1234
Specialia
of 5*Fe in b o n e falls b e l o w t h e COlitrol value b u t s h o w s no a d d i t i o n a l r e d u c t i o n w i t h increasing e x p o s u r e 4. I t is a s s u m e d t h a t t h i s r e p r e s e n t s ~~ t a k e n u p in s t o r a g e or o t h e r r a d i o r e s i s t a n t sites, a n d t h e s e u p t a k e s will be referred to as 'baseline ~ u p t a k e s . Therefore, t h e n e t u p t a k e will be t h e t o t a l u p t a k e m i n u s t h e baseline u p t a k e . The results will be p r e s e n t e d as t w o factions : t h e ratio of t h e t o t a l u p t a k e s of i r r a d i a t e d to c o n t r o l a n d t h e s a m e r a t i o of t h e n e t u p t a k e s . Results. R a t b o n e s m a y be d i v i d e d into 2 classes; t h o s e w h i c h s h o w a r e d u c t i o n in SSFe u p t a k e following e x p o s u r e a n d t h o s e i n s e n s i t i v e b o n e s w h i c h s h o w no r e d u c t i o n following i r r a d i a t i o n 4. All of t h e sensitive b o n e s will be p r e s e n t e d here. The c o n t r o l iron u p t a k e s a n d t h e baseline u p t a k e s for t h e s e s a m p l e s are p r e s e n t e d in t h e t a b l e in t e r m s of p e r c e n t a g e s of t h e i n j e c t e d 59Fe. A p l o t of t h e f r a c t i o n of t o t a l c o n t r o l u p t a k e w i t h t i m e p o s t - e x p o s u r e s is p r e s e n t e d in figure A. I t m a y be n o t e d t h a t a m o n g t h e b o n e s considered, t h e f e m u r a n d s t e r n u m s h o w g r e a t e r r e d u c t i o n s in u p t a k e t h a n t h e s c a p u l a or cervical spinal s e g m e n t s . This is similar t o t h e results r e p o r t e d b y Hol~2. H o w e v e r , w h e n t h e r a t i o s of t h e n e t u p t a k e s are p l o t t e d , as in figure B, t h e s e differences in r e s p o n s e of t h e m i n i m a are r e d u c e d .
Immunostimulation
by a formula-defined
EXPERIENTIA 33/9
Also, it m a y be n o t e d t h a t t h e m i n i m u m in u p t a k e f o r e a c h b o n e occurs b e t w e e n 16 a n d 24 h p o s t - e x p o s u r e . This t y p e of r e s p o n s e was n o t e d for all r a d i o s e n s i t i v e b o n e s even w i t h differing dose r a t e s (8.2 R / m i n . a n d 116 t~/min.). W h o l e - b o d y e x p o s u r e s of 100 R a n d 300 R g a v e similar results. T h e o n l y n o t a b l e d i f f e r e n c e was a m o r e p r o n o u n c e d m i n i m u m a t 24 h p o s t - i r r a d i a t i o n , a n d a d e l a y e d r e t u r n t o w a r d n o r m a l u p t a k e values. Discussion. The u p t a k e of ~SFe in t h e f e m u r or t i b i a m a y be c o n s i d e r e d as being r e p r e s e n t a t i v e of t h e e r y t h r o poietic a c t i v i t y of t h e t o t a l s k e l e t o n in i r r a d i a t e d r a t s as long as t h e baseline a c t i v i t y h a s b e e n s u b t r a c t e d . T h e m i n i m a in u p t a k e in t h i s s t u d y occur earlier t h a n t h o s e r e p o r t e d b y Hol~ et al. H o w e v e r , o t h e r s h a v e r e p o r t e d m a x i m u m r e s p o n s e s a t 24 h p o s t i r r a d i a t i o n 5,s. This difference m a y be due to t h e m o d e of injection. Hol~ used an i n t r a p e r i t o n e a l i n j e c t i o n r o u t e while a n i n t r a v e n o u s r o u t e was used in t h i s s t u d y . H.D. Maillie and H. Mermagen, Health Physics 20, 179 (1971). W.A. Rambach, J. A. D. Cooper, H. L. Alt, H. H. Vogel, J. W. Clark and D. L. Jordan, Radiat. Res. 10, 148 (1959). 6 E.H. Belcher, I. G. F. Gilbert and L. F. Lamerton, Brit. J. Radiol. 27, 387 (1954). 4 5
diet I
R. P a g e a u , G. B o u n o u s a n d R. Lallier
Department of Nuclear Medicine and Radiobiology, Department o/ Surgery, University o[ Sherbrooke Medical Center, Sherbrooke (Quebec, Canada), and Department o/ Pathology and Microbiology, School o[ Veterinary Medicine o[ the University o[ Montreal, St-Hyacinthe (Quebec, Canada), 2 March 1977 Summary. A f o r m u l a - d e f i n e d d i e t (diet 3, t a b l e 1) a c t e d as a n a d j u v a n t in t h e r e s p o n s e of t h e i m m u n e s y s t e m to S R B C in t h e rat. Similar m i n i m u m a n d m a x i m u m a n t i b o d y levels were m e a s u r e d in b o t h males a n d females. I n t h e males, t h e m a x i m u m was r e a c h e d w i t h d i e t 3 alone while females r e q u i r e d t h e c o m p l e m e n t a r y a c t i o n of d i e t 3 a n d L P S m R 595. A s e x - d e p e n d e n t e n h a n c e m e n t of t h e u p t a k e of t r i t i a t e d t h y m i d i n e (aH-TdR) b y h e m a t o p o i e t i c tissues h a s been r e p o r t e d w h e n r a t s were fed c e r t a i n f o r m u l a - d e f i n e d diets ~. T h o s e r e s u l t s s u g g e s t e d t h a t such f o r m u l a - d e f i n e d d i e t s could also a f f e c t t h e r e s p o n s e of t h e i m m u n e s y s t e m in a s e x - r e l a t e d m a n n e r . W e n o w r e p o r t t h e effect of e l e m e n t a l d i e t 3 3 on t h e r e s p o n s e of t h e r a t i m m u n e s y s t e m to s h e e p e r y t h r o c y t e s (SRBC). T h e c o m p o s i t i o n of t h e diet h a s a l r e a d y b e e n d e s c r i b e d elsewhere *. A n t i - S R B C a n t i b o d y levels were m e a s u r e d in t h e s e r u m of m a l e a n d virgin female S p r a g u e -
Table 1. Detailed composition of diet 3
D a w l e y r a t s 6-7 weeks old, fed e i t h e r t h e e l e m e n t a l d i e t or P u r i n a l a b o r a t o r y chow. I n a d d i t i o n , we i n v e s t i g a t e d t o w h a t e x t e n t d i e t a n d sex m o d i f y t h e a d j u v a n t effect of l i p o p o l y s a c c h a r i d e (LPS) m R 5955. R a n d o m i z e d g r o u p s of 6 a n i m a l s w e r e fed a s p r e v i o u s l y described 4. S R B C s were r e s u s p e n d e d a t a 1:10 dilution in p h o s p h a t e b u f f e r e d saline (PBS) a n d i n j e c t e d i.p. (1 ml, a p p r o x i m a t e l y l0 s cells). L P S w a s p r e p a r e d in P B S a n d 40 ~g i n j e c t e d ip s i m u l t a n e o u s l y to SRBC. A n t i - S R B C a n t i b o d y c o n t e n t of s e r u m was d e t e r m i n e d b y passive h e m a g g l u t i n a t i o n 7 d a y s later. R e s u l t s are p r e s e n t e d in t a b l e 2. I n c o n t r o l s fed l a b o r a t o r y chow, b o t h m a l e s a n d females h a d c o m p a r a b l e a n t i - S R B C a n t i b o d y levels. R e m a r k a b l y h i g h e r levels (by a factor of 23 on t h e a v e r a g e in t h e m a l e s a n d 7 in t h e females) were f o u n d in a n i m a l s fed d i e t 3. B y c o n t r a s t , L P S was f o u n d
Grams per 100 g 1 Casein hydrolysate Sucrose Corn syrup solids (glucose oligosaccharides ) Long-chain triglycerides Medium-chain triglycerides Tapioca starch Minerals Free amino acids Choline chloride Water Sodium citrate hydrous Potassium chloride (from casein)
12.560 46.839 16.812 11.982 2.997 5.234 2.611 0.384 0.028 0.553 0.656 0.248
2 3 4 5 6
Acknowledgments. This work was made possible in part through a grant from the Medical Research Council of Canada. The authors are grateful to Mrs S. Baehand for her technical assistance and to Dr Gordon J. Fisher for his advice offered in the preparation of the manuscript. Mr P. Charland took care of the animals. R. Pageau, Radiation Res. Soc. Meeting, 27 June to 2 July 1976, San Francisco, Calif., USA (abstract). Mead Johnson Canada Limited. R. Pageau and G. Bounous, Radiat. Res. 66, 267 (1976). Lipopolysacearide of Salmonella minnesota mR 595, graciously provided by Dr O. Lfideritz, Max-Planck-Institut ffir Immunobiologie, Freiburg, Federal Republic of Germany. Institut Armand Frappier, Laval des Rapides, Quebec, Canada.
15. 9. 1977
Specialia
t o i n c r e a s e t h e a n t i b o d y c o n t e n t only b y a f a c t o r of 2 in t h e m a l e s fed l a b o r a t o r y c h o w a n d a p p r o x i m a t e l y 5 in t h e females. This i n d i c a t e s t h a t t h e a d j u v a n t effect of diet 3 o n t h e r e s p o n s e of t h e i m m u n e s y s t e m to S R B C is cons i d e r a b l y g r e a t e r in t h e m a l e s b u t n o t t h e females t h a n t h a t o b t a i n e d w i t h L P S . W h i l e t h e c o m b i n a t i o n of diet 3 a n d L P S did n o t s i g n i f i c a n t l y a f f e c t a n t i b o d y levels in t h e males, i t h a d a n a d j u v a n t effect (by a f a c t o r of 4) in t h e females. This s h o w s t h a t a c o m p l e m e n t a r y action of d i e t 3 a n d L P S is f o u n d in t h e f e m a l e s only. N o t w i t h s t a n d i n g t h e s e s e x - r e l a t e d differences, b o t h m a l e s a n d females
Table 2. Effects of diet 3, sex and LPS mR 595 on the response of the immune system to SRBC ill the rat Anti-SRBC antibody levels determined in serum by passive hemag.glutination Sex Animals fed laboratory chow Animals fed diet 3 SRBC 9 SRBC-LPSb SRBC SRBC-LPS
Males
3.50 -4- 0.85 c 11a (6-20) e
4.67 • 0.61 25 (17-39)
8.00 -t- 0.32 256 (205-320)
7.00 ~- 0.45 128 (94-175)
Females
3.61 -t- 0.51 12 (9-17)
5.83 ~- 0.38 57 (44-74)
6.45 4- 0.37 87 (68-113)
8.50 4- 0.22 362 (311-422)
9SRBC: sheep erythrocytes (1 ml, 108 cells) injected i.p. bLPS: 40~tg injected i.p. simultaneously to SRBC. cMean number of wells (x) 4- SE, 2 groups of 6 animals, aTitre as obtained by 2x. eTitre range as obtained from SE of x.
/ Persistance of ~ a t e r n ~ l l y derived~__~stradiol
1235
s h o w e d similar m i n i m u m a n d m a x i m u m a n t i b o d y levels. These f i n d i n g s m a y be i n t e r p r e t e d in t e r m s of a sexr e l a t e d s t i m u l a t i o n of t h e h e m a t o p o i e t i c s y s t e m b y d i e t 3. I n t h e males, a g r e a t e r u p t a k e of 3 H - T d R b y t h e h e m a t o poietic tissues h a s a l r e a d y b e e n s h o w n w h e n a n i m a l s were fed t h e e l e m e n t a l d i e t i n s t e a d of l a b o r a t o r y c h o w 4, 7. I n t h e females, o n t h e o t h e r h a n d , a s i g n i f i c a n t l y lower upt a k e of 3 H - T d R b y t h e spleen a n d t h e l y m p h n o d e s was o b t a i n e d u n d e r t h e s a m e n u t r i t i o n a l c o n d i t i o n s 2. I t app e a r s t h e n t h a t d i e t 3 affects t h e cellular s y s t e m s r e s p o n sible for t h e i m m u n e r e s p o n s e t o a d i f f e r e n t e x t e n t in t h e males a n d t h e females. S u c h a d i e t a r y effect w o u l d also a c c o u n t for t h e s e x - r e l a t e d differences o b s e r v e d in t h e a d j u v a n t a c t i o n of L P S . A n a l t e r n a t i v e i n t e r p r e t a t i o n t h a t a n t i b o d y p r o d u c t i o n or t u r n o v e r is m o d i f i e d b y t h e e l e m e n t a l diet is p r e s e n t l y u n d e r i n v e s t i g a t i o n . The influence of f o r m u l a - d e f i n e d diets on t h e r e s p o n s e of t h e i m m u n e s y s t e m is likely to h a v e clinical i m p o r t a n c e . S u c h d i e t s are a l r e a d y fed t u m o u r - b e a r i n g p a t i e n t s to assure p r o p e r n u t r i t i o n a l a n d s y s t e m i c b a l a n c e d u r i n g c a n c e r o t h e r a p y s-l~ Of p a r t i c u l a r i n t e r e s t is our finding t h a t d i e t 3 affects a n t i - S R B C a n t i b o d y p r o d u c t i o n to a similar a n d e v e n g r e a t e r e x t e n t t h a n L P S . L i p o p o l y s a c c h a r i d e s h a v e b e e n a n d are still i n v e s t i g a t e d as promising a d j u v a n t s in t h e t r e a t m e n t of c a n c e r n.
7 8
R. Pageau, R. Lallier and G. Bounous, Radiat. Res. 62, 357 (1975). G. Bounous, j . Gentile and J. Hugon, Can. J. Surg. 14, 312 (1971). 9 G. Bounous, E. LeBel, J. Shuster, P. Gold and W. T. Tahan, Strahlentherapie 149, 476 (1975). 10 J . V . Rivard and R. Lapointe, Can. J. Surg. 18, 90 (1975). 11 V.N. Nigam, Cancer Res, 35, 628 (1975).
in ~__~al and n ~ o n a t a l ~ s
~
J. P. F r i e n d
Department of Anatomy, Health Sciences Center, School of Medicine, University of Louisville, Louisville (Kentucky 40201, USA), 7 February 1977 Summary. I n j e c t i o n of 3 H - e s t r a d i o l i n t o p r e g n a n t r a t s r e s u l t e d in fetal b l o o d r a d i o a c t i v i t y 5 t i m e s h i g h e r t h a n in m a t e r n a l blood. Significant a m o u n t s of ~ H - e s t r a d i o l were f o u n d in fetal b l o o d 24 h l a t e r a n d in t h e offspring 5 d a y s a f t e r birth. D i s t r i b u t i o n of 3H-estradiol in fetal a n d n e o n a t a l r a t tissues h a s b e e n s t u d i e d e x t e n s i v e l y 2-5, b u t no r e p o r t on t h e long t e r m r e t e n t i o n of t h i s s t e r o i d in t h e blood a p p e a r s in t h e l i t e t a t u r e . F e t a l a n d n e o n a t a l r a t p l a s m a c o n t a i n s a n e s t r a d i o l b i n d i n g p l a s m a p r o t e i n (EBP) w h i c h h a s a c a p a c i t y for b i n d i n g large q u a n t i t i e s o f e s t r a d i o l ~ I t s f u n c t i o n a l significance is n o t well u n d e r stood. H i g h c o n c e n t r a t i o n s are f o u n d in fetal rats, w i t h a rising t i t e r as t h e y a p p r o a c h t e r m . E B P d i s p l a y s a half-life in p o s t n a t a l blood of a b o u t 4 d a y s a n d d i s a p p e a r s b y t h e 29th day. E B P is f o u n d in low c o n c e n t r a t i o n s in p r e g n a n t rats, b u t is a b s e n t in n o n p r e g n a n t a d u l t s L R e m o v a l of 3H-estradiol f r o m t h e blood of n o n p r e g n a n t a d u l t s is fairly r a p i d 8, b u t b e c a u s e of t h e p r e s e n c e of E B P one m i g h t s u s p e c t t h e r e t e n t i o n of t h i s steroid in t h e blood of f e t a l a n d n e o n a t a l r a t s could b e longer. D d h l e r a n d W u t t k e f o u n d s e r u m e s t r a d i o l was g r e a t e r in 1- a n d 2-day-old n e o n a t e s t h a n in a n y o t h e r age group, i n c l u d i n g a d u l t s ~ E s t r a d i o l values declined slowly for several days, a n d t h e n rose t o m o d e r a t e l y high levels b y d a y 10. The a u t h o r s were u n a b l e to e x p l a i n t h i s p a t t e r n . T h e declining estradiol levels parallel a k n o w n d r o p in c i r c u l a t i n g E B P a n d m a y be c a s u a l l y r e l a t e d to it. The e s t r o g e n in t h e offspring could result f r o m p l a c e n t a l
t r a n s f e r of m a t e r n a l e s t r o g e n p r i o r t o p a r t u r i t i o n . The p r e s e n t s t u d y r e p o r t s t h e long t e r m r e t e n t i o n of 3He s t r a d i o l in t h e blood of fetal a n d n e o n a t a l r a t s following i n j e c t i o n of t h i s steroid i n t o t h e m o t h e r . Materials and methods. S p r a g u e - D a w l e y rats, 20 d a y s p r e g n a n t , r e c e i v e d i.v. 0.5 txCi ~H-estradiol/g of b . w t . The e s t r a d i o l (91.3 Ci/mM, New E n g l a n d Nuclear) was c h e c k e d for p u r i t y b y TLC prior to use. I n j e c t e d animals were d e c a p i t a t e d a n d t h e i r fetuses r e m o v e d a n d k e p t cold u n t i l b l o o d s a m p l e s could be t a k e n . A d u l t a n d fetal blood 1 2 3 4 5
Supported by NIH Grant HD 08844. B. Vannier and J. P. Raynaud, Molee. Cell Endocr. 3, 323 (1975). T. Nakal, S. Sakamoto, T. Kigawa and A. Shigematsu, Endocr. jap. 19, 47 (1972). J. Presl, S. R~hhling,J. Horsk~ and J. Herznlann, Endocrinology 86, 899 (1970). E . O . Alvarez and V. D. Ramirez, Neuroendoerinology 6, 349
(197o). 6 7 8 9
J . P . Raynaud, Steroids 21,249 (1973). E. Nunez, L. Savu, F. Engelmann, C. Benassayag, O. Crepy and M. Jayle, C. r. Acad. Sei., Paris 273, 242 (1971). J . L . MeGuire and R. D. Lisk, Proe. nat. Acad. Sci. USA 67, 497 (1968). K.D. D~ihler and W. Wuttke, Endoerinoldgy 97, 898 (1975).
Specialia
of 5*Fe in b o n e falls b e l o w t h e COlitrol value b u t s h o w s no a d d i t i o n a l r e d u c t i o n w i t h increasing e x p o s u r e 4. I t is a s s u m e d t h a t t h i s r e p r e s e n t s ~~ t a k e n u p in s t o r a g e or o t h e r r a d i o r e s i s t a n t sites, a n d t h e s e u p t a k e s will be referred to as 'baseline ~ u p t a k e s . Therefore, t h e n e t u p t a k e will be t h e t o t a l u p t a k e m i n u s t h e baseline u p t a k e . The results will be p r e s e n t e d as t w o factions : t h e ratio of t h e t o t a l u p t a k e s of i r r a d i a t e d to c o n t r o l a n d t h e s a m e r a t i o of t h e n e t u p t a k e s . Results. R a t b o n e s m a y be d i v i d e d into 2 classes; t h o s e w h i c h s h o w a r e d u c t i o n in SSFe u p t a k e following e x p o s u r e a n d t h o s e i n s e n s i t i v e b o n e s w h i c h s h o w no r e d u c t i o n following i r r a d i a t i o n 4. All of t h e sensitive b o n e s will be p r e s e n t e d here. The c o n t r o l iron u p t a k e s a n d t h e baseline u p t a k e s for t h e s e s a m p l e s are p r e s e n t e d in t h e t a b l e in t e r m s of p e r c e n t a g e s of t h e i n j e c t e d 59Fe. A p l o t of t h e f r a c t i o n of t o t a l c o n t r o l u p t a k e w i t h t i m e p o s t - e x p o s u r e s is p r e s e n t e d in figure A. I t m a y be n o t e d t h a t a m o n g t h e b o n e s considered, t h e f e m u r a n d s t e r n u m s h o w g r e a t e r r e d u c t i o n s in u p t a k e t h a n t h e s c a p u l a or cervical spinal s e g m e n t s . This is similar t o t h e results r e p o r t e d b y Hol~2. H o w e v e r , w h e n t h e r a t i o s of t h e n e t u p t a k e s are p l o t t e d , as in figure B, t h e s e differences in r e s p o n s e of t h e m i n i m a are r e d u c e d .
Immunostimulation
by a formula-defined
EXPERIENTIA 33/9
Also, it m a y be n o t e d t h a t t h e m i n i m u m in u p t a k e f o r e a c h b o n e occurs b e t w e e n 16 a n d 24 h p o s t - e x p o s u r e . This t y p e of r e s p o n s e was n o t e d for all r a d i o s e n s i t i v e b o n e s even w i t h differing dose r a t e s (8.2 R / m i n . a n d 116 t~/min.). W h o l e - b o d y e x p o s u r e s of 100 R a n d 300 R g a v e similar results. T h e o n l y n o t a b l e d i f f e r e n c e was a m o r e p r o n o u n c e d m i n i m u m a t 24 h p o s t - i r r a d i a t i o n , a n d a d e l a y e d r e t u r n t o w a r d n o r m a l u p t a k e values. Discussion. The u p t a k e of ~SFe in t h e f e m u r or t i b i a m a y be c o n s i d e r e d as being r e p r e s e n t a t i v e of t h e e r y t h r o poietic a c t i v i t y of t h e t o t a l s k e l e t o n in i r r a d i a t e d r a t s as long as t h e baseline a c t i v i t y h a s b e e n s u b t r a c t e d . T h e m i n i m a in u p t a k e in t h i s s t u d y occur earlier t h a n t h o s e r e p o r t e d b y Hol~ et al. H o w e v e r , o t h e r s h a v e r e p o r t e d m a x i m u m r e s p o n s e s a t 24 h p o s t i r r a d i a t i o n 5,s. This difference m a y be due to t h e m o d e of injection. Hol~ used an i n t r a p e r i t o n e a l i n j e c t i o n r o u t e while a n i n t r a v e n o u s r o u t e was used in t h i s s t u d y . H.D. Maillie and H. Mermagen, Health Physics 20, 179 (1971). W.A. Rambach, J. A. D. Cooper, H. L. Alt, H. H. Vogel, J. W. Clark and D. L. Jordan, Radiat. Res. 10, 148 (1959). 6 E.H. Belcher, I. G. F. Gilbert and L. F. Lamerton, Brit. J. Radiol. 27, 387 (1954). 4 5
diet I
R. P a g e a u , G. B o u n o u s a n d R. Lallier
Department of Nuclear Medicine and Radiobiology, Department o/ Surgery, University o[ Sherbrooke Medical Center, Sherbrooke (Quebec, Canada), and Department o/ Pathology and Microbiology, School o[ Veterinary Medicine o[ the University o[ Montreal, St-Hyacinthe (Quebec, Canada), 2 March 1977 Summary. A f o r m u l a - d e f i n e d d i e t (diet 3, t a b l e 1) a c t e d as a n a d j u v a n t in t h e r e s p o n s e of t h e i m m u n e s y s t e m to S R B C in t h e rat. Similar m i n i m u m a n d m a x i m u m a n t i b o d y levels were m e a s u r e d in b o t h males a n d females. I n t h e males, t h e m a x i m u m was r e a c h e d w i t h d i e t 3 alone while females r e q u i r e d t h e c o m p l e m e n t a r y a c t i o n of d i e t 3 a n d L P S m R 595. A s e x - d e p e n d e n t e n h a n c e m e n t of t h e u p t a k e of t r i t i a t e d t h y m i d i n e (aH-TdR) b y h e m a t o p o i e t i c tissues h a s been r e p o r t e d w h e n r a t s were fed c e r t a i n f o r m u l a - d e f i n e d diets ~. T h o s e r e s u l t s s u g g e s t e d t h a t such f o r m u l a - d e f i n e d d i e t s could also a f f e c t t h e r e s p o n s e of t h e i m m u n e s y s t e m in a s e x - r e l a t e d m a n n e r . W e n o w r e p o r t t h e effect of e l e m e n t a l d i e t 3 3 on t h e r e s p o n s e of t h e r a t i m m u n e s y s t e m to s h e e p e r y t h r o c y t e s (SRBC). T h e c o m p o s i t i o n of t h e diet h a s a l r e a d y b e e n d e s c r i b e d elsewhere *. A n t i - S R B C a n t i b o d y levels were m e a s u r e d in t h e s e r u m of m a l e a n d virgin female S p r a g u e -
Table 1. Detailed composition of diet 3
D a w l e y r a t s 6-7 weeks old, fed e i t h e r t h e e l e m e n t a l d i e t or P u r i n a l a b o r a t o r y chow. I n a d d i t i o n , we i n v e s t i g a t e d t o w h a t e x t e n t d i e t a n d sex m o d i f y t h e a d j u v a n t effect of l i p o p o l y s a c c h a r i d e (LPS) m R 5955. R a n d o m i z e d g r o u p s of 6 a n i m a l s w e r e fed a s p r e v i o u s l y described 4. S R B C s were r e s u s p e n d e d a t a 1:10 dilution in p h o s p h a t e b u f f e r e d saline (PBS) a n d i n j e c t e d i.p. (1 ml, a p p r o x i m a t e l y l0 s cells). L P S w a s p r e p a r e d in P B S a n d 40 ~g i n j e c t e d ip s i m u l t a n e o u s l y to SRBC. A n t i - S R B C a n t i b o d y c o n t e n t of s e r u m was d e t e r m i n e d b y passive h e m a g g l u t i n a t i o n 7 d a y s later. R e s u l t s are p r e s e n t e d in t a b l e 2. I n c o n t r o l s fed l a b o r a t o r y chow, b o t h m a l e s a n d females h a d c o m p a r a b l e a n t i - S R B C a n t i b o d y levels. R e m a r k a b l y h i g h e r levels (by a factor of 23 on t h e a v e r a g e in t h e m a l e s a n d 7 in t h e females) were f o u n d in a n i m a l s fed d i e t 3. B y c o n t r a s t , L P S was f o u n d
Grams per 100 g 1 Casein hydrolysate Sucrose Corn syrup solids (glucose oligosaccharides ) Long-chain triglycerides Medium-chain triglycerides Tapioca starch Minerals Free amino acids Choline chloride Water Sodium citrate hydrous Potassium chloride (from casein)
12.560 46.839 16.812 11.982 2.997 5.234 2.611 0.384 0.028 0.553 0.656 0.248
2 3 4 5 6
Acknowledgments. This work was made possible in part through a grant from the Medical Research Council of Canada. The authors are grateful to Mrs S. Baehand for her technical assistance and to Dr Gordon J. Fisher for his advice offered in the preparation of the manuscript. Mr P. Charland took care of the animals. R. Pageau, Radiation Res. Soc. Meeting, 27 June to 2 July 1976, San Francisco, Calif., USA (abstract). Mead Johnson Canada Limited. R. Pageau and G. Bounous, Radiat. Res. 66, 267 (1976). Lipopolysacearide of Salmonella minnesota mR 595, graciously provided by Dr O. Lfideritz, Max-Planck-Institut ffir Immunobiologie, Freiburg, Federal Republic of Germany. Institut Armand Frappier, Laval des Rapides, Quebec, Canada.
15. 9. 1977
Specialia
t o i n c r e a s e t h e a n t i b o d y c o n t e n t only b y a f a c t o r of 2 in t h e m a l e s fed l a b o r a t o r y c h o w a n d a p p r o x i m a t e l y 5 in t h e females. This i n d i c a t e s t h a t t h e a d j u v a n t effect of diet 3 o n t h e r e s p o n s e of t h e i m m u n e s y s t e m to S R B C is cons i d e r a b l y g r e a t e r in t h e m a l e s b u t n o t t h e females t h a n t h a t o b t a i n e d w i t h L P S . W h i l e t h e c o m b i n a t i o n of diet 3 a n d L P S did n o t s i g n i f i c a n t l y a f f e c t a n t i b o d y levels in t h e males, i t h a d a n a d j u v a n t effect (by a f a c t o r of 4) in t h e females. This s h o w s t h a t a c o m p l e m e n t a r y action of d i e t 3 a n d L P S is f o u n d in t h e f e m a l e s only. N o t w i t h s t a n d i n g t h e s e s e x - r e l a t e d differences, b o t h m a l e s a n d females
Table 2. Effects of diet 3, sex and LPS mR 595 on the response of the immune system to SRBC ill the rat Anti-SRBC antibody levels determined in serum by passive hemag.glutination Sex Animals fed laboratory chow Animals fed diet 3 SRBC 9 SRBC-LPSb SRBC SRBC-LPS
Males
3.50 -4- 0.85 c 11a (6-20) e
4.67 • 0.61 25 (17-39)
8.00 -t- 0.32 256 (205-320)
7.00 ~- 0.45 128 (94-175)
Females
3.61 -t- 0.51 12 (9-17)
5.83 ~- 0.38 57 (44-74)
6.45 4- 0.37 87 (68-113)
8.50 4- 0.22 362 (311-422)
9SRBC: sheep erythrocytes (1 ml, 108 cells) injected i.p. bLPS: 40~tg injected i.p. simultaneously to SRBC. cMean number of wells (x) 4- SE, 2 groups of 6 animals, aTitre as obtained by 2x. eTitre range as obtained from SE of x.
/ Persistance of ~ a t e r n ~ l l y derived~__~stradiol
1235
s h o w e d similar m i n i m u m a n d m a x i m u m a n t i b o d y levels. These f i n d i n g s m a y be i n t e r p r e t e d in t e r m s of a sexr e l a t e d s t i m u l a t i o n of t h e h e m a t o p o i e t i c s y s t e m b y d i e t 3. I n t h e males, a g r e a t e r u p t a k e of 3 H - T d R b y t h e h e m a t o poietic tissues h a s a l r e a d y b e e n s h o w n w h e n a n i m a l s were fed t h e e l e m e n t a l d i e t i n s t e a d of l a b o r a t o r y c h o w 4, 7. I n t h e females, o n t h e o t h e r h a n d , a s i g n i f i c a n t l y lower upt a k e of 3 H - T d R b y t h e spleen a n d t h e l y m p h n o d e s was o b t a i n e d u n d e r t h e s a m e n u t r i t i o n a l c o n d i t i o n s 2. I t app e a r s t h e n t h a t d i e t 3 affects t h e cellular s y s t e m s r e s p o n sible for t h e i m m u n e r e s p o n s e t o a d i f f e r e n t e x t e n t in t h e males a n d t h e females. S u c h a d i e t a r y effect w o u l d also a c c o u n t for t h e s e x - r e l a t e d differences o b s e r v e d in t h e a d j u v a n t a c t i o n of L P S . A n a l t e r n a t i v e i n t e r p r e t a t i o n t h a t a n t i b o d y p r o d u c t i o n or t u r n o v e r is m o d i f i e d b y t h e e l e m e n t a l diet is p r e s e n t l y u n d e r i n v e s t i g a t i o n . The influence of f o r m u l a - d e f i n e d diets on t h e r e s p o n s e of t h e i m m u n e s y s t e m is likely to h a v e clinical i m p o r t a n c e . S u c h d i e t s are a l r e a d y fed t u m o u r - b e a r i n g p a t i e n t s to assure p r o p e r n u t r i t i o n a l a n d s y s t e m i c b a l a n c e d u r i n g c a n c e r o t h e r a p y s-l~ Of p a r t i c u l a r i n t e r e s t is our finding t h a t d i e t 3 affects a n t i - S R B C a n t i b o d y p r o d u c t i o n to a similar a n d e v e n g r e a t e r e x t e n t t h a n L P S . L i p o p o l y s a c c h a r i d e s h a v e b e e n a n d are still i n v e s t i g a t e d as promising a d j u v a n t s in t h e t r e a t m e n t of c a n c e r n.
7 8
R. Pageau, R. Lallier and G. Bounous, Radiat. Res. 62, 357 (1975). G. Bounous, j . Gentile and J. Hugon, Can. J. Surg. 14, 312 (1971). 9 G. Bounous, E. LeBel, J. Shuster, P. Gold and W. T. Tahan, Strahlentherapie 149, 476 (1975). 10 J . V . Rivard and R. Lapointe, Can. J. Surg. 18, 90 (1975). 11 V.N. Nigam, Cancer Res, 35, 628 (1975).
in ~__~al and n ~ o n a t a l ~ s
~
J. P. F r i e n d
Department of Anatomy, Health Sciences Center, School of Medicine, University of Louisville, Louisville (Kentucky 40201, USA), 7 February 1977 Summary. I n j e c t i o n of 3 H - e s t r a d i o l i n t o p r e g n a n t r a t s r e s u l t e d in fetal b l o o d r a d i o a c t i v i t y 5 t i m e s h i g h e r t h a n in m a t e r n a l blood. Significant a m o u n t s of ~ H - e s t r a d i o l were f o u n d in fetal b l o o d 24 h l a t e r a n d in t h e offspring 5 d a y s a f t e r birth. D i s t r i b u t i o n of 3H-estradiol in fetal a n d n e o n a t a l r a t tissues h a s b e e n s t u d i e d e x t e n s i v e l y 2-5, b u t no r e p o r t on t h e long t e r m r e t e n t i o n of t h i s s t e r o i d in t h e blood a p p e a r s in t h e l i t e t a t u r e . F e t a l a n d n e o n a t a l r a t p l a s m a c o n t a i n s a n e s t r a d i o l b i n d i n g p l a s m a p r o t e i n (EBP) w h i c h h a s a c a p a c i t y for b i n d i n g large q u a n t i t i e s o f e s t r a d i o l ~ I t s f u n c t i o n a l significance is n o t well u n d e r stood. H i g h c o n c e n t r a t i o n s are f o u n d in fetal rats, w i t h a rising t i t e r as t h e y a p p r o a c h t e r m . E B P d i s p l a y s a half-life in p o s t n a t a l blood of a b o u t 4 d a y s a n d d i s a p p e a r s b y t h e 29th day. E B P is f o u n d in low c o n c e n t r a t i o n s in p r e g n a n t rats, b u t is a b s e n t in n o n p r e g n a n t a d u l t s L R e m o v a l of 3H-estradiol f r o m t h e blood of n o n p r e g n a n t a d u l t s is fairly r a p i d 8, b u t b e c a u s e of t h e p r e s e n c e of E B P one m i g h t s u s p e c t t h e r e t e n t i o n of t h i s steroid in t h e blood of f e t a l a n d n e o n a t a l r a t s could b e longer. D d h l e r a n d W u t t k e f o u n d s e r u m e s t r a d i o l was g r e a t e r in 1- a n d 2-day-old n e o n a t e s t h a n in a n y o t h e r age group, i n c l u d i n g a d u l t s ~ E s t r a d i o l values declined slowly for several days, a n d t h e n rose t o m o d e r a t e l y high levels b y d a y 10. The a u t h o r s were u n a b l e to e x p l a i n t h i s p a t t e r n . T h e declining estradiol levels parallel a k n o w n d r o p in c i r c u l a t i n g E B P a n d m a y be c a s u a l l y r e l a t e d to it. The e s t r o g e n in t h e offspring could result f r o m p l a c e n t a l
t r a n s f e r of m a t e r n a l e s t r o g e n p r i o r t o p a r t u r i t i o n . The p r e s e n t s t u d y r e p o r t s t h e long t e r m r e t e n t i o n of 3He s t r a d i o l in t h e blood of fetal a n d n e o n a t a l r a t s following i n j e c t i o n of t h i s steroid i n t o t h e m o t h e r . Materials and methods. S p r a g u e - D a w l e y rats, 20 d a y s p r e g n a n t , r e c e i v e d i.v. 0.5 txCi ~H-estradiol/g of b . w t . The e s t r a d i o l (91.3 Ci/mM, New E n g l a n d Nuclear) was c h e c k e d for p u r i t y b y TLC prior to use. I n j e c t e d animals were d e c a p i t a t e d a n d t h e i r fetuses r e m o v e d a n d k e p t cold u n t i l b l o o d s a m p l e s could be t a k e n . A d u l t a n d fetal blood 1 2 3 4 5
Supported by NIH Grant HD 08844. B. Vannier and J. P. Raynaud, Molee. Cell Endocr. 3, 323 (1975). T. Nakal, S. Sakamoto, T. Kigawa and A. Shigematsu, Endocr. jap. 19, 47 (1972). J. Presl, S. R~hhling,J. Horsk~ and J. Herznlann, Endocrinology 86, 899 (1970). E . O . Alvarez and V. D. Ramirez, Neuroendoerinology 6, 349
(197o). 6 7 8 9
J . P . Raynaud, Steroids 21,249 (1973). E. Nunez, L. Savu, F. Engelmann, C. Benassayag, O. Crepy and M. Jayle, C. r. Acad. Sei., Paris 273, 242 (1971). J . L . MeGuire and R. D. Lisk, Proe. nat. Acad. Sci. USA 67, 497 (1968). K.D. D~ihler and W. Wuttke, Endoerinoldgy 97, 898 (1975).
