1592
Specialia
EXPERI E N'I'IA 32/12
Relative number of mast cells (MC) in pinna of nude and normal Balb/c mice nu/nu a
Total count of MC
No. of scale projected
Average No. of MC per 1 scale unit (250 pm~)
1115 641 789 638 1263 639 638
96.0 56.2 52.0 53.0 104.5 70.5 64.0
11.60 13.20 15.17 12.78 12.08 9.06 9.96
nu/+~ Total count of MC
No. of scale projected
Average No. of MC per ] scale unit (250 [~m)
864 423 796 435 661
88.8 43.7 69.8 43.0 71.5
9.72 9.67 1.1.40 10.10 9.24
9Mean 11.9 • 1.92; ~tncan 10.02 _q__0.84.
easier to p e r f o r m . C o n t r a r y to earlier r e p o r t s of e l e v a t e d n u m b e r s of s k i n MC in ' n u d e ' micelO, 11 we did n o t f i n d a n y difference in t h e n u m b e r of in t h e p i n n a of ' n u d e ' a n d n o r m a l mice. Materials and methods. 11-week-old B a l b / c ' n u d e ' (nu/ nu) a n d l i t t e r - m a t e , n o r m a l h e t e r o z y g o t e s (nu/q) m i c e of b o t h s e x e s were used. T h e lower o n e - t h i r d of one p i n n a w a s r e m o v e d , fixed 4 - 6 h in B o u i n s o l u t i o n , e m b e d d e d in p a r a f f i n a n d 7 [zm s e c t i o n s were s t a i n e d w i t h 0.1.% a q u e o u s s o l u t i o n of t o l u i d i n e blue, p H 7.6. U n d e r 40 X 10 m a g n i f i c a t i o n , t h e t o t a l n u m b e r of MC on b o t h sides of elastic c a r t i l a g e w a s c o u n t e d a l o n g s i d e t h e p r o j e c t i o n of t h e Zeiss m i c r o p I a t e (1 c m d i v i d e d i n t o 100 units) i n s e r t e d in t h e ocular. B e t w e e n 10 a n d 32 l e n g t h of scale w a s p r o j e c t e d i n t o o n e s e c t i o n of p i n n a a n d , f r o m e a c h specim e n , t h e n u m b e r of MC w a s c o u n t e d i~n 3-6 s e c t i o n s . T o t a l n u m b e r of ~ C f r o m e a c h s e c t i o n wa.s pooled, d i v i d e d b y t h e n u m b e r of scale p r o j e c t e d , a n d t h e v a l u e o b t a i n e d r e p r e s e n t e d t h e a v e r a g e n u m b e r of MC p e r l e n g t h u n i t (250 p.m). T h e m e a n v a l u e s for b o t h g r o u p s of m i c e -were c o u n t e d , a n d d a t a o b t a i n e d were s t a t i s t i c a l l y a n a l y z e d u s i n g S t u d e n t ' s t - t e s t a t t h e level of p 0.05. Results and discussion. T h e t a b l e s h o w s t h e n u m b e r of animals used and the results obtained. The m e a n n u m b e r of MC in p i n n a of n u d e m i c e w a s 11.98 • 1.97, a n d , in t h o s e of n o r m a ! h e t e r o z y g o t e s , 10.02 • 0.84. T h i s difference is n o t s i g n i f i c a n t a t p > 0.05. T h e d i s t r i b u t i o n of MC in b o t h cases w a s similar. T h e y were d i s t r i b u t e d h o m o g e n o u s l y in t h e s k i n of p i n n a . 11 l{. KELLIJ2R, M. W. HESS and J. F. R~I~Y, Experientia 32, 171 (1976).
Foetal Blood Abnormality
T h e r e s u l t s are in c o n t r a s t to t h o s e o b t a i n e d b y o t h ers a0,x~ in s k i n t a k e n f r o m t h e b a c k . KELLER et al. u rep o r t e d n e a r l y 4 t i m e s h i g h e r c o n c e n t r a t i o n of s k i n MC in ' n u d e ' B a l b / c m i c e t h a n in n o r m a l mice. VIKLICKY et al. ~~ f o u n d 3 t i m e s m o r e MC in s k i n of ' n u d e ' B~0LP t h a n in c o n t r o l mice. T h e h i s t o l o g y of p i n n a s in b o t h ' n u d e s ' a n d h e t e r o z y g o t e s w a s similar, a n d t h e n u m b e r of h a i r follicles a n d g l a n d s is equal. T h e o n l y difference w a s l a c k of or ret a r d a t i o n of h a i r d e v e l o p m e n t . T h e m i t o t i c a c t i v i t y in t h e e p i d e r m i s a n d g l a n d u l a r follicIes, a l t h o u g h n o t exa m i n e d s y s t e m a t i c a l l y , s e e m e d to be i d e n t i c a l in b o t h cases.
VVe do n o t h a v e a n e x p l a n a t i o n for t h e d i s c r e p a n c y of s k i n MC c o n t e n t in r e g a r d to t h e site (back skin, p i n n a skin). T h e p r e s e l m e or a b s e n c e of t h e ' n u ' g e n e m a y be m o r e i m p o r t a n ~ in d e t e r m i n i n g t h e n u m b e r of MC t h a n s k i n site. T h e p r e s e n c e of t h i s g e n e in b o t h ' n u d e ' a n d c o n t r o l m i c e m a y be r e s p o n s i b l e for s i m i l a r MC c o n t e n t . K~LLER et al. n u s e d n o r m a l B a l b / c ( + / q - ) for t h e i r controis, while in the p r e s e n t s t u d y ~zu/nu were c o m p a r e d to ~eu/+ a n i m a l s . I t w o u l d be i n t e r e s t i n g to f i n d w h e t h e r t h e p r e s e n c e of large a m o u n t s of c a r t i l a g e in p i n n a t i s s u e . e x e r t s o m e n l o d i f y i n g effect on MC c o n t e n t . T h i s p o s s i b i l i t y will be e x a m i n e d . A t p r e s e n t , we are i n c l i n e d to c o n s i d e r t h e local factor(s) or ' n u ' g e n e in r e g u l a t i o n of s k i n MC n u m b e r s m o r e t h a n t h e i n f l u e n c e of t h e t h y m u s itself. T h i s a s s u m p t i o n is b a s e d on t h e m o r e p r o n o u n c e d h i s tological difference of b a c k ' n u d e ' s k i n a n d h a i r - c o v e r e d s k i n in c o n t r a s t to p i n n a s , a n d on t h e lack of d r a s t i c differences in t h e a b s o l u t e n u m b e r of lVIC in l y m p h n o d e s .
A s s o c i a t e d w i t h H y p o d a c t y l y in t h e h d S t r a i n of R a t
C. PETTER
Laboratoire de Physiolog'ie d~ Ddveloppement du Coll@e de France et de l'Universitd Pierre et Marie Curie, 4, place Jussieu, F-75 230 Paris-Cedez 05 (France), 1 .June 1976. Summary. T h e h o m o z y g o u s f o e t u s e s of h y p o d a c t y l r a t s (hd s t r a i n ) p r e s e n t a n o b v i o u s red blood cell m a c r o c y t o s i s ( d a y 14 of g e s t a t i o n ) . T h i s blood a b n o r m a l i t y could give rise to t h r o m b o s i s l e a d i n g t o e a r l y n e c r o s i s of t h e e x t r e m i t i e s . S o m e m u t a t i o n s are k n o w n to a f f e c t t h e n u m b e r of d i g i t s in m o u s e 1 , or to lead to l i m b a m p u t a t i o n s in r a b b i t 2, c a t 3 a n d m a n ~. A m o n g t h e m , t h e s t r a i n of b r r a b b i t ( b r a c h y d a c t y l i a ) , first d e s c r i b e d b y GREENE a n d SAXTON 2 h a s b e e n s t u d i e d b y s e v e r a l a u t h o r s a a.
In t h a t s t r a i n , a blood a b n o r m a l i t y h a s b e e n s h o w n : t h e f o e t a l p r i m o r d i a l r e d cells are e s p e c i a l l y large a n d n u m e r o u s , a n d could g i v e rise s p o n t a n e o u s l y , b e t w e e n d a y s 15 a n d 16 of g e s t a t i o n , to t h r o m b o s i s , h a e m o r r h a g e s a n d n e c r o s i s of t h e e x t r e m i t i e s L T h e lesions c a n be p r e -
15.12. 1976
Specialia
v e n t e d in u t e r o b y s e v e r a l t r e a t m e n t s a c t i n g u p o n t h e foetal blood ( p h e n y l h y d r a z i n e , h y p e r o x i a 7, foiic acid, folinic acid a n d v i t a m i n B~2s. T h e p r e v e n t i n g a c t i o n on m a c r o c y t o s i s of t h e l a s t t h r e e t r e a t m e n t s s e e m s to indic a t e a failure in t h e folate m e t a b o l i s m , v i a a l t e r a t i o n s in t h e cell division. M o r e o v e r , p y r i m e t h a m i n e , a d r u g w h i c h i n h i b i t s t h e a c t i v i t y of t h e d i h y d r o f o l a t e r e d u c t a s e , a n d t h u s t h e f o r m a t i o n of t e t r a h y d r o f o l a t e , w h e n a d m i n i s t e r e d to p r e g n a n t rats, i n d u c e s foetal m a l f o r m a t i o n s , including brachydactylia and limb defects: syndactylia, o l i g o d a c t y l i a a n d p h o c o m e l i a 9. i t h a s b e e n s h o w n 9 t h a t p y r i m e t h a m i n e could induce both foetal macrocytosis a n d n e c r o s i s of t h e e x t r e m i t i e s in t h e foetus.
:
~
30 %
--Normocytic foetuses ic foetuses
1593
T h u s , in m a n y cases, t h e r e is a p p a r e n t l y a r e l a t i o n s h i p b e t w e e n t h e e x i s t e n c e of a f o e t a l blood m a c r o c y t o s i s a n d t h e f o r m a t i o n of t h r o m b o s i s followed b y necrosis a n d amputations. A s t r a i n of r a t s exists, t h e h d s t r a i n , first d e s c r i b e d b y SABOURDY a n d B o z I c a0, in w h i c h t h e h o m o z y g o n s foet u s e s p r e s e n t d i g i t a b n o r m a l i t i e s m a n i f e s t e d b o t h in t h e i r n u m b e r a n d form. T h e h d g e n e is also r e s p o n s i b l e for s t e r i l i t y o b s e r v e d in t h e h o m o z y g o u s male. H y p o d a c t y l y is i n h e r i t e d as a r e c e s s i v e c h a r a c t e r in b o t h sexes. In t h i s s t u d y , we tried to find o u t w h e t h e r a blood abn o r m a l i t y , s i m i l a r to t h a t o b s e r v e d in t h e b r r a b b i t f o e t u s , could be d i s c o v e r e d in t h e h d r a t s t r a i n , w h a t could p e r m i t a n e x p l a n a t i o n of t h e s e a b n o r m a l i t i e s of t h e extremities. H y p o d a c t y l m u t a t i o n in t h e h d r a t o c c u r r e d in a W'istar s t r a i n of r a t s in w h i c h a f e m a l e carrier h a s b e e n m a t e d w i t h a m a l e of t h e L o n g E v a n s s t r a i n . All t h e a n i mals have the black-hooded pattern. Hypodactyl rats
%\\\ 1 I/. L. GREEN (Ed.), in fliology o/ the Laboratory Mouse, 2nd ed. (McGraw-Hill Book Co., New York ] 966). s H. S. N. GREENE and J. A. SAXTON, Jr., oT. exp. Ned. 69, 301 (1939). a A. G. SEARLE, l-knl3. Eugen., Lond. 17, 279 (1953). A. FREIR~-Ma~A, Ph. D. Thesis, Ribeirao Preto Medicai School (1%s). 5 o. R. Im,~AN, Anat. Rec. 79, 483 (ff941).
20 o~
10
6 A. JOST, J. ROFFI and M. COURTAT, in Lz:lnb
0
8.7 9.6 10.4 11.3 12.1 13 15.9 148 15.7 165prn Diameter
Z)e~,dop~nent and
De/ormity: Problems o/ Evaluation and RehabiEtation. (Charles C. Thomas, Publisher, 1969), p. 187. 7 C. PETTER, J. I~OURBON, J. P. MALTIER and A.
Jos'l',
C. r. A e a d .
Fig. 1. Distribution of nucleated primordial cell diameters in hd rat Ioetus (day 14 of gestation). Two populations are visible. (Mean • s. E. M.).
Sci. Paris 27.3, 2639 (1971b). s C. PETTER, Th~se de Doetorat d'Etat, Paris (1975). 9 C. PETTEa and J. I~ou~BON, Experientia 31, 369 (1975). l0 M. SAI~O~RDYand F,. Bozie, C. r. Acad. Sci. 250, 3397 (1960).
A
/~
Fig. 2. Nucleated primordial cells in hd rat foetuses. (X 1100), (Day 14 of gestation). A Normocytic primordial cells. B Macrocytic primordial cells.
1594
Specialia
show a r e d u c t i o n in t h e n u m b e r of digits of b o t h forefeet a n d h i n d f e e t . T h e forefeet are g e n e r a l l y t r i d a c t y l a n d t h e hindfeet tetradactyl. Material and methods. W e used m a f i n g s b e t w e e n h y p o d a c t y l females (hd/hd) a n d n o r m a l h e t e r o z y g o u s m a l e s ( h d / + ) . I n t h a t t y p e of m a t i n g , t h e r a t i o of n o r m a l to h y p o d a c t y l is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m 1 : 1 ~. N o r m a l h e t e r o z y g o u s males ( h d / + ) were i n t r o d u c e d to t h e cage of females to m a t e , a n d 12 d a y s later, t h e p r e g n a n t females were recognized b y p a l p a t i o n . I n o r d e r to s t u d y t h e foetal blood, d a y 14 of g e s t a t i o n was chosen, since t h e n u c l e a t e d r e d ceils are still n u m e r o u s a t t h a t stage. F i v e p r e g n a n t h d / h d r a t s were o p e r a t e d a n d t h e foetuses r e m o v e d on d a y 14, b l o o d s m e a r s were m a d e a n d s t a i n e d b y t h e p a n o p t i c m e t h o d . T h e microscopic i m a g e of t h e b l o o d ceils was p r o j e c t e d on p a p e r w i t h ' a p r o j e c t i o n microscope. F o r e a c h foetus, t h e outline of 200 n u c l e a t e d red cells was d r a w n a n d t h e d i a m eters d i r e c t l y m e a s u r e d . T h e f r e q u e n c y of e a c h d i a m e t e r was d e t e r m i n e d (36 foetuses f r o m 5 m o t h e r s ) . Results and discussion. A l t h o u g h it seems a h n o s t impossible t o d i s t i n g u i s h t h e a b n o r m a l foetuses m a c r o scopically a t t h a t s t a g e (the a b n o r m a l i t i e s b e c o m e app a r e n t o n d a y 15), t h e s t u d y of t h e foetal b l o o d cells p e r m i t o n e t o o b s e r v e t w o p o p u l a t i o n s in t h e r a t i o 1 : 1 (Figure 1) : in 17 foetuses, a v e r y o b v i o u s m a c r o c y t o s i s is seen: a b o u t 8 0 % of t h e cells h a v e a d i a m e t e r > 12 tzm, while in 19 foetuses, o n l y 36% are > 12 ~zm. T h e difference
EXPERIENTIA 32]12
b e t w e e n t h e t w o p e r c e n t a g e s is s t a t i s t i c a l l y s i g n i f i c a n t ( m e t h o d of Z2: p < 0.001). Moreover, t h e a s p e c t of t h e n u c l e a t e d r e d b l o o d cells is v e r y d i f f e r e n t in t h e m a c r o cyfiie cells t o n o r m a l s ; t h e i r n u c l e u s is g e n e r a l l y large a n d i r r e g u l a r a n d p a l e r t h a n in n o r m o c y t i c cells (Figure 2). T h e m a c r o c y t o s i s o b s e r v e d d u r i n g t h e early foetal d e v e l o p m e n t of h d / h d r a t r e s e m b l e s b o t h t h a t of b r / b r r a b b i t a n d t h e m a c r o c y t o s i s d u e to p y r i m e t h a m i n e . I n t h e l a t t e r cases a n i m p a i r m e n t in t h e folate or v i t a m i n B~2 m e t a b o l i s m seems t o b e i m p l i c a t e d . Moreover, in t h e h d / h d strain, foetal r e d cell m a c r o c y t o s i s is associated w i t h s t e r i l i t y in t h e h o m o z y g o u s males, as h a s a l r e a d y b e e n o b s e r v e d in m a n w i t h s u c h a m e t a b o l i c failure 12. T h e h d gene could be r e s p o n s i b l e for a d i s t u r b a n c e of cell d i v i s i o n w h i c h leads t o m a c r o c y t o s i s . T h e a b n o r m a l n u c l e a t e d r e d cells could t h e n b e d e s t r o y e d too m a s s i v e l y a n d could b l o c k u p t h e s m a l l e s t a r t e r i e s of t h e e x t r e m i t i e s , w h i c h could give rise v e r y soon to necrosis a n d a m p u t a t i o n of t h e digits. F u r t h e r w o r k w i t h v i t a m i n t r e a t m e n t to t h e p r e g n a n t r a t s h o u l d d e t e r m i n e t h e t r u t h of t h i s hypothesis.
11 R, MOUTIER, •. TOYAMAand M. F. CHARRIER,J. Hered. 6~, 99 (1973). 12 I. M. D. JACKSON, W. B. Dora and G. Mc DONALD,Lancet 2, 1159 (1967).
R o s e t t e F o r m a t i o n by H u m a n T and B L y m p h o c y t e s in t h e P r e s e n c e of A d r e n e r g i c and C h o l i n e r g i c Drugs 1 G. G. R. FERREIRA, H. K. MASSUDA BRASCHER,M. Q. JAVIERRE, W. A. SASSINE a n d A. O. LIMA
Division o/ Immunopathology, School o/ Medicine, U. F. R. J., Rio de faneiro (Brazil), 28 June 1976. Summary. I t was s h o w n t h a t a d r e n e r g i c drugs, w h i c h increase t h e i n t r a c e l l u l a r levels of cAMP, i n h i b i t t h e r o s e t t e f o r m a t i o n b y T - l y m p h o c y t e s , b u t s t i m u l a t e t h e r o s e t t e s p r o d u c e d b y B - l y m p h o c y t e s . Cholinergic drugs, w h i c h increase tile levels of cGMP, o n t h e c o n t r a r y , s t i m u l a t e t h e f o r m a t i o n of r o s e t t e s b y T - l y m p h o c y t e s b u t i n h i b i t t h o s e produced by B-lymphocytes. I t was r e c e n t l y d e m o n s t r a t e d t h a t E - r o s e t t e f o r m a t i o n b y h u m a n T l y m p h o c y t e s is e i t h e r i n h i b i t e d b y d r u g s w h i c h increase t h e i n t r a c e t l u l a r levels of cyclic a d e n o s i n e m o n o p h o s p h a t e (cAMP) 2=4, or s t i m u l a t e d b y d r u g s w h i c h raise t h e levels of cyclic g u a n o s i n e m o n o p h o s p h a t e (cGMP) 5, 6. I n t h i s p a p e r we p r e s e n t e v i d e n c e s u g g e s t i n g that human T and B lymphocytes bear adrenergic and cholinergic r e c e p t o r s w h o s e s t i m u l a t i o n r e s u l t s in a n t a g onistic effects on r o s e t t e f o r m a t i o n . Material and methods. L y m p h o c y t e s . L y m p h o c y t e susp e n s i o n s o b t a i n e d f r o m p e r i p h e r a l b l o o d of n o r m a l subj e c t s b y F i c o l l - H y p a q u e were a d j u s t e d to c o n t a i n 2 - 106 cells/ml. Red blood cells: U n s e n s i t i z e d (E) sheep red blood cells (SRBC) were p r e p a r e d w e e k l y in H u n k ' s solution. Sensitized (EAC) S R B C were p r e p a r e d a c c o r d i n g to LAY a n d NUSSENZWEIG v, u s i n g r a b b i t a n t i s e r u m a n d h u m a n complement. Drugs. T h e following d r u g s (Sigma C h e m i c a l Co.) were a s s a y e d : t h e o p h i l l i n e 10 -a M , p a p a v e r i n e 10 -a M , isop r o t e r e n o I 2 9 10 -4 M , d i b u t y r y l c A M P 2 9 1.0-4 M , carb a m y l c h o l i n e 10 -6 M , p i l o c a r p i n e 10 -a M , d i b u t y r y l c G M P 2 9 10 -4 M , a t r o p i n e sulfate 10 -G M . Solutions of t h e d r u g s were p r e p a r e d j u s t before use in H a n k s ' s o l u t i o n w i t h final p H a d j u s t e d to 7.4.
Rosette assays. Drug effects on rosette formation were assayed by incubation 0.45 ml of the lymphocyte suspension containing 2 9 106 viable cells/ml with 0.05 ml of the drug dilution, for 60 min at 37~ The mixture was separated in two tubes, each receiving, respectively, 0.5 ml of a 0.25% suspension of unsensitized (E) and sensitized (EAC) SRBC. Control tubes received no drugs. The tubes were then centrifuged at room temperature for 5 rain at 200 g and incubated at 37 ~ for 30 min (for B-lymphocyte rosettes) or at 4~ for 60 min (for T-lymphocytes rosettes). The resulting pellets were gently resuspended and counted in a hemocytometer. The viability 1 Acknowledgments. This investigation was supported by Grant from National Council for Scientific and Technological Development (CNPq), Rio de Janeiro, Brazil. F. V. CHISARIand T. S. EDGINOTON, J. Exp. Med. 140, 1122 (1974). S. P. GALANTand R. A. REMO, J. hnmun. 714, 512 (1975). 4 M. H. GRIECO, I. SmEL and Z. GOEL, Abstr. Am. Aead. Allergy, 31st Ann. Meeting (1975). 5 M. H. GRIECO,I. SIGEL and Z. GOEL, Abstr. Am. Aead. Allergy, 32nd Ann. Meeting (1976). 6 R. LUNDAK, L. EATON and S. GALANT,Abstr. Am. Aead. Alergy, 32rid Ann. Meeting (1976). 7 W. H. LAY and V. NUSSENZWEIG, J. exp. Med. 728, 991 (1968).
Specialia
EXPERI E N'I'IA 32/12
Relative number of mast cells (MC) in pinna of nude and normal Balb/c mice nu/nu a
Total count of MC
No. of scale projected
Average No. of MC per 1 scale unit (250 pm~)
1115 641 789 638 1263 639 638
96.0 56.2 52.0 53.0 104.5 70.5 64.0
11.60 13.20 15.17 12.78 12.08 9.06 9.96
nu/+~ Total count of MC
No. of scale projected
Average No. of MC per ] scale unit (250 [~m)
864 423 796 435 661
88.8 43.7 69.8 43.0 71.5
9.72 9.67 1.1.40 10.10 9.24
9Mean 11.9 • 1.92; ~tncan 10.02 _q__0.84.
easier to p e r f o r m . C o n t r a r y to earlier r e p o r t s of e l e v a t e d n u m b e r s of s k i n MC in ' n u d e ' micelO, 11 we did n o t f i n d a n y difference in t h e n u m b e r of in t h e p i n n a of ' n u d e ' a n d n o r m a l mice. Materials and methods. 11-week-old B a l b / c ' n u d e ' (nu/ nu) a n d l i t t e r - m a t e , n o r m a l h e t e r o z y g o t e s (nu/q) m i c e of b o t h s e x e s were used. T h e lower o n e - t h i r d of one p i n n a w a s r e m o v e d , fixed 4 - 6 h in B o u i n s o l u t i o n , e m b e d d e d in p a r a f f i n a n d 7 [zm s e c t i o n s were s t a i n e d w i t h 0.1.% a q u e o u s s o l u t i o n of t o l u i d i n e blue, p H 7.6. U n d e r 40 X 10 m a g n i f i c a t i o n , t h e t o t a l n u m b e r of MC on b o t h sides of elastic c a r t i l a g e w a s c o u n t e d a l o n g s i d e t h e p r o j e c t i o n of t h e Zeiss m i c r o p I a t e (1 c m d i v i d e d i n t o 100 units) i n s e r t e d in t h e ocular. B e t w e e n 10 a n d 32 l e n g t h of scale w a s p r o j e c t e d i n t o o n e s e c t i o n of p i n n a a n d , f r o m e a c h specim e n , t h e n u m b e r of MC w a s c o u n t e d i~n 3-6 s e c t i o n s . T o t a l n u m b e r of ~ C f r o m e a c h s e c t i o n wa.s pooled, d i v i d e d b y t h e n u m b e r of scale p r o j e c t e d , a n d t h e v a l u e o b t a i n e d r e p r e s e n t e d t h e a v e r a g e n u m b e r of MC p e r l e n g t h u n i t (250 p.m). T h e m e a n v a l u e s for b o t h g r o u p s of m i c e -were c o u n t e d , a n d d a t a o b t a i n e d were s t a t i s t i c a l l y a n a l y z e d u s i n g S t u d e n t ' s t - t e s t a t t h e level of p 0.05. Results and discussion. T h e t a b l e s h o w s t h e n u m b e r of animals used and the results obtained. The m e a n n u m b e r of MC in p i n n a of n u d e m i c e w a s 11.98 • 1.97, a n d , in t h o s e of n o r m a ! h e t e r o z y g o t e s , 10.02 • 0.84. T h i s difference is n o t s i g n i f i c a n t a t p > 0.05. T h e d i s t r i b u t i o n of MC in b o t h cases w a s similar. T h e y were d i s t r i b u t e d h o m o g e n o u s l y in t h e s k i n of p i n n a . 11 l{. KELLIJ2R, M. W. HESS and J. F. R~I~Y, Experientia 32, 171 (1976).
Foetal Blood Abnormality
T h e r e s u l t s are in c o n t r a s t to t h o s e o b t a i n e d b y o t h ers a0,x~ in s k i n t a k e n f r o m t h e b a c k . KELLER et al. u rep o r t e d n e a r l y 4 t i m e s h i g h e r c o n c e n t r a t i o n of s k i n MC in ' n u d e ' B a l b / c m i c e t h a n in n o r m a l mice. VIKLICKY et al. ~~ f o u n d 3 t i m e s m o r e MC in s k i n of ' n u d e ' B~0LP t h a n in c o n t r o l mice. T h e h i s t o l o g y of p i n n a s in b o t h ' n u d e s ' a n d h e t e r o z y g o t e s w a s similar, a n d t h e n u m b e r of h a i r follicles a n d g l a n d s is equal. T h e o n l y difference w a s l a c k of or ret a r d a t i o n of h a i r d e v e l o p m e n t . T h e m i t o t i c a c t i v i t y in t h e e p i d e r m i s a n d g l a n d u l a r follicIes, a l t h o u g h n o t exa m i n e d s y s t e m a t i c a l l y , s e e m e d to be i d e n t i c a l in b o t h cases.
VVe do n o t h a v e a n e x p l a n a t i o n for t h e d i s c r e p a n c y of s k i n MC c o n t e n t in r e g a r d to t h e site (back skin, p i n n a skin). T h e p r e s e l m e or a b s e n c e of t h e ' n u ' g e n e m a y be m o r e i m p o r t a n ~ in d e t e r m i n i n g t h e n u m b e r of MC t h a n s k i n site. T h e p r e s e n c e of t h i s g e n e in b o t h ' n u d e ' a n d c o n t r o l m i c e m a y be r e s p o n s i b l e for s i m i l a r MC c o n t e n t . K~LLER et al. n u s e d n o r m a l B a l b / c ( + / q - ) for t h e i r controis, while in the p r e s e n t s t u d y ~zu/nu were c o m p a r e d to ~eu/+ a n i m a l s . I t w o u l d be i n t e r e s t i n g to f i n d w h e t h e r t h e p r e s e n c e of large a m o u n t s of c a r t i l a g e in p i n n a t i s s u e . e x e r t s o m e n l o d i f y i n g effect on MC c o n t e n t . T h i s p o s s i b i l i t y will be e x a m i n e d . A t p r e s e n t , we are i n c l i n e d to c o n s i d e r t h e local factor(s) or ' n u ' g e n e in r e g u l a t i o n of s k i n MC n u m b e r s m o r e t h a n t h e i n f l u e n c e of t h e t h y m u s itself. T h i s a s s u m p t i o n is b a s e d on t h e m o r e p r o n o u n c e d h i s tological difference of b a c k ' n u d e ' s k i n a n d h a i r - c o v e r e d s k i n in c o n t r a s t to p i n n a s , a n d on t h e lack of d r a s t i c differences in t h e a b s o l u t e n u m b e r of lVIC in l y m p h n o d e s .
A s s o c i a t e d w i t h H y p o d a c t y l y in t h e h d S t r a i n of R a t
C. PETTER
Laboratoire de Physiolog'ie d~ Ddveloppement du Coll@e de France et de l'Universitd Pierre et Marie Curie, 4, place Jussieu, F-75 230 Paris-Cedez 05 (France), 1 .June 1976. Summary. T h e h o m o z y g o u s f o e t u s e s of h y p o d a c t y l r a t s (hd s t r a i n ) p r e s e n t a n o b v i o u s red blood cell m a c r o c y t o s i s ( d a y 14 of g e s t a t i o n ) . T h i s blood a b n o r m a l i t y could give rise to t h r o m b o s i s l e a d i n g t o e a r l y n e c r o s i s of t h e e x t r e m i t i e s . S o m e m u t a t i o n s are k n o w n to a f f e c t t h e n u m b e r of d i g i t s in m o u s e 1 , or to lead to l i m b a m p u t a t i o n s in r a b b i t 2, c a t 3 a n d m a n ~. A m o n g t h e m , t h e s t r a i n of b r r a b b i t ( b r a c h y d a c t y l i a ) , first d e s c r i b e d b y GREENE a n d SAXTON 2 h a s b e e n s t u d i e d b y s e v e r a l a u t h o r s a a.
In t h a t s t r a i n , a blood a b n o r m a l i t y h a s b e e n s h o w n : t h e f o e t a l p r i m o r d i a l r e d cells are e s p e c i a l l y large a n d n u m e r o u s , a n d could g i v e rise s p o n t a n e o u s l y , b e t w e e n d a y s 15 a n d 16 of g e s t a t i o n , to t h r o m b o s i s , h a e m o r r h a g e s a n d n e c r o s i s of t h e e x t r e m i t i e s L T h e lesions c a n be p r e -
15.12. 1976
Specialia
v e n t e d in u t e r o b y s e v e r a l t r e a t m e n t s a c t i n g u p o n t h e foetal blood ( p h e n y l h y d r a z i n e , h y p e r o x i a 7, foiic acid, folinic acid a n d v i t a m i n B~2s. T h e p r e v e n t i n g a c t i o n on m a c r o c y t o s i s of t h e l a s t t h r e e t r e a t m e n t s s e e m s to indic a t e a failure in t h e folate m e t a b o l i s m , v i a a l t e r a t i o n s in t h e cell division. M o r e o v e r , p y r i m e t h a m i n e , a d r u g w h i c h i n h i b i t s t h e a c t i v i t y of t h e d i h y d r o f o l a t e r e d u c t a s e , a n d t h u s t h e f o r m a t i o n of t e t r a h y d r o f o l a t e , w h e n a d m i n i s t e r e d to p r e g n a n t rats, i n d u c e s foetal m a l f o r m a t i o n s , including brachydactylia and limb defects: syndactylia, o l i g o d a c t y l i a a n d p h o c o m e l i a 9. i t h a s b e e n s h o w n 9 t h a t p y r i m e t h a m i n e could induce both foetal macrocytosis a n d n e c r o s i s of t h e e x t r e m i t i e s in t h e foetus.
:
~
30 %
--Normocytic foetuses ic foetuses
1593
T h u s , in m a n y cases, t h e r e is a p p a r e n t l y a r e l a t i o n s h i p b e t w e e n t h e e x i s t e n c e of a f o e t a l blood m a c r o c y t o s i s a n d t h e f o r m a t i o n of t h r o m b o s i s followed b y necrosis a n d amputations. A s t r a i n of r a t s exists, t h e h d s t r a i n , first d e s c r i b e d b y SABOURDY a n d B o z I c a0, in w h i c h t h e h o m o z y g o n s foet u s e s p r e s e n t d i g i t a b n o r m a l i t i e s m a n i f e s t e d b o t h in t h e i r n u m b e r a n d form. T h e h d g e n e is also r e s p o n s i b l e for s t e r i l i t y o b s e r v e d in t h e h o m o z y g o u s male. H y p o d a c t y l y is i n h e r i t e d as a r e c e s s i v e c h a r a c t e r in b o t h sexes. In t h i s s t u d y , we tried to find o u t w h e t h e r a blood abn o r m a l i t y , s i m i l a r to t h a t o b s e r v e d in t h e b r r a b b i t f o e t u s , could be d i s c o v e r e d in t h e h d r a t s t r a i n , w h a t could p e r m i t a n e x p l a n a t i o n of t h e s e a b n o r m a l i t i e s of t h e extremities. H y p o d a c t y l m u t a t i o n in t h e h d r a t o c c u r r e d in a W'istar s t r a i n of r a t s in w h i c h a f e m a l e carrier h a s b e e n m a t e d w i t h a m a l e of t h e L o n g E v a n s s t r a i n . All t h e a n i mals have the black-hooded pattern. Hypodactyl rats
%\\\ 1 I/. L. GREEN (Ed.), in fliology o/ the Laboratory Mouse, 2nd ed. (McGraw-Hill Book Co., New York ] 966). s H. S. N. GREENE and J. A. SAXTON, Jr., oT. exp. Ned. 69, 301 (1939). a A. G. SEARLE, l-knl3. Eugen., Lond. 17, 279 (1953). A. FREIR~-Ma~A, Ph. D. Thesis, Ribeirao Preto Medicai School (1%s). 5 o. R. Im,~AN, Anat. Rec. 79, 483 (ff941).
20 o~
10
6 A. JOST, J. ROFFI and M. COURTAT, in Lz:lnb
0
8.7 9.6 10.4 11.3 12.1 13 15.9 148 15.7 165prn Diameter
Z)e~,dop~nent and
De/ormity: Problems o/ Evaluation and RehabiEtation. (Charles C. Thomas, Publisher, 1969), p. 187. 7 C. PETTER, J. I~OURBON, J. P. MALTIER and A.
Jos'l',
C. r. A e a d .
Fig. 1. Distribution of nucleated primordial cell diameters in hd rat Ioetus (day 14 of gestation). Two populations are visible. (Mean • s. E. M.).
Sci. Paris 27.3, 2639 (1971b). s C. PETTER, Th~se de Doetorat d'Etat, Paris (1975). 9 C. PETTEa and J. I~ou~BON, Experientia 31, 369 (1975). l0 M. SAI~O~RDYand F,. Bozie, C. r. Acad. Sci. 250, 3397 (1960).
A
/~
Fig. 2. Nucleated primordial cells in hd rat foetuses. (X 1100), (Day 14 of gestation). A Normocytic primordial cells. B Macrocytic primordial cells.
1594
Specialia
show a r e d u c t i o n in t h e n u m b e r of digits of b o t h forefeet a n d h i n d f e e t . T h e forefeet are g e n e r a l l y t r i d a c t y l a n d t h e hindfeet tetradactyl. Material and methods. W e used m a f i n g s b e t w e e n h y p o d a c t y l females (hd/hd) a n d n o r m a l h e t e r o z y g o u s m a l e s ( h d / + ) . I n t h a t t y p e of m a t i n g , t h e r a t i o of n o r m a l to h y p o d a c t y l is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m 1 : 1 ~. N o r m a l h e t e r o z y g o u s males ( h d / + ) were i n t r o d u c e d to t h e cage of females to m a t e , a n d 12 d a y s later, t h e p r e g n a n t females were recognized b y p a l p a t i o n . I n o r d e r to s t u d y t h e foetal blood, d a y 14 of g e s t a t i o n was chosen, since t h e n u c l e a t e d r e d ceils are still n u m e r o u s a t t h a t stage. F i v e p r e g n a n t h d / h d r a t s were o p e r a t e d a n d t h e foetuses r e m o v e d on d a y 14, b l o o d s m e a r s were m a d e a n d s t a i n e d b y t h e p a n o p t i c m e t h o d . T h e microscopic i m a g e of t h e b l o o d ceils was p r o j e c t e d on p a p e r w i t h ' a p r o j e c t i o n microscope. F o r e a c h foetus, t h e outline of 200 n u c l e a t e d red cells was d r a w n a n d t h e d i a m eters d i r e c t l y m e a s u r e d . T h e f r e q u e n c y of e a c h d i a m e t e r was d e t e r m i n e d (36 foetuses f r o m 5 m o t h e r s ) . Results and discussion. A l t h o u g h it seems a h n o s t impossible t o d i s t i n g u i s h t h e a b n o r m a l foetuses m a c r o scopically a t t h a t s t a g e (the a b n o r m a l i t i e s b e c o m e app a r e n t o n d a y 15), t h e s t u d y of t h e foetal b l o o d cells p e r m i t o n e t o o b s e r v e t w o p o p u l a t i o n s in t h e r a t i o 1 : 1 (Figure 1) : in 17 foetuses, a v e r y o b v i o u s m a c r o c y t o s i s is seen: a b o u t 8 0 % of t h e cells h a v e a d i a m e t e r > 12 tzm, while in 19 foetuses, o n l y 36% are > 12 ~zm. T h e difference
EXPERIENTIA 32]12
b e t w e e n t h e t w o p e r c e n t a g e s is s t a t i s t i c a l l y s i g n i f i c a n t ( m e t h o d of Z2: p < 0.001). Moreover, t h e a s p e c t of t h e n u c l e a t e d r e d b l o o d cells is v e r y d i f f e r e n t in t h e m a c r o cyfiie cells t o n o r m a l s ; t h e i r n u c l e u s is g e n e r a l l y large a n d i r r e g u l a r a n d p a l e r t h a n in n o r m o c y t i c cells (Figure 2). T h e m a c r o c y t o s i s o b s e r v e d d u r i n g t h e early foetal d e v e l o p m e n t of h d / h d r a t r e s e m b l e s b o t h t h a t of b r / b r r a b b i t a n d t h e m a c r o c y t o s i s d u e to p y r i m e t h a m i n e . I n t h e l a t t e r cases a n i m p a i r m e n t in t h e folate or v i t a m i n B~2 m e t a b o l i s m seems t o b e i m p l i c a t e d . Moreover, in t h e h d / h d strain, foetal r e d cell m a c r o c y t o s i s is associated w i t h s t e r i l i t y in t h e h o m o z y g o u s males, as h a s a l r e a d y b e e n o b s e r v e d in m a n w i t h s u c h a m e t a b o l i c failure 12. T h e h d gene could be r e s p o n s i b l e for a d i s t u r b a n c e of cell d i v i s i o n w h i c h leads t o m a c r o c y t o s i s . T h e a b n o r m a l n u c l e a t e d r e d cells could t h e n b e d e s t r o y e d too m a s s i v e l y a n d could b l o c k u p t h e s m a l l e s t a r t e r i e s of t h e e x t r e m i t i e s , w h i c h could give rise v e r y soon to necrosis a n d a m p u t a t i o n of t h e digits. F u r t h e r w o r k w i t h v i t a m i n t r e a t m e n t to t h e p r e g n a n t r a t s h o u l d d e t e r m i n e t h e t r u t h of t h i s hypothesis.
11 R, MOUTIER, •. TOYAMAand M. F. CHARRIER,J. Hered. 6~, 99 (1973). 12 I. M. D. JACKSON, W. B. Dora and G. Mc DONALD,Lancet 2, 1159 (1967).
R o s e t t e F o r m a t i o n by H u m a n T and B L y m p h o c y t e s in t h e P r e s e n c e of A d r e n e r g i c and C h o l i n e r g i c Drugs 1 G. G. R. FERREIRA, H. K. MASSUDA BRASCHER,M. Q. JAVIERRE, W. A. SASSINE a n d A. O. LIMA
Division o/ Immunopathology, School o/ Medicine, U. F. R. J., Rio de faneiro (Brazil), 28 June 1976. Summary. I t was s h o w n t h a t a d r e n e r g i c drugs, w h i c h increase t h e i n t r a c e l l u l a r levels of cAMP, i n h i b i t t h e r o s e t t e f o r m a t i o n b y T - l y m p h o c y t e s , b u t s t i m u l a t e t h e r o s e t t e s p r o d u c e d b y B - l y m p h o c y t e s . Cholinergic drugs, w h i c h increase tile levels of cGMP, o n t h e c o n t r a r y , s t i m u l a t e t h e f o r m a t i o n of r o s e t t e s b y T - l y m p h o c y t e s b u t i n h i b i t t h o s e produced by B-lymphocytes. I t was r e c e n t l y d e m o n s t r a t e d t h a t E - r o s e t t e f o r m a t i o n b y h u m a n T l y m p h o c y t e s is e i t h e r i n h i b i t e d b y d r u g s w h i c h increase t h e i n t r a c e t l u l a r levels of cyclic a d e n o s i n e m o n o p h o s p h a t e (cAMP) 2=4, or s t i m u l a t e d b y d r u g s w h i c h raise t h e levels of cyclic g u a n o s i n e m o n o p h o s p h a t e (cGMP) 5, 6. I n t h i s p a p e r we p r e s e n t e v i d e n c e s u g g e s t i n g that human T and B lymphocytes bear adrenergic and cholinergic r e c e p t o r s w h o s e s t i m u l a t i o n r e s u l t s in a n t a g onistic effects on r o s e t t e f o r m a t i o n . Material and methods. L y m p h o c y t e s . L y m p h o c y t e susp e n s i o n s o b t a i n e d f r o m p e r i p h e r a l b l o o d of n o r m a l subj e c t s b y F i c o l l - H y p a q u e were a d j u s t e d to c o n t a i n 2 - 106 cells/ml. Red blood cells: U n s e n s i t i z e d (E) sheep red blood cells (SRBC) were p r e p a r e d w e e k l y in H u n k ' s solution. Sensitized (EAC) S R B C were p r e p a r e d a c c o r d i n g to LAY a n d NUSSENZWEIG v, u s i n g r a b b i t a n t i s e r u m a n d h u m a n complement. Drugs. T h e following d r u g s (Sigma C h e m i c a l Co.) were a s s a y e d : t h e o p h i l l i n e 10 -a M , p a p a v e r i n e 10 -a M , isop r o t e r e n o I 2 9 10 -4 M , d i b u t y r y l c A M P 2 9 1.0-4 M , carb a m y l c h o l i n e 10 -6 M , p i l o c a r p i n e 10 -a M , d i b u t y r y l c G M P 2 9 10 -4 M , a t r o p i n e sulfate 10 -G M . Solutions of t h e d r u g s were p r e p a r e d j u s t before use in H a n k s ' s o l u t i o n w i t h final p H a d j u s t e d to 7.4.
Rosette assays. Drug effects on rosette formation were assayed by incubation 0.45 ml of the lymphocyte suspension containing 2 9 106 viable cells/ml with 0.05 ml of the drug dilution, for 60 min at 37~ The mixture was separated in two tubes, each receiving, respectively, 0.5 ml of a 0.25% suspension of unsensitized (E) and sensitized (EAC) SRBC. Control tubes received no drugs. The tubes were then centrifuged at room temperature for 5 rain at 200 g and incubated at 37 ~ for 30 min (for B-lymphocyte rosettes) or at 4~ for 60 min (for T-lymphocytes rosettes). The resulting pellets were gently resuspended and counted in a hemocytometer. The viability 1 Acknowledgments. This investigation was supported by Grant from National Council for Scientific and Technological Development (CNPq), Rio de Janeiro, Brazil. F. V. CHISARIand T. S. EDGINOTON, J. Exp. Med. 140, 1122 (1974). S. P. GALANTand R. A. REMO, J. hnmun. 714, 512 (1975). 4 M. H. GRIECO, I. SmEL and Z. GOEL, Abstr. Am. Aead. Allergy, 31st Ann. Meeting (1975). 5 M. H. GRIECO,I. SIGEL and Z. GOEL, Abstr. Am. Aead. Allergy, 32nd Ann. Meeting (1976). 6 R. LUNDAK, L. EATON and S. GALANT,Abstr. Am. Aead. Alergy, 32rid Ann. Meeting (1976). 7 W. H. LAY and V. NUSSENZWEIG, J. exp. Med. 728, 991 (1968).
