e t o t a l number o f c e l l s i s m o st i m p o r t a n t ( 3 6 ) .
a l t e r n a t i v e p o s s i b i l i t y they o ffe re d is t h a t th e Ig
The
cells
t h a t th e y found a c t i v e in a c c e l e r a t i n g e x p u ls io n o f a l a r v a l
91
i n f e c t i o n was a h e l p e r c e l l t h a t c o l l a b o r a t e d w i t h t h e B
c e l l s o f th e r e c i p i e n t anim al to produce th e a c c e l e r a t e d
a n tib o d y resp o n se needed to g e t e a r l y e x p u ls io n .
They n e v e r
c o n s id e r e d the p o s s i b i l i t y o f a T c e l l p ro d u c t c a u s in g the
damage.
Two e x p e r i m e n t s ' r e p o r t e d h e r e s u p p o r t t h e f a c t t h a t
m ic e w i t h l i m i t e d a n t i b o d y f o r m i n g p o t e n t i a l , b o t h
i a t e d (350 o r 450 r a d s ) a n i m a l s n o t g i v e n any c e l l s
irrad ­
( T ab le
XV) and t h e 550 r a d ^ - i r r a d i a t e d m ic e g i v e n t h y m o c y t e s
( T a b l e s XVII, X V I I I ) , c a n s t i l l e x p e l t h e i r worm b u r d e n a t
ab o u t th e normal tim e .
These m i c e , t h o u g h h a v i n g a low
num ber o f s p e c i f i c a n t i b o d y f o r m i n g c e l l s
( a s shown by SRBC-
s p e c i f i c PFC s e e n ( T a b l e X V I II ) a n d p e r s o n a l c o m m u n i c a ti o n
w i t h B rad B r o o k s ) , e x p e l l e d t h e i r worms by day I 4 w h ich
shows t h a t t h e d a m a g in g f a c t o r was p r o d u c e d a t a l m o s t t h e
n o r m a l r a t e e v e n t h o u g h t h e B c e l l c o m p e te n ce o f t h e s e a n i ­
m a l s i s v e r y low.
One m a j o r p r o b l e m w i t h my k i n e t i c e x p e r i m e n t s ( T a b l e s
XI, X I I , X I I I ) was t h a t e a c h day c e l l d o n o r s w ere immunized
w i t h a d i f f e r e n t l a r v a l b a t c h w h i c h i n t r o d u c e d .a l o t o f
v a r i a n c e b e c a u s e e a c h o f t h e im m u n i z i n g i n f e c t i o n s b e h a v e d
a little
d ifferen tly .
I d e a l l y , m ice i n f e c t e d w i t h one ,
l a r v a l p r e p a r a t i o n s h o u l d be k i l l e d on d i f f e r e n t day s and
92
c e l l s t r a n s f e r r e d t o g r o u p s o f m ic e w i t h c o n t r o l s i n f e c t e d
t h e same t i m e a s e a c h g r o u p t h a t r e c e i v e d c e l l s .
The mes­
e n t e r i c lymphadenopathy c o r r e l a t e d v e ry w e ll w ith th e a b i l ­
i t y o f c e l l s t o a d o p t i v e l y t r a n s f e r im m u n ity t o Njl b r a s i l ien sis.
B o th t h e p e a k o f l y m p h a d e n o p a t h y a n d t h e a c t i v i t y
o f t h e c e l l s was j u s t a t o r j u s t a f t e r t h e t i m e t h a t t h e
worms a r e b e i n g damaged and e x p e l l e d from t h e d o n o r m ic e .
A c o n s t a n t num ber o f c e l l s
( I x 10
n u c l e a t e d c e l l s ) was .
t r a n s f e r r e d so t h e r e l a t i v e num bers o f t h e d a m a g in g f a c t o r p r o d u c i n g c e l l s m u s t h ave i n c r e a s e d d u r i n g t h e l y m p h a d e n o ­
p a t h y w h ic h wou ld s u g g e s t a l o c a l i n c r e a s e i n t h e c e l l .
c a p a b l e o f t r a n s f e r r i n g t h e a b i l i t y t o c a u s e damage t o a
l a r v a l i n f e c t i o n to a naiv e anim al.
The p r o l i f e r a t i o n o f
t h e s p e c i f i c c e l l s may have b e e n i n t h e MLN o r t h e y may have
p r o l i f e r a t e d e l s e w h e r e and t h e i r m i g r a t i o n p a t t e r n c a r r i e d
them t o t h e MLN.
B l a s t c e l l s f ro m t h e MLN and t h o r a c i c d u c t
lymph p r e f e r e n t i a l l y l o c a l i z e i n t h e s m a l l i n t e s t i n e o f r a t s
when i n j e c t e d i n t o a r e c i p i e n t a n i m a l .
In flam m a tio n such as
t h a t c a u s e d by a N_j_ b r a s i l i e n s i s i n f e c t i o n c a u s e s an even
l a r g e r p e rc e n ta g e o f th e s e b l a s t c e l l s to l o c a l i z e i n the
sm all i n t e s t i n e
( 3 0 ) w h ich w o u ld s u g g e s t t h a t t h e MLN may
be i n t h e n o r m a l t r a f f i c p a t t e r n o f b l a s t c e l l s s p e c i f i c f o r
a n tig e n s in the gut.
93
A f t e r I f o u n d t h a t some p r e p a r a t i o n s o f c e l l s c o u l d
a d o p t i v e l y t r a n s f e r a h i g h l e v e l o f p r o t e c t i v e im m u n ity t o
a l a r v a l c h a lle n g e i n n a iv e m ice,
I t r i e d g i v i n g seru m a l o n e
t o m ice b e f o r e a l a r v a l i n f e c t i o n ( T a b l e XIX).
The r e s u l t s
were u n e x p e c t e d b e c a u s e Love ( 2 9 ) h a d shown p r e v i o u s l y t h a t
t o g e t t h e same h i g h l e v e l o f p r o t e c t i o n t h a t I f o u n d , b o t h
immune c e l l s and immune se rum h a d t o be g i v e n .
I found a
l o t o f v a r i a b i l i t y i n t h e num ber o f worms r e c o v e r e d a f t e r
g i v i n g immune seru m a l o n e t o m i c e .
W ith good p p o l s o f mouse
p r o t e c t i v e se rum t h e eg g c o u n t s w ere a l w a y s v e r y d e p r e s s e d
on day 6, t h e n p o s s i b l y t h e worm e s c a p e d and p r o d u c e d a few
e g g s by da y 7 o r 8 an d t h e mouse d e v e l o p e d a n immune r e s - v
p o n s e a nd c a u s e d t h e worms t o be e x p e l l e d .
I believe th a t
t h i s may be a phenomenon o f t h e p a s s i v e se rum f a c t o r b e i n g
d e p l e t e d b e f o r e t h e mouse makes en o u g h o f a n immune r e s p o n s e
- - e i t h e r d a m agin g f a c t o r o r e x p e l l i n g m ec h an ism — t o p r e v e n t
eg g p r o d u c t i o n a n d / o r c a u s e e x p u l s i o n .
T h i s phenomenon o f
e g g p r o d u c t i o n b e i n g d e l a y e d I day was s e e n s e v e r a l t i m e s i n
lo w se rum d o s e s a nd t w i c e i n immunocompromised a n i m a l s —nude
a n d c y c l o p h o s p h a m i d e - t r e a t e d n o r m a l m ic e ( T a b l e s XXV, XXVI).
The d a t a s u g g e s t i n g t h a t n u d e s g i v e n b o t h immune c e l l s and
mouse p r o t e c t i v e s e ru m c a n d e l a y p a t e n c y by one day s u g g e s t s
t h a t s o m e t h i n g fro m t h e c e l l s m u s t s y n e r g i z e w i t h t h e serum
. 94
To g e t damage o r d e l a y e d p a t e n c y i n a l a r v a l i n f e c t i o n i n
n u d e s ( T a b l e XXVI) .
The o b s e r v a t i o n t h a t immune se rum i s more e f f i c i e n t a t
c a u s i n g e x p u l s i o n when g i v e n on day 3 t h a n when g i v e n on day
0 c o u l d be e x p l a i n e d i n s e v e r a l ways.
One p o s s i b i l i t y is.
t h a t t h e immune se rum p r e s e n t when t h e l a r v a e a r e m i g r a t i n g
c a u s e s t h e worms t o become " a d a p t e d " ( 4 4 ) an d s u r v i v e l o n g e r
e v e n i n a n immunoc ompetent h o s t .
A second p o s s i b i l i t y i s
t h a t t h e worm a n t i g e n s a r e b l o c k e d by t h e a n t i b o d i e s i n t h e
p a s s i v e l y t r a n s f e r r e d immune seru m and i t t a k e s a l o n g e r
t i m e f o r t h e mouse t o r e a c t a c t i v e l y t o t h e worm i n f e c t i o n
an d d e l a y s t h e worm e x p u l s i o n .
tie s
E ith e r of these p o s s i b i l i ­
c o u l d a c c o u n t f o r t h e worm p r o t e c t i v e se ru m s s e e n i n
F ig u re 4.
I d o n ' t know i f t h e worm p r o t e c t i v e f a c t o r i s t h e
same a s t h e mouse p r o t e c t i v e f a c t o r o r i f t h e t i m e o f admin­
i s t r a t i o n o r t h e do se i s t h e d e c i d i n g f a c t o r a s t o w h e t h e r .
t h e end r e s u l t w i l l be p r o l o n g e d o r a b l a t e d p a t e n c y . ■ I f t h e
2 f a c t o r s a re d i f f e r e n t , th e r e l a t i v e c o n c e n t r a t i o n could
be i m p o r t a n t and t o t a l dose o f e a c h may a l t e r t h e en d o u t ­
come.
I h a v e some u n r e p o r t e d d a t a t h a t would s u g g e s t t h a t
one se rum s p e c i m e n c a n be b o t h worm p r o t e c t i v e a n d mouse
p r o t e c t i v e a t d i f f e r e n t doses b u t, because o f th e sh o rta g e
o f worm p r o t e c t i v e s e r u m , a l l o f t h e p r o p e r c o n t r o l s w e r e n ' t .
95
r u n a t one t i m e .
T h e r e f o r e , no d e f i n i t i v e s t a t e m e n t can be
made.
How t h e se rum f a c t o r a c t s on t h e worm i s unknown.
IL^
i n c u b a t e d i n immune se rum i n v i t r o l i v e a s l o n g a s an d a r e
i n d i s t i n g u i s h a b l e fr o m n o r m a l worms ( 2 1 ) .
I t has been found
t h a t a d u l t worms m a i n t a i n e d u n d e r t h e b e s t known c o n d i t i o n s
a r e damaged i n t h e same way t h a t " a n t i b o d y damaged" worms
a r e damaged i n v i v o , w h ic h s u g g e s t s t h a t t h e f a c t o r may j u s t
c a u s e t h e worm t o move t o a l e s s optimum e n v i r o n m e n t i n t h e
sm all i n t e s t i n e
(31).
I f o u n d t h a t i n c u b a t i n g IL^ i n a c t i v e mouse p r o t e c t i v e
se rum d i d n ' t a l t e r t h e i r i n f e c t i v i t y e v en when t h e y were
u s e d t o i n f e c t a mouse g i v e n IMLNC ( T a b l e I X ) .
I also in ­
j e c t e d mouse p r o t e c t i v e s e r u m . i n t o t h e duodenum on day 4
when t h e worms were t h e r e and t h e se ru m d i d n ' t seem t o have
any d e t r i m e n t a l a f f e c t on t h e worms ( T a b l e I X ) .
More d o s e s
o f .serum g i v e n i n t r a d u o d e n a l l y would be n e e d e d t o d e f i n i ­
t i v e l y show t h a t s e ru m g i v e n i n t h e g u t lumen w o u l d n ' t c a u s e
damage t o t h e worm, b u t t h i s would be t e c h n i c a l l y d i f f i c u l t .
.
I t h a s b e e n shown t h a t Worms I n t h e g u t a r e l a b e l e d w i t h p
when g i v e n t o t h e h o s t a n i m a l IV b e t t e r t h a n when t h e p -'
g iven in the g ut.
32
is
T h i s o b s e r v a t i o n s u g g e s t s t h a t t h e worms
may n e e d t o i n g e s t t h e serum f a c t o r o r t h e seru m f a c t o r may
96
a c t on an unknown c e l l o r t i s s u e
vs.
site
i n t h e mouse when g i v e n IV
ID w h ic h c o u l d c a u s e t h e worm t o move from i t s p r e f e r r e d
in the gut.
I f o u n d t h e f a c t o r r e s p o n s i b l e f o r worm damage i n v i v o
t o be r e l a t i v e l y s t a b l e .
I f o u n d t h e mouse p r o t e c t i v e f a c ­
t o r t o be much l e s s h e a t l a b i l e
r e p o r t e d (Table X X III).
(56 C) t h a n O g i l v i e (3 8 )
The p r e v i o u s r e p o r t o f t h e h e a t
l a b i l i t y o f th e p r o t e c t i v e f a c t o r (38) a ls o dem onstrated
t h a t t h e PCA a c t i v i t y was d e s t r o y e d , a s would be e x p e c t e d ,
b u t t h e r e p o r t a l s o s t a t e d t h a t worm s p e c i f i c p r e c i p i t i n i n
g e l was d i m i n i s h e d , w h ic h i s u n e x p l a i n e d .
I a l s o found th e
f a c t o r t o be r e s i s t a n t t o f r e e z e - t h a w i n g ( T a b l e X X I I I ) w h ic h
sug g ests t h a t i t is d i f f e r e n t than Ja c k s o n 's l a r v a l l e t h a l
f a c t o r f o u n d i n se ru m ( 1 4 ) .
The a c t i v i t y i n immune serum
was f o u n d i n t h e g l o b u l i n - r i c h f r a c t i o n o f ammonium s u l f a t e
p r e c i p i t a t i o n f r a c t i o n s and w a s n ' t i r r e v e r s i b l y d e n a t u r e d by
t h e p r e c i p i t a t i o n p r o c e d u r e o r l o s t d u r i n g d i a l y s i s ( T a b le
V III).
. My a t t e m p t s t o a d s o r b t h e f a c t o r o n t o IL^ s u g g e s t t h a t
th e f a c t o r d i d n ' t adhere to the l a r v a e ,
in t h a t the larv ae
w e r e n ' t n o t i c e a b l y damaged e v en a f t e r i n j e c t i o n i n t o a h o s t
t h a t h a d b e e n g i v e n IMLNC ( T a b l e I X ) .
t h a t ILo i n j e c t e d i n as l i t t l e
J
I t has been r e p o r te d
a s 0 . 1 ml o f immune serum
97
c o u l d c a u s e d e c r e a s e d numbers o f a d u l t worms ( 5 9 ) •
T h is
same w o r k e r a l s o f o u n d t h a t l a r v a l c u l t u r e f l u i d — e x c r e t i o n
an d s e c r e t i o n a n t i g e n s - - c o u l d n e u t r a l i z e t h e p r o t e c t i v e
a c t i o n o f t h e se ru m , w h i l e l a r v a l e x t r a c t w o u l d n ' t ( 5 9 ) None o f t h e c h e m i c a l c h a r a c t e r i z a t i o n p a r a m e t e r s I have
d e f i n e d have s u g g e s t e d t h a t th e p r o t e c t i v e f a c t o r ' c o u l d n 't
be a n ' i m m u n o g l o b u l i n .
,
I b e l i e v e t h a t I h ave t h e s y s t e m
s u f f i c i e n t l y w orked o u t so t h a t by u s i n g a f f i n i t y c h r o m a t o ­
g r a p h y and a n t i - i m m u n o g l o b u l i n s e r u m ,
i t c o u l d be d e t e r m i n e d
i f th e f a c t o r i s immunoglobulin and i f i t i s ,
the c la s s '
responsible f o r passive p ro te c tio n .
T h ere i s o n l y one c h a r a c t e r i z e d c h e m i c a l . t h a t h a s b e e n
r e p o r t e d t o a c c e l e r a t e damage i n t h e
b rasilien sis rat
s y s t e m a n d t h a t i s p r o s t a g l a n d i n E (PGE^ & PGE^).
The d a t a
t h a t s u p p o r t t h e c l a i m t h a t PGE i s i m p o r t a n t i n v i v o a r e :
f r a c t i o n s o f semen c o n t a i n i n g p r o s t a g l a n d i n s , i n j e c t e d i n t r a d u o d e n a l l y d u r i n g a n i n f e c t i o n i n c r e a s e d e x p u l s i o n o f worms
(5^); p ro s ta g la n d in in h ib ito r s
( a c e t y l s a l i c i l i c a c i d arid
d-propoxyphene) delayed ex p u lsio n ;
the le v e l o f p r o s ta g la n ­
dins in c re a s e d in the sm all i n t e s t i n e during e x p u lsio n (7);
s y n t h e t i c PGE^ and PGEg i n j e c t e d i n t r a d u o d e n a l l y d u r i n g an
i n f e c ti o n caused e a r l i e r e x p u lsio n .
More r e c e n t l y a d u l t worms h a v e b e e n i n c u b a t e d i n v i t r o
98
w ith p r o s ta g la n d in s .
A f t e r i n c u b a t i o n as s h o r t a s I 8 h o u r s ,
a d u l t worms i n c u b a t e d i n PGE^ were e x p e l l e d by r e c i p i e n t
r a t s a s i f t h e y were damaged, w h i l e c o n t r o l worms were e x ­
p e l l e d l a t e r (5°)*
While f a r fro m b e i n g d e f i n i t i v e ,
experi­
m en ts r e p o r t e d h e r e f a i l e d t o d e m o n s t r a t e any d i f f e r e n c e i n
e x p u l s i o n t i m e s i n mice g i v e n e i t h e r p r o s t a g l a n d i n i n h i b i t o r s - - a s p i r i n (T able IV),
i n d o m e t h a c i n ( T a b l e V)— o r s y n ­
t h e t i c p r o s t a g l a n d i n s ( T a b l e s VI, V I I ) .
I u s e d d o s e s and
t r e a t m e n t s c h e d u l e s c o m p a r a b l e t o t h o s e u s e d i n t h e r a t work
f o r a s p i r i n ( 3 , 2 4 ) and p r o s t a g l a n d i n s ( 2 4 ) .
Indom ethacin
was g i v e n a s i t h a s b e e n r e p o r t e d t o d e l a y e x p u l s i o n i n t h e
T r i c h u r i s m u r i s mouse s y s t e m ( 2 ) .
Some l o w - d o s e i m m u n o s u p p r e s s i v e a g e n t s u s e d i n t h i s
work c a u s e d h i g h e r EPG c o u n t s t h a n c o n t r o l s e a r l y i n t h e
i n f e c t i o n e v e n t h o u g h t h e m ic e w e r e n ' t a n o r e x i c a n d e x p e l l e d
t h e i r worms on t i m e .
T h i s s u g g e s t s t h a t more worms r e a c h e d
t h e g u t o r damage o c c u r r e d l a t e r .
of Y -irradiation,
The a g e n t s a r e low d o s e s
100 -3 5 0 r a d s ( T a b l e XV), and c y c l o p h o s ­
p h a m i d e , a s shown i n T a b le XXV.
The EPG d a t a i n t h e s e
s t u d i e s r e s e m b l e somewhat t h e l a r v a l i n f e c t i o n s i n t r y p a n
b l u e - t r e a t e d mice i n w h ic h t h e i n c r e a s e d EPG a p p e a r t o be
c a u s e d by more l a r v a e r e a c h i n g t h e a d u l t s t a g e ( T a b l e I I ,
day 6 ) .
The p r o b l e m i s t o d e c i d e w h e t h e r t r y p a n b l u e
99
i n h i b i t s t h e h o s t r e s p o n s e i n more ways t h a n j u s t t h e m a c r o ­
ph ag e e f f e c t o r f u n c t i o n o r w h e t h e r t h e o t h e r a g e n t s u s e d
( c y c l o p h o s p h a m i d e an d ^ ^ i r r a d i a t i o n ) a l s o i n h i b i t m acrophage
e ffe c to r function.
S t r a u s e ( 5 7 ) r e c e n t l y p u b l i s h e d work
sh o w in g d e p r e s s i o n o f s p e c i f i c p l a q u e f o r m i n g r e s p o n s e i n
m ice g i v e n d o s e s o f t r y p a n b l u e much l o w e r t h a n t h e on es I
was u s i n g .
I c a n ' t make a f i n a l d e c i s i o n on t h e i m p o r t a n c e
o f m a c r o p h a g e s a s e f f e c t o r c e l l s i n Njl b r a s i l i e n s i s i n f e c ­
tions .
A l l I can r e p o r t i s t h a t h ig h doses o f t r y p a n b lu e
a l l o w s i n c r e a s e d num bers o f l a r v a e t o d e v e l o p e i n t o a d u l t s
and d e l a y s t h e i r e x p u l s i o n a l i t t l e
T ry p an b l u e a p p a r e n t l y h a d l i t t l e
( T a b le I I ,
F i g u r e I ).
o r no e f f e c t on t h e e x p u l ­
s i o n o f n o r m a l a d u l t worms t r a n s f e r r e d i n t o t r y p a n b l u e t r e a t e d m ic e ( F i g u r e s 2 & 3)■
In conclusion,
I f e e l t h a t I h ave shown a n a s s a y s y s t e m
f o r c h a r a c t e r i z i n g t h e h u m o r a l worm damaging f a c t o r and a l s o
t o r a p i d l y e v a l u a t e se rum p o o l s f o r t h e i r worm damaging
ab ility .
I a l s o have found a s o u r c e o f s e n s i t i z e d c e l l s
w h i c h , when g i v e n t o a n o r m a l a n i m a l ,
can p r o d u c e o r s t i m u ­
l a t e t h e a n i m a l t o p r o d u c e damage i n a l a r v a l i n f e c t i o n
e a r l i e r th an norm al.
E i t h e r o r b o t h o f t h e s e m e th o d s o f
da magin g worms s h o u l d be a p p l i e d t o immunod e f i c i e n t a n i m a l s
( ^ - i r r a d i a t e d a n d / o r n u d e ) t o i d e n t i f y t h e c o m ponents
100
n e c e s s a r y t o c o - o p e r a t e w i t h t h e f a c t o r i n c a u s i n g damage,
i f any i s n e c e s s a r y .
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