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The influence of intestinal bilharziasis (Schistosoma mansoni) on serum thyroxine-binding prealbumin (TBPA) in rat
NUTRITION RESEARCH, Vol. 8, pp. 57-64, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All rights reserved.
THE
INFLUENCE ON SERUM
OF I N T E S T I N A L B I L H A R Z I A S I S (SCHISTOSOMA MANSONI) T H Y R O X I N E - B I N D I N G P R E A L B U M I N (TBPA) IN RAT
F. B l e i b e r g - D a n i e l I, S. Wade, E. Borel, B. Le Moullac, P.Gouache, D. Biou, F. Gauthier and D. L e m o n n i e r Unit~ de R e c h e r c h e s sur la N u t r i t i o n et l ' A l i m e n t a t i o n , U.I INSERM, H6pital Bichat, 170 bd Ney, 75877 Paris Cedex 18 ; L a b o r a t o i r e de P a r a s i t o l o g i e , F a c u l t 4 de M4decine, Lyon ; L a b o r a t o i r e de Biochimie, H6pital Bretonneau, Paris ; L a b o r a t o i r e de Biochimie, F a c u l t 4 de M4decine, Tours.
ABSTRACT The i n f l u e n c e of S c h i s t o s o m a m a n s o n i i n f e c t i o n on the c i r c u l a t i n g level of t h y r o x i n e b i n d i n g p r e a l b u m i n (TBPA) was s t u d i e d in 4 - w e e k - o l d male Wistar rats fed ad libitum a 18~ p r o t e i n diet. The i n f e c t i o n was carried out by c u t a n e o u s p e n e t r a t i o n of 700 S c h i s t o s o m a mansoni cercariae. Groups of 8 i n f e c t e d rats and their c o n t r o l s were s a c r i f i e d 4, 6 and 12 weeks f o l l o w i n g the infection. T h r o u g h o u t the study, body w e i g h t and food intake of the infected rats were similar to those of their m a t c h e d controls. Serum TBPA c o n c e n t r a t i o n d e c r e a s e d s l i g h t l y but s i g n i f i c a n t l y (p <0.05) in the infected rats c o m p a r e d to their r e s p e c t i v e c o n t r o l s only from w e e k 4 onwards. Antib i l h a r z i a l a n t i b o d i e s were d e t e c t e d by d a y 15. The level reached maximal value by w e e k 6. H i s t o p a t h o l o g i c a l e x a m i n a t i o n of the liver showed g r a n u l o m a f o r m a t i o n s by weeks 6 and 12. D e s p i t e this i n f l a m m a t o r y reaction, b i l h a r z i a s i s did not induce any rise in the serum levels of o r o s o m u c o i d and e 2 - m a c r o g l o b u l i n , two main a c u t e - p h a s e reactant p r o t e i n s (APRP). As for serum TBPA, a slight d e c r e a s e in a l b u m i n and t r a n s f e r r i n also occurred. These results suggest that i n f e c t i o n w i t h S c h i s t o s o m a mansoni does n e i t h e r induce m a l n u t r i t i o n in rats nor p r o d u c e an increase in their serum APRP. In these c i r c u m s t a n c e s , serum TBPA level is not a f f e c t e d until occurence of severe liver damage. KEY WORDS : T h y r o x i n e - b i n d i n g prealbumin, intestinal b i l h a r z i a s i s , inflammation, n u t r i t i o n a l markers, a c u t e - p h a s e reactant proteins.
IAuthor
to whom
all c o r r e s p o n d e n c e 57
should
be directed.
58
F. BLEIBERG-DANIEL et al. INTRODUCTION
F o r many years, TBPA, r e c e n t l y n a m e d t r a n s t h y r e t i n was c o n s i d e r e d as one of the most s e n s i t i v e n u t r i t i o n a l m a r k e r s (1-3). Its c i r c u l a t i n g level is now w i d e l y u s e d to d e t e c t s u b c l i n i c a l signs of p r o t e i n - e n e r g y m a l n u t r i t i o n and to assess the e f f i c i e n c y of n u t r i t i o n a l r e h a b i l i t a t i o n in i n d u s t r i a l i z e d as well as in d e v e l o p i n g c o u n t r i e s (I, 3-6). However, a l t h o u g h it is c l e a r l y d e m o n s t r a t e d that n u t r i t i o n a l d e f i c i e n c i e s d e c r e a s e s e r u m T B P A concens (2, 7, 8) it is also r e p o r t e d that i n f e c t i o n s a n d / o r i n f l a m m a t i o n can affect its c i r c u l a t i n g level in m a n and in rat (9-11). D u r i n g i n f l a m m a t i o n , liver p r e f e r e n t i a l s y n t h e s i s of a c u t e - p h a s e r e a c t a n t p r o t e i n s o c c u r s at the e x p e n s e of that of n u t r i t i o n a l markers such as a l b u m i n and T B P A (12). This r e g u l a t o r y m e c h a n i s m seems to o c c u r at the m R N A level (ii, 13). E x p e r i m e n t a l m o d e l s are p a r t i c u l a r l y s u i t a b l e to assess the r e l a t i v e i n f l u e n c e of i n f e c t i o n and/or i n f l a m m a t i o n on n u t r i t i o n a l markers, e s p e c i a l l y on TBPA, i n d e p e n d e n t l y f r o m that of f o o d d e f i c i e n c i e s . We s e l e c t e d the rat for two m a i n r e a s o n s : firstly, the p r o p e r t i e s of rat T B P A c l o s e l y r e s e m b l e those of the human p r o t e i n (14), s e c o n d l y , b i l h a r z i a s i s , one of the most w i d e s p r e a d p a t h o l o g i e s in d e v e l o p i n g c o u n t r i e s can be e a s i l y a c h i e v e d in this model. F u r t h e r m o r e , S c h i s t o s o m a m a n s o n i i n f e c t i o n does not induce p r o t e i n l o s s e s t h r o u g h the rat gut. The p r e s e n t study was d e s i g n e d to i n v e s t i g a t e the effect of i n t e s t i n a l b i l h a r z i a s i s on serum T B P A as w e l l as on other n u t r i t i o n a l and i n f l a m m a t o r y markers. MATERIAL
AND M E T H O D S
4 - w e e k - o l d male W i s t a r rats w e r e u s e d for the e x p e r i m e n t . All a n i m a l s were h o u s e d in i n d i v i d u a l p o l y p r o p y l e n e cages and m a i n t a i n e d at 22 + 2~ on a 12h l i g h t / 1 2 d a r k cycle. The rats were fed ad l i b i t u m a ~8 % p r o t e i n diet (control diet) and had free a c c e s s to w a t e r t h r o u g h o u t the study. T h e y were r a n d o m l y a s s i g n e d to c o n t r o l and i n f e c t e d groups. I n f e c t i o n was c a r r i e d out by skin p e n e t r a t i o n of 700 S c h i s t o s o m a c e r c a r i a e per animal : the rats w e r e a n e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (Clin-Midy, Paris, France) and their abdomens w e r e s h a v e d w i t h a fine e l e c t r i c clipper. A ring was p l a c e d on the a b d o m e n and was p a r t i a l l y f i l l e d w i t h the c e r c a r i a l s u s p e n s i o n . After a 30 minute e x p o s u r e period, the s h a v e d skin was rinsed w i t h a l c o h o l and the rats w e r e p l a c e d b a c k in their cages. F o o d i n t a k e and body w e i g h t w e r e r e c o r d e d e v e r y two days. Blood was c o l l e c t e d by o r b i t a l p u n c t u r e on d a y o and at weekly i n t e r v a l s for 4 weeks. Groups of 8 i n f e c t e d rats and their r e s p e c t i v e c o n t r o l s w e r e s a c r i f i e d on w e e k s 4,6 a n d 12. Blood was c o l l e c t e d from the a b d o m i n a l aorta, the liver was perfused, removed, w e i g h e d and f i x e d in b o u i n m e d i u m for h i s t o p a t h o l o g i c a l examination. The s p e c i f i c immune r e s p o n s e to b i l h a r z i a s i s was i n v e s t i g a t e d by m e a s u r i n g the a n t i - S c h i s t o s o m a m a n s o n i a n t i b o d i e s by i n d i r e c t i m m u n o f l u o r e s c e n c e using frozen s e c t i o n s of S c h i s t o s o m a m a n s o n i adult w o r m s as antigens. Serum TBPA, a l b u m i n a n d e p m a c r o g l o b u l i n c o n c e n t r a t i o n s were m e a s u r e d by e l e c t r 0 i m m u n o a s s a y - w h i l e
TBPA IN INTESTINAL BILHARZIASIS
59
t r a n s f e r r i n and o r o s o m u c o i d c o n c e n t r a t i o n s w e r e d e t e r m i n e d by radial i m m u n o d i f f u s i o n . Rat t r a n s f e r r i n and albumin as well as anti rat a l b u m i n and anti rat t r a n s f e r r i n were s u p p l i e d by C a p p e l L a b o r a t o r i e s (West Chester, U.S.A.). Rat T B P A and a m o n o s p e c i f i c anti rat TBPA a n t i b o d y w e r e o b t a i n e d a c c o r d i n g to a p r o c e d u r e p r e v i o u s l y d e s c r i b e d (15). H e m o g l o b i n c o n c e n t r a t i o n was d e t e r m i n e d by the c y a n m e t h e m o g l o b i n m e t h o d (kit N ~ 525 s u p p l i e d by Sigma C h e m i c a l Company, ST. Louis, U.S.A). D a t a from the i n f e c t e d and the r e s p e c t i v e c o n t r o l groups were s t a t i s t i c a l l y a n a l y s e d by the s t u d e n t ' s t test.
RESULTS
AND D I S C U S S I O N
I n t e s t i n a l b i l h a r z i a s i s i n d u c e d a s p e c i f i c immune response: a n t i - S c h i s t o s o m a mansoni a n t i b o d i e s were d e t e c t e d by the second week f o l l o w i n g i n f e c t i o n and the a n t i b o d y titers reached the highest values by w e e k 6, then d e c l i n e d s l i g h t l y by week 12 (FIG.l)
ANTI-SCHISTOSOMA MANSONI ANTIBODY LEVEL ( l o g 2 o f the dilution)
10.3
9.3
8.3
7.3
6.3
5.3
4.3 lit
t
/
- J
1
2
3
4
5
6
I WEEKS 12
infection
FIG. I. A n t i - S c h i s t o s o m a m a n s o n i a n t i b o d y levels d u r i n g the d e v e l o p m e n t of intestinal b i l h a r z i a s i s .
60
F. BLEIBERG-DANIEL et al.
Host d e f e n s e also i n v o l v e d a d r a m a t i c i n f l a m m a t o r y r e a c t i o n w h i c h r e s u l t e d in n u m e r o u s g r a n u l o m a f o r m a t i o n s around the eggs. Small m a c r o s c o p i c l e s i o n s w e r e o b s e r v e d as s o o n a s w e e k 4 but m a r k e d g r a n u l o m a f o r m a t i o n s only by w e e k 6 (FIG. 2a). T w e l v e w e e k s f o l l o w i n g e x p o s u r e to c e r c a r i a e , g r a n u l o m a size was r e d u c e d as well as c e l l u l a r d e n s i t y a r o u n d t h e eggs (FIG. 2b). This p h e n o m e n o n has a l r e a d y b e e n d e s c r i b e d in d e t a i l in the rat (16). W a r r e n et al (17) e s t a b l i s h e d that g r a n u l o m a f o r m a t i o n was a m a n i f e s t a t i o n of d e l a y e d h y p e r s e n s i t i v i t y .
A
B
FIG.2 egg in liver of rat Representative granuloma around schistosome i n f e c t e d with S c h i s t o s o m a m a n s o n i : A- for 6 w e e k s ; B- for 12 weeks ; H & E stain x25 (x.25)
TBPA IN INTESTINAL BILHARZIASIS
61
D e s p i t e the m a r k e d i n f l a m m a t o r y liver r e a c t i o n o b s e r v e d in the i n f e c t e d rats, the serum levels of two main a c u t e - p h a s e r e a c t a n t proteins, o r o s o m u c o i d and e g - m a c r o g l o b u l i n d i d not exhibit p a t h o l o g i c a l values during th~ d e v e l o p m e n t of b i l h a r z i a s i s . Thus, e l e v a t i o n of acute phase r e a c t a n t p r o t e i n s does not seem to be i n v o l v e d in the natural r e s i s t a n c e o f rat to S c h i s t o s o m a mansoni infection. A l t h o u g h the rats w e r e e x p o s e d to an i m p o r t a n t number of cercariae, little effect has been o b s e r v e d on their n u t r i t i o n a l status. Body weight g a i n and food intake as well as h e m a t o c r i t and h e m o g l o b i n c o n c e n t r a t i o n s were c o m p a r a b l e in the infected animals and in their m a t c h e d c o n t r o l s t h r o u g h o u t the study. In addition, T B P A c o n c e n t r a t i o n was very m o d e r a t e l y i n f l u e n c e d by S c h i s t o s o m a m a n s o n i i n f e c t i o n : serum TBPA levels started to d e c r e a s e s l i g h t l y but s i g n i f i c a n t l y by w e e k 4 (4 % d r o p c o m p a r e d to the c o n t r o l g r o u p ; p < 0.05) and c o n t i n u e d to d e c l i n e m o d e r a t e l y by w e e k 6 (11% d r o p ; p < 0.05). By week 12, the d i f f e r e n c e in T B P A c o n c e n t r a t i o n s b e t w e e n the i n f e c t e d and the control rats r e m a i n e d c o n s t a n t w i t h o u t any s i g n i f i c a n t d i f f e r e n c e (FIG. 3).
TB
PA (mg/Iooml) CONTROL ....
,~t
INFECTED
9 P',~ 0 . 0 5
T
60
50
40
~
'
1
2
3
4
5
6
4U v
12
I-WEEKS
infection
FIG.3 Serum t h y r o x i n e - b i n d i n g p r e a l b u m i n (TBPA) c o n c e n t r a t i o n s i n f e c t e d rats and in their r e s p e c t i v e controls
in the
62
F. BLEIBERG-DANIEL et al.
As s h o w n in T A B L E 1 serum c o n c e n t r a t i o n s of two o t h e r n u t r i t i o n a l markers, a l b u m i n and t r a n s f e r r i n , w e r e r e l a t i v e l y less a f f e c t e d than T B P A d u r i n g the d e v e l o p m e n t of b i l h a r z i a s i s . S e r u m a l b u m i n level d e c r e a s e d b y 6 and 9 ~ by weeks 4 a n d 6, r e s p e c t i v e l y (p < 0.05 versus the c o n t r o l group) w h i l e t r a n s f e r r i n levels only d e c r e a s e d by w e e k 4 (ii ~ drop, p < 0.05).
TABLE 1 S e r u m A l b u m i n and T r a n s f e r r i n C o n c e n t r a t i o n s in the control (C) and i n f e c t e d (I) Rats d u r i n g the D e v e l o p m e n t of B i l h a r z i a s i s .
WEEKS F O L L O W I N G INFECTION
ALBUMIN (g/l)
TRANSFERRIN (g/l)
C 32.1 + 0.6
4.4 + 0.07
I 33.2 + 0.8
4.3 + 1.13
0.6
4.5 + 0.08
I 33.3 + 0.2
4.3 + 0.13
C 32.6 + 0.6
4.5 + 0.05
I 30.8 + 0.4 %
4.0 + 0.05 ~
C 30.8 + 0.8
4.2 + 0.08
0.8
4.3 + 0.08
C 28.3 + 0.6
4.4 + 0.17
0.7
4.1 + 0 . 0 9
0
C
I
32.3
28.1
+
+
12 I
30.0
* D a t a are e x p r e s s e d % p < 0.05 v e r s u s c ~ p < 0.001 v e r s u s c
+
as mean ~ SEM
In the a b s e n c e of w e i g h t loss and r e d u c t i o n in food intake, the m o d e r a t e d e c r e a s e in the n u t r i t i o n a l markers might b e e x p l a i n e d either by liver d a m a g e due to g r a n u l o m a f o r m a t i o n a n d / o r by an e n h a n c e d s y n t h e s i s of other a c u t e - p h a s e r e a c t a n t p r o t e i n s than o r o s o m u c o i d or e 2 - m a c r o g l o b u l i n . However, as both p r o t e i n s count among the major a c u t e - p h a s e r e a c t a n t p r o t e i n s in the rat, the a b s e n c e of i n c r e m e n t in their serum levels s u g g e s t that the a l t e r a t i o n in n u t r i t i o n a l m a r k e r c o n c e n t r a t i o n s , e s p e c i a l l y in TBPA, might be m a i n l y i n t e r p r e t e d as a c o n s e q u e n c e of liver impairment.
TBPA IN INTESTINAL BILHARZIASIS
63
The d a t a s u g g e s t that i n f l a m m a t i o n , b e s i d e s its adverse effect on liver, does not n e c e s s a r i l y a f f e c t T B P A levels when the f o l l o w i n g c o n d i t i o n s are f u l f i l l e d : i- m a i n t e n a n c e of adequate food intake. 2- absence of i n c r e a s e d s y n t h e s i s of a c u t e - p h a s e r e a c t a n t proteins.
References. 1. I n g e n b l e e k Y, Van den S c h r i e c k HG, De Nayer Ph, De V i s s c h e r Albumin, t r a n s f e r r i n and the t h y r o x i n e - b i n d i n g p r e a l b u m i n / r e t i n o l - b i n d i n g p r o t e i n (TBPA-RBP) c o m p l e x in a s s e s s m e n t of m a l n u t r i t i o n . Clin. Chim. Acta 1975; 63:61-67.
M.
2. Shetty PS, W a t r a s i e w i c z KE, Yung RT and James WPT. Rapidt u r n o v e r t r a n s p o r t p r o t e i n s : an index of s u b c l i n i c a l p r o t e i n - e n e r g y m a l n u t r i t i o n . Lancet 1978; 2:230-232.
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S c h r e i b e r G, H o w l e t t G, N a g a s h i m a M, M i l l e r s h i p A, Martin H, Urban J and K o t l t e r L. The acute phase response of plasma protein s y n t h e s i s d u r i n g e x p e r i m e n t a l inflammation. J. Biol. Chem. 1982: 2 5 7 : 1 0 2 7 1 - 1 0 2 7 7 .
13.
Dickson PW, H o w l e t t GJ and S c h r e i b e r G. Rat t r a n s t h y r e t i n (prealbumin). M o l e c u l a r cloning, n u c l e o t i d e sequence, and g e n e e x p r e s s i o n in liver and brain. J. Biol. Chem. 1985; 260:82148219.
14.
Navab, M, M a l l i a AK, K a n d a Y and G o o d m a n DS. Rat plasma prealbumin. I s o l a t i o n and partial c h a r a c t e r i z a t i o n . J. Biol. Chem. 1977; 2 5 2 : 5 1 0 0 - 5 1 0 6 .
15.
B l e i b e r g F, Vranckx, Wade S and Nunez EA. A s i m p l i f i e d method for the p r e p a r a t i o n of rat t h y r o x i n e - b i n d i n g prealbumin. F a c t o r s i n f l u e n c i n g its c i r c u l a t i n g level. B.B.A. 1985; 828:270-278.
16. Joky A and G a r i n JP. H i s t o p a t h o l o g i e h 4 p a t i q u e et u l t r a s t r u c t u r e du g r a n u l o m e dans la b i l h a r z i o s e du rat blanc due ~ S c h i s t o s o m a mansoni. Ann. Soc. Belge Med. Trop. 1980; 60:355-368.
17. W a r r e n KS, D o m i n g o EO and Cowan RBT. G r a n u l o m a f o r m a t i o n around S c h i s t o s o m e eggs as a m a n i f e s t a t i o n of d e l a y e d hypersensitivity. Am. J. Pathol. 1967; 51:737-756.
Accepted for publication July 12, 1987
THE
INFLUENCE ON SERUM
OF I N T E S T I N A L B I L H A R Z I A S I S (SCHISTOSOMA MANSONI) T H Y R O X I N E - B I N D I N G P R E A L B U M I N (TBPA) IN RAT
F. B l e i b e r g - D a n i e l I, S. Wade, E. Borel, B. Le Moullac, P.Gouache, D. Biou, F. Gauthier and D. L e m o n n i e r Unit~ de R e c h e r c h e s sur la N u t r i t i o n et l ' A l i m e n t a t i o n , U.I INSERM, H6pital Bichat, 170 bd Ney, 75877 Paris Cedex 18 ; L a b o r a t o i r e de P a r a s i t o l o g i e , F a c u l t 4 de M4decine, Lyon ; L a b o r a t o i r e de Biochimie, H6pital Bretonneau, Paris ; L a b o r a t o i r e de Biochimie, F a c u l t 4 de M4decine, Tours.
ABSTRACT The i n f l u e n c e of S c h i s t o s o m a m a n s o n i i n f e c t i o n on the c i r c u l a t i n g level of t h y r o x i n e b i n d i n g p r e a l b u m i n (TBPA) was s t u d i e d in 4 - w e e k - o l d male Wistar rats fed ad libitum a 18~ p r o t e i n diet. The i n f e c t i o n was carried out by c u t a n e o u s p e n e t r a t i o n of 700 S c h i s t o s o m a mansoni cercariae. Groups of 8 i n f e c t e d rats and their c o n t r o l s were s a c r i f i e d 4, 6 and 12 weeks f o l l o w i n g the infection. T h r o u g h o u t the study, body w e i g h t and food intake of the infected rats were similar to those of their m a t c h e d controls. Serum TBPA c o n c e n t r a t i o n d e c r e a s e d s l i g h t l y but s i g n i f i c a n t l y (p <0.05) in the infected rats c o m p a r e d to their r e s p e c t i v e c o n t r o l s only from w e e k 4 onwards. Antib i l h a r z i a l a n t i b o d i e s were d e t e c t e d by d a y 15. The level reached maximal value by w e e k 6. H i s t o p a t h o l o g i c a l e x a m i n a t i o n of the liver showed g r a n u l o m a f o r m a t i o n s by weeks 6 and 12. D e s p i t e this i n f l a m m a t o r y reaction, b i l h a r z i a s i s did not induce any rise in the serum levels of o r o s o m u c o i d and e 2 - m a c r o g l o b u l i n , two main a c u t e - p h a s e reactant p r o t e i n s (APRP). As for serum TBPA, a slight d e c r e a s e in a l b u m i n and t r a n s f e r r i n also occurred. These results suggest that i n f e c t i o n w i t h S c h i s t o s o m a mansoni does n e i t h e r induce m a l n u t r i t i o n in rats nor p r o d u c e an increase in their serum APRP. In these c i r c u m s t a n c e s , serum TBPA level is not a f f e c t e d until occurence of severe liver damage. KEY WORDS : T h y r o x i n e - b i n d i n g prealbumin, intestinal b i l h a r z i a s i s , inflammation, n u t r i t i o n a l markers, a c u t e - p h a s e reactant proteins.
IAuthor
to whom
all c o r r e s p o n d e n c e 57
should
be directed.
58
F. BLEIBERG-DANIEL et al. INTRODUCTION
F o r many years, TBPA, r e c e n t l y n a m e d t r a n s t h y r e t i n was c o n s i d e r e d as one of the most s e n s i t i v e n u t r i t i o n a l m a r k e r s (1-3). Its c i r c u l a t i n g level is now w i d e l y u s e d to d e t e c t s u b c l i n i c a l signs of p r o t e i n - e n e r g y m a l n u t r i t i o n and to assess the e f f i c i e n c y of n u t r i t i o n a l r e h a b i l i t a t i o n in i n d u s t r i a l i z e d as well as in d e v e l o p i n g c o u n t r i e s (I, 3-6). However, a l t h o u g h it is c l e a r l y d e m o n s t r a t e d that n u t r i t i o n a l d e f i c i e n c i e s d e c r e a s e s e r u m T B P A concens (2, 7, 8) it is also r e p o r t e d that i n f e c t i o n s a n d / o r i n f l a m m a t i o n can affect its c i r c u l a t i n g level in m a n and in rat (9-11). D u r i n g i n f l a m m a t i o n , liver p r e f e r e n t i a l s y n t h e s i s of a c u t e - p h a s e r e a c t a n t p r o t e i n s o c c u r s at the e x p e n s e of that of n u t r i t i o n a l markers such as a l b u m i n and T B P A (12). This r e g u l a t o r y m e c h a n i s m seems to o c c u r at the m R N A level (ii, 13). E x p e r i m e n t a l m o d e l s are p a r t i c u l a r l y s u i t a b l e to assess the r e l a t i v e i n f l u e n c e of i n f e c t i o n and/or i n f l a m m a t i o n on n u t r i t i o n a l markers, e s p e c i a l l y on TBPA, i n d e p e n d e n t l y f r o m that of f o o d d e f i c i e n c i e s . We s e l e c t e d the rat for two m a i n r e a s o n s : firstly, the p r o p e r t i e s of rat T B P A c l o s e l y r e s e m b l e those of the human p r o t e i n (14), s e c o n d l y , b i l h a r z i a s i s , one of the most w i d e s p r e a d p a t h o l o g i e s in d e v e l o p i n g c o u n t r i e s can be e a s i l y a c h i e v e d in this model. F u r t h e r m o r e , S c h i s t o s o m a m a n s o n i i n f e c t i o n does not induce p r o t e i n l o s s e s t h r o u g h the rat gut. The p r e s e n t study was d e s i g n e d to i n v e s t i g a t e the effect of i n t e s t i n a l b i l h a r z i a s i s on serum T B P A as w e l l as on other n u t r i t i o n a l and i n f l a m m a t o r y markers. MATERIAL
AND M E T H O D S
4 - w e e k - o l d male W i s t a r rats w e r e u s e d for the e x p e r i m e n t . All a n i m a l s were h o u s e d in i n d i v i d u a l p o l y p r o p y l e n e cages and m a i n t a i n e d at 22 + 2~ on a 12h l i g h t / 1 2 d a r k cycle. The rats were fed ad l i b i t u m a ~8 % p r o t e i n diet (control diet) and had free a c c e s s to w a t e r t h r o u g h o u t the study. T h e y were r a n d o m l y a s s i g n e d to c o n t r o l and i n f e c t e d groups. I n f e c t i o n was c a r r i e d out by skin p e n e t r a t i o n of 700 S c h i s t o s o m a c e r c a r i a e per animal : the rats w e r e a n e s t h e t i z e d by i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (Clin-Midy, Paris, France) and their abdomens w e r e s h a v e d w i t h a fine e l e c t r i c clipper. A ring was p l a c e d on the a b d o m e n and was p a r t i a l l y f i l l e d w i t h the c e r c a r i a l s u s p e n s i o n . After a 30 minute e x p o s u r e period, the s h a v e d skin was rinsed w i t h a l c o h o l and the rats w e r e p l a c e d b a c k in their cages. F o o d i n t a k e and body w e i g h t w e r e r e c o r d e d e v e r y two days. Blood was c o l l e c t e d by o r b i t a l p u n c t u r e on d a y o and at weekly i n t e r v a l s for 4 weeks. Groups of 8 i n f e c t e d rats and their r e s p e c t i v e c o n t r o l s w e r e s a c r i f i e d on w e e k s 4,6 a n d 12. Blood was c o l l e c t e d from the a b d o m i n a l aorta, the liver was perfused, removed, w e i g h e d and f i x e d in b o u i n m e d i u m for h i s t o p a t h o l o g i c a l examination. The s p e c i f i c immune r e s p o n s e to b i l h a r z i a s i s was i n v e s t i g a t e d by m e a s u r i n g the a n t i - S c h i s t o s o m a m a n s o n i a n t i b o d i e s by i n d i r e c t i m m u n o f l u o r e s c e n c e using frozen s e c t i o n s of S c h i s t o s o m a m a n s o n i adult w o r m s as antigens. Serum TBPA, a l b u m i n a n d e p m a c r o g l o b u l i n c o n c e n t r a t i o n s were m e a s u r e d by e l e c t r 0 i m m u n o a s s a y - w h i l e
TBPA IN INTESTINAL BILHARZIASIS
59
t r a n s f e r r i n and o r o s o m u c o i d c o n c e n t r a t i o n s w e r e d e t e r m i n e d by radial i m m u n o d i f f u s i o n . Rat t r a n s f e r r i n and albumin as well as anti rat a l b u m i n and anti rat t r a n s f e r r i n were s u p p l i e d by C a p p e l L a b o r a t o r i e s (West Chester, U.S.A.). Rat T B P A and a m o n o s p e c i f i c anti rat TBPA a n t i b o d y w e r e o b t a i n e d a c c o r d i n g to a p r o c e d u r e p r e v i o u s l y d e s c r i b e d (15). H e m o g l o b i n c o n c e n t r a t i o n was d e t e r m i n e d by the c y a n m e t h e m o g l o b i n m e t h o d (kit N ~ 525 s u p p l i e d by Sigma C h e m i c a l Company, ST. Louis, U.S.A). D a t a from the i n f e c t e d and the r e s p e c t i v e c o n t r o l groups were s t a t i s t i c a l l y a n a l y s e d by the s t u d e n t ' s t test.
RESULTS
AND D I S C U S S I O N
I n t e s t i n a l b i l h a r z i a s i s i n d u c e d a s p e c i f i c immune response: a n t i - S c h i s t o s o m a mansoni a n t i b o d i e s were d e t e c t e d by the second week f o l l o w i n g i n f e c t i o n and the a n t i b o d y titers reached the highest values by w e e k 6, then d e c l i n e d s l i g h t l y by week 12 (FIG.l)
ANTI-SCHISTOSOMA MANSONI ANTIBODY LEVEL ( l o g 2 o f the dilution)
10.3
9.3
8.3
7.3
6.3
5.3
4.3 lit
t
/
- J
1
2
3
4
5
6
I WEEKS 12
infection
FIG. I. A n t i - S c h i s t o s o m a m a n s o n i a n t i b o d y levels d u r i n g the d e v e l o p m e n t of intestinal b i l h a r z i a s i s .
60
F. BLEIBERG-DANIEL et al.
Host d e f e n s e also i n v o l v e d a d r a m a t i c i n f l a m m a t o r y r e a c t i o n w h i c h r e s u l t e d in n u m e r o u s g r a n u l o m a f o r m a t i o n s around the eggs. Small m a c r o s c o p i c l e s i o n s w e r e o b s e r v e d as s o o n a s w e e k 4 but m a r k e d g r a n u l o m a f o r m a t i o n s only by w e e k 6 (FIG. 2a). T w e l v e w e e k s f o l l o w i n g e x p o s u r e to c e r c a r i a e , g r a n u l o m a size was r e d u c e d as well as c e l l u l a r d e n s i t y a r o u n d t h e eggs (FIG. 2b). This p h e n o m e n o n has a l r e a d y b e e n d e s c r i b e d in d e t a i l in the rat (16). W a r r e n et al (17) e s t a b l i s h e d that g r a n u l o m a f o r m a t i o n was a m a n i f e s t a t i o n of d e l a y e d h y p e r s e n s i t i v i t y .
A
B
FIG.2 egg in liver of rat Representative granuloma around schistosome i n f e c t e d with S c h i s t o s o m a m a n s o n i : A- for 6 w e e k s ; B- for 12 weeks ; H & E stain x25 (x.25)
TBPA IN INTESTINAL BILHARZIASIS
61
D e s p i t e the m a r k e d i n f l a m m a t o r y liver r e a c t i o n o b s e r v e d in the i n f e c t e d rats, the serum levels of two main a c u t e - p h a s e r e a c t a n t proteins, o r o s o m u c o i d and e g - m a c r o g l o b u l i n d i d not exhibit p a t h o l o g i c a l values during th~ d e v e l o p m e n t of b i l h a r z i a s i s . Thus, e l e v a t i o n of acute phase r e a c t a n t p r o t e i n s does not seem to be i n v o l v e d in the natural r e s i s t a n c e o f rat to S c h i s t o s o m a mansoni infection. A l t h o u g h the rats w e r e e x p o s e d to an i m p o r t a n t number of cercariae, little effect has been o b s e r v e d on their n u t r i t i o n a l status. Body weight g a i n and food intake as well as h e m a t o c r i t and h e m o g l o b i n c o n c e n t r a t i o n s were c o m p a r a b l e in the infected animals and in their m a t c h e d c o n t r o l s t h r o u g h o u t the study. In addition, T B P A c o n c e n t r a t i o n was very m o d e r a t e l y i n f l u e n c e d by S c h i s t o s o m a m a n s o n i i n f e c t i o n : serum TBPA levels started to d e c r e a s e s l i g h t l y but s i g n i f i c a n t l y by w e e k 4 (4 % d r o p c o m p a r e d to the c o n t r o l g r o u p ; p < 0.05) and c o n t i n u e d to d e c l i n e m o d e r a t e l y by w e e k 6 (11% d r o p ; p < 0.05). By week 12, the d i f f e r e n c e in T B P A c o n c e n t r a t i o n s b e t w e e n the i n f e c t e d and the control rats r e m a i n e d c o n s t a n t w i t h o u t any s i g n i f i c a n t d i f f e r e n c e (FIG. 3).
TB
PA (mg/Iooml) CONTROL ....
,~t
INFECTED
9 P',~ 0 . 0 5
T
60
50
40
~
'
1
2
3
4
5
6
4U v
12
I-WEEKS
infection
FIG.3 Serum t h y r o x i n e - b i n d i n g p r e a l b u m i n (TBPA) c o n c e n t r a t i o n s i n f e c t e d rats and in their r e s p e c t i v e controls
in the
62
F. BLEIBERG-DANIEL et al.
As s h o w n in T A B L E 1 serum c o n c e n t r a t i o n s of two o t h e r n u t r i t i o n a l markers, a l b u m i n and t r a n s f e r r i n , w e r e r e l a t i v e l y less a f f e c t e d than T B P A d u r i n g the d e v e l o p m e n t of b i l h a r z i a s i s . S e r u m a l b u m i n level d e c r e a s e d b y 6 and 9 ~ by weeks 4 a n d 6, r e s p e c t i v e l y (p < 0.05 versus the c o n t r o l group) w h i l e t r a n s f e r r i n levels only d e c r e a s e d by w e e k 4 (ii ~ drop, p < 0.05).
TABLE 1 S e r u m A l b u m i n and T r a n s f e r r i n C o n c e n t r a t i o n s in the control (C) and i n f e c t e d (I) Rats d u r i n g the D e v e l o p m e n t of B i l h a r z i a s i s .
WEEKS F O L L O W I N G INFECTION
ALBUMIN (g/l)
TRANSFERRIN (g/l)
C 32.1 + 0.6
4.4 + 0.07
I 33.2 + 0.8
4.3 + 1.13
0.6
4.5 + 0.08
I 33.3 + 0.2
4.3 + 0.13
C 32.6 + 0.6
4.5 + 0.05
I 30.8 + 0.4 %
4.0 + 0.05 ~
C 30.8 + 0.8
4.2 + 0.08
0.8
4.3 + 0.08
C 28.3 + 0.6
4.4 + 0.17
0.7
4.1 + 0 . 0 9
0
C
I
32.3
28.1
+
+
12 I
30.0
* D a t a are e x p r e s s e d % p < 0.05 v e r s u s c ~ p < 0.001 v e r s u s c
+
as mean ~ SEM
In the a b s e n c e of w e i g h t loss and r e d u c t i o n in food intake, the m o d e r a t e d e c r e a s e in the n u t r i t i o n a l markers might b e e x p l a i n e d either by liver d a m a g e due to g r a n u l o m a f o r m a t i o n a n d / o r by an e n h a n c e d s y n t h e s i s of other a c u t e - p h a s e r e a c t a n t p r o t e i n s than o r o s o m u c o i d or e 2 - m a c r o g l o b u l i n . However, as both p r o t e i n s count among the major a c u t e - p h a s e r e a c t a n t p r o t e i n s in the rat, the a b s e n c e of i n c r e m e n t in their serum levels s u g g e s t that the a l t e r a t i o n in n u t r i t i o n a l m a r k e r c o n c e n t r a t i o n s , e s p e c i a l l y in TBPA, might be m a i n l y i n t e r p r e t e d as a c o n s e q u e n c e of liver impairment.
TBPA IN INTESTINAL BILHARZIASIS
63
The d a t a s u g g e s t that i n f l a m m a t i o n , b e s i d e s its adverse effect on liver, does not n e c e s s a r i l y a f f e c t T B P A levels when the f o l l o w i n g c o n d i t i o n s are f u l f i l l e d : i- m a i n t e n a n c e of adequate food intake. 2- absence of i n c r e a s e d s y n t h e s i s of a c u t e - p h a s e r e a c t a n t proteins.
References. 1. I n g e n b l e e k Y, Van den S c h r i e c k HG, De Nayer Ph, De V i s s c h e r Albumin, t r a n s f e r r i n and the t h y r o x i n e - b i n d i n g p r e a l b u m i n / r e t i n o l - b i n d i n g p r o t e i n (TBPA-RBP) c o m p l e x in a s s e s s m e n t of m a l n u t r i t i o n . Clin. Chim. Acta 1975; 63:61-67.
M.
2. Shetty PS, W a t r a s i e w i c z KE, Yung RT and James WPT. Rapidt u r n o v e r t r a n s p o r t p r o t e i n s : an index of s u b c l i n i c a l p r o t e i n - e n e r g y m a l n u t r i t i o n . Lancet 1978; 2:230-232.
3. Michel L, S e r r a n o A and Malt RA. N u t r i t i o n a l support h o s p i t a l i z e d patients. New. Engl. J. Med. 1981; 304: 1152.
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4. Smith FR, S u s k i n d R, T h a n a n g k u l O, L e i t z m a n n C, G o o d m a n DS and Olson RE. Plasma v i t a m i n A, r e t i n o l - b i n d i n g protein and p r e a l b u m i n c o n c e n t r a t i o n s in p r o t e i n - c a l o r i e malnutrition. III. R e s p o n s e to v a r y i n g dietary treatments. Am. J. Clin. Nutr. 1975; 29:732-738.
5. Young G, C h e m C, C o l l i n s JP and Hill GL. Plasma proteins in patients r e c e i v i n g i n t r a v e n o u s amino acids or i n t r a v e n o u s h y p e r a l i m n t a t i o n after major surgery. Am. J. Clin. Nutr. 1979; 32:1192-1199.
6. Bourry J, M i l a n o G, C a l d a n i C and S c h n e i d e r M. Assessment of n u t r i t i o n a l p r o t e i n s d u r i n g the p a r e n t a l n u t r i t i o n of cancer patients. Ann. Clin. Lab. Sci. 1982; 12:158-162.
7. K e l l e h e r PC, P h i n n e y SD, Sims EAH, B o g a r d u s C, Horton ES, B i s t r i a n BR, A m a t r u d a JM and L o c k w o o d DH. Effects of c a r b o h y d r a t e - c o n t a i n i n g and c a r b o h y d r a t e - r e s t r i c t e d h y p o c a l o r i c and e u c a l o r i c diets on serum c o n c e n t r a t i o n s of r e t i n o l - b i n d i n g protein, t h y r o x i n e - b i n d i n g p r e a l b u m i n and transferrin. M e t a b o l i s m 1983; 32:95-101.
8. Bates J and M c C l a i n CJ. The effect of severe zinc d e f i c i e n c y on serum levels of albumin, transferrin, and p r e a l b u m i n in man. Am. J. Clin. Nutr. 1981; 3 4 : 1 6 5 5 - 1 6 6 0 .
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Accepted for publication July 12, 1987