15.3. 1975

Specialia

r e c e p t o r s for n o r m a l a n d s t i m u l a t e d m a c r o p h a g e s is similar in t h e t w o groups. A p p a r e n t l y , a c t i v a t i o n of m a c r o p h a g e s b y e n d o t o x i n does n o t s i g n i f i c a n t l y a l t e r t h e n u m b e r of available c o n - A b i n d i n g sites on t h e m e m b r a n e surface. Since c o n - A b i n d s specifically to a - m e t h y l m a n n o s i d e a n d t o a lesser degree a-D-glucose like residues 7, it is n o t possible t o s p e c u l a t e if o t h e r g l y c o p r o t e i n re-

Table II. The ratio of (vz~I)-con-Abound (cpm) to the content of DNA (/xg/ml) from normal and endotoxin stimulated macrophages. (125I) (cpm) Normal 7321

DNA [zg/ml

(DNA/nSI cpm) X 10s

7911 8003 7422 7984 8682 7917

0.94 1.19 1.08 1.19 0.98 0.98 1.22 1.14

7.80 8.01 7.35 7.20 7.50 8.13 7.10 6.95 x = 7.514-0.51

Stimulated 5696 5372 6117 6178 7182 6131 7303 6088 6016

0.92 0.74 0.82 0.75 0.96 0.78 0.90 0.83 0.75

6.30 7.28 7.50 8.25 7.50 7.82 8.10 7.35 8.05 x = 7.574-0.19

9525

371

c e p t o r s are involved. S u c h a l t e r a t i o n s in g l y c o p r o t e i n m i g h t be e x p e c t e d if t h e m u c o p o l y s a c c h a r i d e c o a t of t h e s t i m u l a t e d m a c r o p h a g e were q u a n i t a t i v e l y or q u a l i t a t i v e l y d i f f e r e n t f r o m t h a t of t h e n o r m a l m a c r o p h a g e . I t is of i n t e r e s t t h a t e x t e n s i v e m e m b r a n e s p r e a d i n g of n o r m a l m a c r o p h a g e s does n o t a l t e r t h e b i n d i n g of t h e a g g l u t i n i n 8. R e s u l t s i n d i c a t e d t h a t t h e n u m b e r s of m e m b r a n e r e c e p t o r s for con-A were similar for m a c r o p h a g e s in t h e r o u n d as in t h e s p r e a d configurations. Such c o n f i g u r a t i o n s are t h o u g h t t o involve c h a n g e s in t h e reserve m e m b r a n e . A similar c o n d i t i o n m a y exist for t h e e n d o t o x i n s t i m u l a t e d m a c r o p h a g e . This is e v i d e n t since t h e a m o u n t of con-A b o u n d (or t h e n u m b e r of con-A receptors) was t h e s a m e for both g r o u p s e v e n t h o u g h s t i m u l a t e d m a c r o p h a g e s are k n o w n t o possess g r e a t e r ruffling (reserve m e m b r a n e ) a n d a g r e a t e r ability to s p r e a d ~.

Zusammen/assung. Nachweis, dass n o r m a l e u n d m i t E n d o t o x i n s t i m u l i e r t e M a k r o p h a g e n des M/iusebauchfells in ihrer B i n d u n g s k a p a z i t / i t y o n C o n c a n a v a l i n - A n i c h t s i g n i f i k a n t divergieren, was d a r a u f hinweist, dass d u r c h ihre A k t i v i e r u n g die A n z a h l y o n G l y k o p r o t e i n - R e z e p t o r e n Iiir Con-A n i c h t ~indert. j . D. LUTI"ON az

50 [zg/ml (1~5I)-con-Awas used in all binding assays, x ~ SE.

Department o[ Physiology, Division o/Hematology, Mount Sinai School o/ Medicine, l OOth Street and 5th Avenue, New York (N. Y. 70029), 18 September 1974.

11 The author wishes to thank the support and advice of Prof. M. R. RAmNOVITCHof New York University.

/

Synthesis of a Pregnancy-Associated a-Macroglobulin by Human Leucocytes The e x i s t e n c e of a p r e g n a n c y associated s e r u m p r o t e i n was first n o t e d b y SMITHIES 1 in 1959 using s t a r c h gel electrophoresis. Since t h a t t i m e m a n y i n v e s t i g a t o r s h a v e similarly r e p o r t e d t h a t h u m a n p r e g n a n c y s e r u m c o n t a i n s one a n d s o m e t i m e s m o r e t h a n one a d d i t i o n a l p r o t e i n (for e x a m p l e refs3-1~ The s e r u m p r o t e i n m o s t c o m m o n l y d e t e c t a b l e in late p r e g n a n c y s e r u m was r e c e n t l y isolated a n d characterized ~. I t was s h o w n to be a h i g h m o l e c u l a r w e i g h t eglobulin c o n t a i n i n g 10% c a r b o h y d r a t e . T h i s p r e g n a n c y associated r (PAM) h a s b e e n q u a n t i t a t e d in t h e blood of s u b j e c t s in m a n y physiological s t a t e s a n d f o u n d to be q u i t e w i d e l y d i s t r i b u t e d s,~~ ~a. I t can be r e a d i l y i d e n t i f i e d of b o t h m a l e s a n d females receiving o e s t r o g e n t r e a t m e n t b u t it does n o t a p p e a r to be i n v o l v e d in t h e t r a n s p o r t of s t e r o i d s ~ - ~ % T h e biological role of P A M has n o t as y e t b e e n clarified a l t h o u g h it has b e e n s u g g e s t e d t h a t it m a y f u n c t i o n in t h e r e g u l a t i o n of t h e i m m u n e r e s p o n s e 6. The sites of s y n t h e s i s of m a n y i n d i v i d u a l s e r u m p r o t e i n s h a v e a l r e a d y b e e n e s t a b l i s h e d 17-2~ a n d this r e p o r t p r o v i d e s s t r o n g l y s u g g e s t i v e e v i d e n c e for t h e s y n t h e s i s of P A M b y p e r i p h e r a l b l o o d leucocytes. H e p a r i n i z e d blood, o b t a i n e d f r o m h e a l t h y males, cont r a c e p t i v e s t e r o i d t r e a t e d females (Norinyl) a n d p r e g n a n t w o m e n (38-40 weeks p r e g n a n t ) , was allowed t o s e d i m e n t a t 37 ~ for 90 rain, L e u c o c y t e s were r e c o v e r e d f r o m t h e

10. SMITHIES, Adv. Protein Chem. 74, 65 (1959). 2 j. A. MAcLAREN, R. D. THORNES, C. C. RoBY and D. E. REID, Am. J. Obstet. Gynec. 78, 939 (1959). 3 j . HIRSCHFELD and U. S6DERBERO, Nature, Lond. 787, 332 (1960). 4 J. F. AFONSOand R. R. DE ALVAREZ,Am. J. Obstet. Gynec., 89, 204 (1964). 5 j. C. ROBINSON, W. T. LONDOn"and J. E. PIERCE, Am. J. Obstet. Gynec. 96, 226 (1966). W. H. STI~SON, Laucet, 1, 684 (1972). 7 S. A. GaLL and S. P. HALBERT, Int. Arch. Allergy 42, 503 (1972). s B. H. B~RNE, Fedn. Proc. 32, 677 (1973). 9 B. VON SCHOULTZand T. STIOBRAND, Acta obstet, gynec, stand. 52, 51 (1973). i0 C. H. W. HORNE, A. L. C. McLAY, H. ]3. TAVADIA,I. CARMICHAEL, A. C. MALLINSON, A. A. C. YEUNO LAIWAH, M~. A. THOMAS and

R. N. M. MAcSwEEN,Clin. exp. Immun. 13, 603 (1973). n W. H. STIMSONand L. EUBANK-SCoTT, FEBS Lett. 23, 298 (1972). /2 W. H. STI~SON, IRCS Med. Sci. 7973, 3. 13 W. H. STIMSO~, J. Endocr. 67, 30 (1974). 14 W. I-I. STIMSON, IRCS Med. Sci. 7973, 10. 15 I~. HOFMANN~ H. A. SCHULZE, W. STRAUBE and 13. I{LAUSCH,

Arch. Gynfik. 213, 86 (1972). is If. BOHN and T. KRANZ,Arch. Gyn/ik. 215, 63 (1973). 17 M. E. PHILLIPS and G. J. TI~OgBECKE, Int. Arch. Allergy 29, 553 (1966). 18 V. J. STEGHER and G. J. THORBEOKE, J. Immun. 99, 643 (1967). ;9 V. J. STEeliER and G. J. THORBECKE, J. Immun. 99, 653 (1967). 20 V. J. STEeliER and G. J. TEORBECKE, J. Immure 99, 660 (1967).

372

EXPERIENTIA 31/3

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Table I. Incorporation of labelled glutamine into cell cultures Type and source of cultured cells

Additions to culture medium

1. No additions

Males (dpm]106 cells)

M 17 ( 15, 2, 34) MS 11 ( 18, 1, 13) 2. Mestranol M 63 ( 48, 56, 85) MS 115 (122,123,101) 3. Mestranol(labelledglutamine M 7 ( 1, 9, 10) addedafterincubationeompleted) MS 5 ( 8, 5, 3) 4. Mestranol + puromyein ~ S --

Leucocytes

Lymphocytes

Contraceptive steroid treated females Pregnant females (dpm]10~cells) (dpm[10s cells)

Pregnant females (dpm]10~ ceils)

48 ( 42, 52, 49) 58 ( 41, 71, 62) 73 ( 91, 41, 86) 133 (117, 150, 132) 9( 15, 1, 12) 13( 6, 25, 9) --

21 (15, 40, 8) 18 (4, 14,30,23) 25 (17, 44, 13) 15 (1, 3, 24, 30) ----

48 ( 41, 34, 68) 76 ( 72, 66, 90) 94( 71, 98,113) 141 (115: 137, 170) 4( 2, 9, 2) 8 ( 5, 11, 7) 5 ( 3, 4, 8)

Figures in parentheses are the results of individual experiments carried out in duplicate. M, cultured in medium alone; MS, cultured in medium + 15% autologous serum.

p l a s m a l a y e r b y c e n t r i f u g a t i o n (150 g, 10 mill) a n d l y m p h o c y t e s were p r e p a r e d b y use of Ficoll-I-Iypaque g r a d i e n t ~t. B o t h were t h e n w a s h e d 6 t i m e s in W a y m o u t h ' s M13 752/1 c u l t u r e m e d i u m . Cells (2 • 10G/ml) were c u l t u r e d w i t h 3 ~Ci, U-*4C g l u t a m i n e or U-SH leucine e i t h e r in c u l t u r e m e d i u m alone or in m e d i u m c o n t a i n i n g 15% a u t o l o g o u s serum. F u r t h e r a d d i t i o n s of m e s t r a n o l (0.25 ~M) or p u r o m y c i n (1 m M ) 2~- were m a d e to a p p r o p r i a t e cultures. I n c u b a t i o n was t h e n c a r r i e d o u t a t 37~ in a 5 % CO2/95 % air a t m o s p h e r e for 5 days. P u r e P A M ~1 a n d F r e u n d ' s c o m p l e t e a d j u v a n t were used for i m m u n i z i n g r a b b i t s a n d t h e r e s u l t a n t a n t i s e r u m was p r e p a r e d b y 50% a m m o n i u m s u l p h a t e p r e c i p i t a t i o n a n d D E A E - c e l l n l o s e f r a c t i o n a t i o n of t h e s e r u m o b t a i n e d 23. T h e a n t i - P A M was a b s o r b e d 4 t i m e s w i t h 413 S e p h a r o s e c o u p l e d b y t h e c y a n o g e n b r o m i d e m e t h o d =~t o male s e r u m p r o t e i n s . T h e specificity of t h e a n t i s e r u m was c h e c k e d b y t w o d i m e n s i o n a l i m m u n o e l e c t r o p h o r e s i s 25 a n d KRAUSE a n d R~-UNZo's i m m n n o d i f f u s i o n technique2% O n c o m p l e t i o n of t h e i n c u b a t i o n p e r i o d t h e c u l t u r e s were c e n t r i f u g e d a n d t h e s u p e r n a t a n t s t r e a t e d w i t h NaC1 to a c o n c e n t r a t i o n of 0.5 M . 1 5 % a u t o l o g o u s s e r u m or 1 5 % p r e g n a n c y s e r u m was t h e n a d d e d to t h e supern a t a n t s of t h e cells c u l t u r e d in m e d i u m a l o n e or m a l e leucocytes respectively. F i n a l l y , a p r e d e t e r m i n e d q u a n t i t y of specific a n t i s e r u m was a d d e d so as t o a t t a i n t h e a n t i g e n a n t i b o d y e q u i v a l e n c e p o i n t in e a c h case. t h e p r e c i p i t a t e s t h a t f o r m e d on i n c u b a t i o n (37~ 1 h) were w a s h e d 3 t i m e s in e i t h e r 0.5 % g l u t a m i n e or leucine solution, c o n t a i n ing 0.5 M NaC1 a n d t h e n c o u n t e d on a liquid s c i n t i l l a t i o n counter.

Autoradiograph of immunoelectrophoretie pattern prepared with culture fluid from the leucocytes of a pregnant woman. The pattern was developed with specific antiserum to PAM.

A u t o r a d i o g r a p h y was p e r f o r m e d as p r e v i o u s l y describe d l L L e u c o c y t e s (5X106/ml) f r o m a p r e g n a n t w o m a n were c u l t u r e d ,as i n d i c a t e d a b o v e , in t h e p r e s e n c e of 1 5 % a u t o l o g o u s serum, 4 ~Ci U-14C g l u t a m i n e a n d m e s t r a n o l . T h e P A M in t h e s u p e r n a t a n t was c o n c e n t r a t e d b y precipit a t i o n w i t h a m m o n i u m s u l p h a t e 11 a n d t h e n sulojected t o i m m u n o e l e c t r o p h o r e t i c a n a l y s i s a g a i n s t t h e specific a n t i serum. I t h a s a l r e a d y b e e n s h o w n t h a t levels of P A M in t h e b l o o d are a t least p a r t i a l l y d e p e n d e n t u p o n o e s t r o g e n c o n c e n t r a t i o n , specifically t h a t oral c o n t r a c e p t i v e s a n d m e s t r a n o l r e s u l t s in t h e a p p e a r a n c e of t h e p r o t e i n in females while s t i l b o e s t r o l causes a n i n c r e a s e d c o n c e n t r a t i o n in m a l e s u n d e r g o i n g t r e a t m e n t for p r o s t a t e c a n c e r 10,1a. T a b l e I s u m m a r i z e s t h e r e s u l t s o b t a i n e d f r o m a series of e x p e r i m e n t s e m p l o y i n g labelled g l u t a m i n e . L e u c o c y t e s f r o m b o t h p r e g n a n t a n d c o n t r a c e p t i v e steroid t r e a t e d w o m e n gave s i g n i f i c a n t l y m o r e s u p e r n a t a n t c o u n t s comp a r e d w i t h t h e m a l e cells, w h o s e levels were s i m i l a r t o t h o s e o b t a i n e d if labelled g l u t a m i n e was a d d e d o n comp l e t i o n of t h e i n c u b a t i o n (i.e. b a c k g r o u n d level). T h i s m o s t p r o b a b l y o c c u r r e d b e c a u s e t h e f e m a l e leucocytes were a l r e a d y ' p r i m e d ' t o p r o d u c e P A M b y b e i n g ill c o n t a c t w i t h h i g h c o n c e n t r a t i o n s of o e s t r o g e n in vivo, w h e r e a s t h e m a l e cells n a t u r a l l y h a d not. H o w e v e r , w h e n l e u c o c y t e s f r o m all t h r e e sources were c u l t u r e d in t h e p r e s e n c e of m e s t r a n o l s u b s t a n t i a l rises in all t h e s u p e r n a t a n t c o u n t s were achieved. T h i s oestrogenic s t i m u l a t i o n of P A M p r o d u c t i o n is t y p i c a l of t h a t o b t a i n e d in t h e in v i v o studies m e n t i o n e d a b o v e a n d t h e f a c t t h a t t h e m a l e leucocytes also p r o d u c e d h i g h c o u n t s ill t h e s u p e r n a t a n t s after 5 days culturing correlates with the observation that the glycoprotein becomes immunochemically detectable in v i v o a p p r o x i m a t e l y 7 d a y s a f t e r o e s t r o g e n a d m i n i s t r a t i o n 13. T h i s e v i d e n c e is s u b s t a n t i a t e d b y t h e r e s u l t s f r o m t h e t r i t i a t e d lencine i n c o r p o r a t i o n e x p e r i m e n t s (Table II), w h e r e a s i m i l a r p a t t e r n t o t h a t n o t e d w i t h labelled gluta-

21 A. B6Yu~, Scand. J. clin. Lab. Invest. Suppl. 21, 97 (1968). 22 j. j. FERGiJSON, J. biol. Chem. 238, 2754 (1963). 23 H. A. SOBER and E. A. P~TERSON, Fedn. Proe. 77, 1116 (1958). 2~ j. PORAT~, R. AXEN and S. ERNBACE, Nature, Lond. 275, 1491 (1967). ~ C. t3. LAm~EBB,Analyt. Bioehem. 70, 358 (1965). 36 U. KRAus~ and V. RAlJiqlO, Acta path. microbiol, seand. 71, 328 (1967).

15.3. 1975

Specialia

373

Table II. Incorporation of labelled leucine into celI cultures Additions to culture medium

1. No additions 2. Mestranol 3. Mestranol (labelled leucine added after incubation completed)

Leucocyte source

M KS M MS M MS

~ales (dpm/106 cells)

Contraceptive steroid treated females (dpm/106 cells)

14 20 68 90 8 11

34 58 62 93 7 4

Each experiment was carried out in duplicate. M, cultured in medium alone; MS, cultured in medium + 15% autologous serum.

m i n e was o b t a i n e d . Also t h e a u t o r a d i o g r a p h (Figure) i l l u s t r a t e s t h a t t h e c u l t u r e fluid f r o m m e s t r a n o l t r e a t e d leucocytes f o r m e d a l a b e l l e d p r e c i p i t i n line a g a i n s t t h e specific a n t i - P A M serum. I n o r d e r t o a s c e r t a i n w h e t h e r it was t h e l y m p h o c y t e p o p u l a t i o n i n t h e leucocytes w h i c h was r e s p o n s i b l e for P A M p r o d u c t i o n , i s o l a t e d cells were cultured with and without mestranol. The results shown in T a b l e I p r o v i d e n o i n d i c a t i o n of t h e p r o t e i n ' s s y n t h e s i s . T h e p o s s i b i l i t y of p r o t e i n - p r o t e i n a n d p r o t e i n - l a b e l l e d a m i n o acid i n t e r a c t i o n s w i t h P A M i n t h e c a r r i e r s e r u m could h a v e lead t o e r r o n e o u s i d e n t i f i c a t i o n of labelled p r o t e i n . F o r e x a m p l e , l a b e l l i n g of e 2 - m a e r o g l o b u l i n a n d l i p o p r o t e i n h a s b e e n o b s e r v e d in m a n y c u l t u r e s of l i v i n g ceils as a r e s u l t of t h e i r c a p a c i t y t o b i n d e n z y m e s ~7,18, 20. T h e s e effects h o w e v e r were n o t c o n s i d e r e d t o h a v e o c c u r r e d t o a n y s i g n i f i c a n t e x t e n t on a c c o u n t of t h e use of a h i g h m o l a r i t y of NaC1 in b o t h t h e a n t i b o d y p r e c i p i t a t i o n a n d cold a m i n o acid w a s h i n g stages. T h u s , p u r o m y c i n i n h i b i t e d cells a n d u n s t i m u l a t e d m a l e leucocytes p r o d u c e d

s i m i l a r c o u n t s to t h e b a c k g r o u n d levels a n d w h e n NaC1 was a d d e d t o t h e c u l t u r e s u p e r n a t a n t s before t h e c a r r i e r serum, t h e e x p e c t e d p a t t e r n s of labelling, i n d i c a t i n g P A M s y n t h e s i s , were a g a i n o b t a i n e d . T h e f a c t t h a t t h e c o u n t s were d i m i n i s h e d in t h e s e r u m free c u l t u r e s is p r o b a b l y a n i n d i c a t i o n t h a t t h e p r o d u c t i o n of t h i s s e r u m p r o t e i n , as w i t h o t h e r s t h a t h a v e b e e n s t u d i e d xg, is e n h a n c e d if s e r u m is a d d e d t o t h e c u l t u r e m e d i u m . The experimental data presented above indicates that t h i s s e r u m ~-globulin, c o m m o n l y associated w i t h pregn a n c y , c a n be s y n t h e s i z e d b y p e r i p h e r a l blood leucocytes.

Rdsumd. Le s a n g des f e m m e s e n c e i n t e s p e u t c o n t e n i r p l u s i e r u s p r o t 6 i n e s s6riques u n i q u e s en leur genre. Celle q u i se p r 6 s e n t e le plus s o u v e n t est u n e a-globuline de g r a n d p o i d s mol6culaire. U n t e s t in v i t r o u t i l i s a n t l'incorp o r a t i o n de 14C-glutamine ou 3tt-leucine darts la glycop r o t 6 i n e a m o n t r 6 q u e celle-ci p e u t gtre s y n t h 6 t i s 6 e p a r des leucocytes. W . I-I. STIMSON a n d J.C. BLACKSTOCK ~7

27 We thank Dr. J. E. O'GRADY and Dr. A. SHEPHARD for obtaining the blood samples. This work was supported by a grant from the Scottish Home and Health Department.

Department o~ Biochemistry, University of Strathdyde, Royal College Building, 204 George Street, Glasgow G7 1XW (Scotland), 27 June 7974.

T r a n s f e r r i n B e h a v i o u r in P r i m a r y H a e m o c h r o m a t o s i s I n P r i m a r y H a e m o c h r o m a t o s i s (PH), a disease d u e t o a c o n g e n i t a l l y a u g m e n t e d iron a b s o r p t i o n , s e r u m t r a n s f e r r i n is o f t e n r e d u c e d t o v e r y low levels 1,2, before a n d i n d e p e n d e n t l y of l i v e r failure d u e t o late cirrhosis 1. Mild r e d u c t i o n s b e l o w n o r m a l r a n g e h a v e also b e e n o b s e r v e d in some r e l a t i v e s 1, 2. I n o r d e r to e x p l a i n t h e s e decreases, t h e e x i s t e n c e of a c o n g e n i t a l l y i m p a i r e d s y n t h e s i s of t r a n s ferrin, i.e. a n i n b o r n e r r o r of m e t a b o l i s m , h a s b e e n p r o p o s e d 2. H o w e v e r , f i r s t l y a decrease of s e r u m t r a n s f e r r i n a n d / o r t o t a l iron b i n d i n g c a p a c i t y (TIBC) is n o t a c o n s t a n t f i n d i n g in P H a n d n o r m a l levels h a v e b e e n r e p o r t e d 1, 3-7, also in 2 m a l e t w i n s w i t h t h e disease 6. Secondly, t h e s t o r e d i r o n itself seems t o affect s e r u m t r a n s f e r r i n : 1. a n e g a t i v e c o r r e l a t i o n b e t w e e n n o n - h e r o i n l i v e r i r o n a n d T I B C exists in n o r m a l subjectsS; 2. i n a n a e m i a d u e to i r o n deficiency, s e r u m t r a n s f e r r i n is i n c r e a s e d b u t i t falls to n o r m a l a f t e r iron stores h a v e b e e n t h e r a p e u t i c a l l y rec o n s t i t u t e d 9,10; 3. p a r e n t e r a l l y iron l o a d i n g is a s s o c i a t e d in t h e r a t w i t h a s u p p r e s s i o n of t r a n s f e r r i n synthesis11;

a n d 4. in h u m a n iron o v e r l o a d s e c o n d a r y to b l o o d t r a n s fusions, to excessive iron t h e r a p y or c h r o n i c h a e m o l y s i s t r a n s f e r r i n or T I B C is lower t h a n in n o r m a l s 1, 3,12. 1 E. FiAscni, L. A. SClJRO, G. DOBRILLA and G. CARTEI, Fisiopatologia e Clinics delle Siderocromatosi (Pozzi, Roma 1972). 2 B. BLANC and A. VANNOTTI, Nature, Lond. 272, 480 (1966). 3 T. H. BOTHWELL and C. A. FINCH, Iron Metabolism (Churchill, London 1962), p. 366. 4 L. W. POWELL and M. J. THOMAS, J. elin. Path. 20, 896 (1967). R. SINNIAH,Arch. intern. ]Vfed. 724, 455 (1969). M. BARRY, G. CARTEl and S. SHERLOCK, Gut 71, 891 (1970). 7 L. A. SCURO, G. ~)OBRILLA, V. LO CASClO, C. BOSELLO, F. D'AxDREA and A. IN~ECCO, Acta hepato-gastroent. 79, 90 (1972). s A. WEmFELD, Acta Med. Scand., suppl. 427 (1964). 9 G. CARTEl, G. PREVIATO and A. INNECCO, Atti. Soc. Med. Clair. Univ. Padova 58, 355 (1968). 10 G. CARTEl, A. MEANI, L. OKOLICSAiqYIand R. NACCARATO, Folia endocrin., Roma 23, 579 (1970). Xl R. S. LANE, Br. J. Haemat. 75, 355 (1968}. 12 M. BARRY, P. J. SeHEUER, S. SHERLOCK, C. F. Ross and R. WILLIAMS, Lancet, 2, 481 (1968).