15.9.1973

Speeialia

tions: 1. A f t e r p l a t e l e t aggregation b y T h r o m b o f a x has occurred, the sample is rapidly centrifuged t o r e m o v e aggregates; t h e n 0.2 m l of t h e s u p e r n a t a n t is a d d e d to 0.8 ml P R P in the a g g r e g o m e t e r : an i m m e d i a t e aggregation is i n v a r i a b l y obtained, indicating the presence of an aggregating a c t i v i t y o t h e r t h a n T h r o m b o f a x ; this a c t i v i t y was found to correspond to a b o u t 1 ~ M A D P / 1 0 s platelets, m e a s u r e d as p l a t e l e t - a g g r e g a t i n g equivalent, according to WEISS and ROGERS 1~. 2. Adenosine, p r o s t a g l a n d i n E~, E D T A , apyrase, t h e e n z y m a t i c s y s t e m phosphoenolp y r u v a t e - p y r u v a t e kinase, 2-de0xy-D-glucose and antim y c i n A (the last 2 substances used simultaneously) all inhibit p l a t e l e t aggregation b y T h r o m b o f a x at the same concentrations which inhibit aggregation b y 2 [zM A D P . 3. B o t h acetylsalicylic acid and i n d o m e t h a c i n , at concent r a t i o n s inhibiting p l a t e l e t aggregation b y 40 ~g/ml collagen (Stago), strongly inhibit aggregation b y T h r o m -

o

1(; I

Thrombofaxalone

20

DPalone f

= 3C .o_ -~ 405C

Thrombofax+ADP

6C

lime Fig. 3. Effect of a non-aggregating concentration of Thrombofax (diluted • 64 in isotonic saline) on human platelet aggregation by ADP (2 • 10-~M, final concentration).

[-1

201 I ~ g 30~

Thrombofax alone

bofax. The aggregation b y T h r o m b o f a x was also inhibited in p l a s m a from n o r m a l people receiving the above antii n f l a m m a t o r y drugs 1~, 13. T h a t T h r o m b o f a x induces the p l a t e l e t 'release reaction' is also suggested by t h e following observations : 1. T h r o m b o f a x releases ~C-serotonin from n o r m a l p l a t e l e t s : a f t e r 10 min aggregation, b e t w e e n 60% and 80% of t h e p l a t e l e t - b o u n d z4C-serotonin is extruded 9 Acetylsalicylic acid and i n d o m e t h a c i n (2 • 10-4M final concentration) almost t o t a l l y inhibit such a release. 2. T h r o m b o f a x also provokes the release of p l a t e l e t factor-4 (PF-4) a c t i v i t y , measured b y the m e t h o d of HARADA and ZUCKER x~ a s described b y DONATI et al. ~5. I n 12 n o r m a l subjects, the m e a n P F - 4 a c t i v i t y released after 10 m i n aggregation b y T h r o m b o f a x was 09 • 0.08 P F - 4 units/ml, which corresponds to a b o u t 80% of the m e a n t o t a l P F - 4 a c t i v i t y (obtained b y i n c u b a t i n g P R P w i t h T r i t o n X-100xs). Release of P F - 4 activity, as well as aggregation b y T h r o m b o f a x , a r e less p r o n o u n c e d at r o o m t e m p e r a t u r e t h a n at 37 ~C. Acetylsalicylic acid and indom e t h a c i n (2 • 10-4M, final concentration) strongly i n h i b i t the release of P F - 4 induced b y T h r o m b o f a x . The ingestion of 500 m g acetylsalicyI!c acid or 50 m g i n d o m e t h a c i n p r o v o k e s the same effect. I n conclusion, T h r o m b o f a x brings a b o u t p l a t e l e t aggregation t h r o u g h t h e release of endogenous p l a t e l e t A D P ; x~C-serotonin and P F I 4 a c t i v i t y are released at t h e s a m e time 9 F F A contained in t h e r e a g e n t seem to be resp o n s i b l e for t h e observed p h e n o m e n a ~.

Rdsumd. Le T h r o m b o f a x O r t h o p r o v o q u e l'agr6gation des p l a q u e t t e s h u m a i n e s (ainsi que celles du chien, du cobaye et du rat) suite ~ la lib6ration d ' A D P endoplaquettaire. L a ~4C-s6rotonine et le facteur p l a q u e t t a i r e 4 sont aussi lib6r6s au cours de la r6action. L a fraction active du p r o d u i t semble r6sider dans les acides gras libres. G. DE GAETANO zT, j . VERMYLEN and M. VERSTRAETE

Laboratory o] Blood Coagulation, Medical Research Department, University of Leuven, Leuven (Belgium), 27 March 1973.

Adrenalineal~ ~

~1 H. J. W~Iss and J. ROGERS, BIood 39, 187 (1972). 1~ G. DE GAETANO, M. B. DONATI and J. VERMYLEN,Int. J. clin. Pharmae. 5, 196 (i971). 1~ G. DE GAETANO, M. CASTEELS-VAN DAELE, J . H . CLAES a n d R.

#- 50

60

1137

Thrombofax+adrenalino

EECKELS, Helv. paediat. Acta 26, 423 (1971). 14 K. HARADAand M. B. ZUCKER,Thromb. Diath. haemorrh. 25, 41 (1971). 15 M. B. DONATI, IV[. PALESTER-CHLEBOWCZYK, G. DE GAET&NO a n d

Time Fig. 4. Effect of a non-aggregating concentration of Thrombofax (diluted • 64 in isotonic saline) on human platelet aggregation by adrenaline (10-6M, final concentration ).

J. VERiYLEN, Adv. expt. Med. Biol. 3d, 295 (1972). 16 The experienced technical assistance of Miss A. VANDENBUSSCHE is gratefully acknowledged. 17 Fellow of the Katholieke Universiteit te Leuven, 1972/73. Present address: Laboratory for Haemostasis and Thrombosis Research, Mario Negri Institute, V. Eritrea, 62, 1-20157 Milano (Italy).

Effect of Phytohaemagglutinin on the Course of Lymphocytic Choriomeningitis Virus Infection in Mice r The effect of p h y t o h a e m a g g l u t i n i n (PHA) on different t y p e s of cellular i m m u n e response has been i n v e s t i g a t e d b y several authors. H o w e v e r , e x p e r i m e n t a l d a t a concerning the influence of P H A on the cellular i m m u n e response seem to be contradictory. This effect of P H A depends on t h e mitogenic a c t i v i t y of the p r e p a r a t i o n as well as on t h e dose and m o d e of its application 1, ~. The course of intra-

cerebral (i.cer.) l y m p h o c y t i c choriomeningitis (LCM) virus infection has been studied in our earlier e x p e r i m e n t s 1 K. ~V[ARKLEY, S. W. TtlORTOIq and E. SMALLMAN, Proc. Soe. exp. Biol. Med. 139, 37 (1972). 2 Gu JkNossY, GY. PETRk~YI JR., and P. ALFOLDY, Folia biol., Praha 15, 453 (1969).

1138

Specialia

10C %

~_~_, ~ __a r---~

10(]

PHA+LCM group . . . . LCM group

%

5[ Virus Ii

i

~

i

6

7

8

9

100F

,

,r___.I - - f- . . . . . . . . . .

dilutiomlO q

r

i

i

,- ~

i

-

10 11 12 13 14days

._#

0

I

__1

50 -//

EXPERIt~NTIA29/9

i

6

Virus dilution

7 8 9

1'1

:10-4

1:4days

100

Ir__" ('a Virusdilution:lO

-2 -

50

Virus dilution

: 10-s

I

100r

6

7

r

i

8

9

%J

'~ 0 0

LCM virus

~

i

10 11

..........

i

t

i

f

0 --/] t 6

12 13 14days

lOOt

r- . . . . .

%]

J

J

'-.---.---,---,---,---,--.

7

8

9

10 11 12 13 14days

Virus dilution : 10-3

,i

i

i

7

8

9

r

t0

i

i

I

11 12 15 14days

Virus dilution:lO -6

0

0

# ..... 6

7

8

9

10 11 12 15 14days

LCM virus

in mice treated with phytohaemagglutinin (Phytoclin, Wellcome Research Laboratories), and it was found t h a t repeatedly given i.p. P H A failed to influence the cellular immune response of mice to i.cer. LCM virus infection 3. In our present work the course of i.cer. LCM virus infection was studied in mice treated with a single i.v. injection of purified PHA. Materials and methods. Experiments were performed on 5-week-old Swiss mice. Purified phytohaemagglutinin (Lot Br~ Wellcome Research Laboratories) was administered and, ill the case of a single i.v. injection, the LDs0 was found to be 20 mg/kg for the mice. 150 mice were given i.v. injection of 10 mg/kg PHA. The following day parallel titrations were performed under identical conditions simultaneously on PHA-pretreated (PHA + LCM group) and untreated (LCM group) animals, using the W E strain of LCM virus. Tenfold dilution between 10 -1 and 10 -~ was used for determination of the virus titer. 16 animals of both groups were infected intracerebrally with each dilution. The remaining P H A - t r e a t e d and an identical number of untreated animals served as P H A and control groups. The development of typical neurological symptoms of LCM virus infection, as well as the mortality rate, were observed in the virus-infected groups. 16 animals were sacrified from both the P H A and control groups 3 and 10 days after P H A treatment. Relative spleen weight of succumbed and sacrificed mice as well as the spleen index were determined in each group. Absolute lymphocyte counts were determined in the peripheral blood o f uninfected animals from the P H A and control groups. Results and discussion. Mortality of P H A tr e a t m e n t was 2% and occurred within 24 h after treatment. No further death was observed during the experiment ill the P H A group. As compared with the untreated controls, P H A treated animals displayed higher absolute lymphocyte counts and significantly increased spleen weight on the 3rd day after treatment. Moderate increase of absolute lymphocyte count and spleen hypertrophy could be observed 10 clays following the P H A treatment. Rate and time course of death observed during the parallel virus titration in the P H A + LCM and LCM

R a t e a n d t i m e course of d e a t h in P H A - t r e a t e d and n o n - t r e a t e d a n i m a l s in consequence of Leer. LCM virus infection, d u r i n g the parallel titration.

Average spleen i n d e x and absolute l y m p h o c y t e c o u n t 3 a n d 10 days following P H A t r e a t m e n t in P H A and control mice groups

Mice

groups

PHA Control

Average absolute l y m p h o c y t e c o u n t (days after t r e a t m e n t )

Spleen i n d e x

3

10

3

10

8000 5000

6600 5000

1.7 1.0

1.2 1.0

groups are summarized in the Figure. Comparing data of the P H A + LCM groups, it appeared t h a t on applying the virus dilutions 10 -1 and 10 -3 death occurred earlier and the dilutions 10 -4 and 10 -5 caused higher mortality rate in the P H A + LCM group than ill the LCM group. Typical neurological symptoms of i.cer. LCM virus infection were apparent before death in each animal succumbing during the parallel titration. Calculating the LDs0 of virus with the REED-MuENCH formula, it was found to be 10 -~m for the P H A + LCM group and 10 -4 for the LCM group on the basis of our results. P H A pretreated and untreated animals thus displayed different response to the same q u a n t i t y of virus. Average spleen weight was higher in animals of the P H A + LCM group succumbing 6 to 9 days after LCM infection than in the LCM group. Spleen index was 1.17 in the P H A + LCM group and 1,0 in the LCM group. A single i.v. injection of purified P H A caused spleen hypertrophy and rise in the absolute lymphocyte count ill the p e r i p h e r a l blood 3 days after the treatment. This observation tallied with the experimental data of other authors 4. Besides this known effect of i.v. P H A treatment, the changed response to i.cer. LCM virus infection of the animals could also be observed. Death of animals

a ZS. B~.NOS, I. SZERI, I ). ANDERLIIr a n d B. RADNAI, E x p e r i e n t i a 25,

1332 (1969). L. B. EPSTEIN a n d CH. W. SMITK, J. I m m u n . 100, 421 (1968}.

15.9.1973

1139

Speeialia

in t h e P H A + L C M g r o u p o c c u r r e d e a r l i e r a n d t h e m o r t a l i t y r a t e w a s also h i g h e r t h a n in a n i m a l s i n f e c t e d w i t h L C M v i r u s b u t n o t p r e t r e a t e d w i t h P H A . I t is k n o w n t h a t , s i m i l a r l y t o h o m o g r a f t r e j e c t i o n , it is t h e c e l l u l a r i m m u n e r e s p o n s e t h a t g i v e s rise t o t h e n e u r o l o g i c a l s y m p t o m s of i.cer. L C M v i r u s i n f e c t i o n a n d t o t h e f a t a l o u t c o m e of l y m p h o c y t i c c h o r i o m e n i n g i t i s ~. F i n d i n g s of our experiment suggest that PHA treatment applied 1 d a y b e f o r e i.cer. L C M v i r u s i n f e c t i o n e n h a n c e s t h e c e l l u l a r i m m u n e r e a c t i o n of m i c e t o t h e v i r u s i n f e c t i o n . O u r r e s u l t s a r e i n a c c o r d w i t h t h e o b s e r v a t i o n t h a t i.v. P H A t r e a t m e n t a c c e l e r a t e s t h e s k i n g r a f t r e j e c t i o n in mice L

Zusammen/assung. E i n m a l i g e i . v . - I n j e k t i o n y o n P h y t o h ~ m a g g l u t i n i n e r h 6 h t die zellul~ire I m m u n r e a k t i o n d e r M~iuse g e g e n L C M - V i r u s i n f e k t i o n . Zs. B.~NOS, I, SZERI a n d P. ANDERLIK

Institute o/ Microbiology, Semmelweis University o/ 2Vfedicine, Hogyes Endre ut. 7-9, Budapest I X (Hungary), 19 March 7973.

s j. HOTCHIN, Monograph in Virology (Ed. J, L. Melniek; S. Karger, Basel 1971), vol. 3, p. 57.

An Incomplete Anti-B Agglutinin in the Eggs of the Prosobranch Snail Pila o v a t a T h e a l b u m i n g l a n d s of s n a i l s h a v e r e c e n t l y b e c o m e h i g h l y a t t r a c t i v e o b j e c t s of r e s e a r c h , s i n c e t h e y h a v e b e e n f o u n d t o c o n t a i n n o t o n l y g a l a c t o g e n a n d e n z y m e s of g a l a c t o s e m e t a b o l i s m 1, b u t also h e t e r o p h i l e a g g l u t i n i n s t o v a r i o u s a n i m a l cells 3, as well as a n u m b e r of p o l y v a l e n t i s o i n h i b i t o r s of p r o t e i n a s e s s. W e h a v e a l r e a d y s t u d i e d t h e a g g l u t i n i n a n d p r o t e i n a s e - i n h i b i t o r a c t i v i t i e s in t h e a l b u m i n g l a n d of Pomacea urceus ~. I n m o r e r e c e n t w o r k o n t h e e g g s of t h e c l o s e l y r e l a t e d p r o s o b r a n c h JPomacea canaliculata, w e h a v e s h o w n t h a t t h e a g g l u t i n a t i o n p a t t e r n is v i r t u a l l y i d e n t i c a l w i t h t h a t of Pomacea urceus. T h e r e s u l t s a r e s h o w n in T a b l e a. I n h i b i t i o n is f o u n d in b o t h cases w i t h pig amnionic mucoid, peptone A sub-

stance,pneumococcus Type XIV polysaccharide and Nacetyl-D-glucosamine, Mthough small differences were f o u n d f o r t h e a g g l u t i n i n s f r o m t h e t w o s o u r c e s ( T a b l e b). N o i m m u n o l o g i c a l c r o s s - r e a c t i o n s w e r e seen, o n t h e o t h e r h a n d , w i t h t h e e g g e x t r a c t f r o m Pomacea canaliculata

1 W. FISCIIER and H. WEINLAND,Der Stof/wechsel der Galaktose und ihrer Derivate (Thieme VerIag, Stuttgart 19651. O. PROKOP, G. UIILENBRUCtr and W. K611LER, VOX Sang. ld, 321 (1968). s G. UHLENBRUCK, I. SPRE~GER and 1. ISliIYAMA, Z. klin. Chem. 9, 361 (1971). a G. UIILENBRUCKand G. STEINIIAUSEN,Blut 25, 335 (1972).

Table a. Agglutination by red cell agglutinins from prosobraixch snails of the genera Pomaeea and Pila OriNn of red cells

Titer against agglutinin extracts Extract from Red cells

Human A O 13 Horse 13ovine Pigeon Pig Cat Sheep Rabbit Bull frog

PU Normal

PC

PO

PU PC Pronase-treated

PO

PU PC RDE-treated

PO

256 256 128 2 8 -256 -128 1000 8

64 64 128 4 4 4 64 8 -500 --

----4 -4 32 -32 4

16,000 8,000 8,000 , 2 512 2,000 500 500 500 500 64

--16 -128 -128 128 -4000 128

1000 4000 1000 2000 4 500 500 1000 1000 500 32

--16 -8 -16 256 -128 16

500 256 500 8 256 500 1000 32 4 2000 4

256 256 128 16 8 64 4000 256 32 1000 2

Table b. Inhibition of haemagglufination by Red cells from

S XIV polysaecharide Peptone A substance Pig amnionic mucoid D-galactose D-melibiose D-glucose L-rhamnose N-acetyl-D-galactosamine

PU

PC

PO

PU

PC

PO

Human A

Human A

Human B pronasetreated

Pig

Pig

Pig pronase treated

4 64 32 "2 8 --2

128 128 64 ------

-8 128 16 32 2 8 --

-32 2 -8 16 -16

2000 128 128 -2 ----

-2 64 2 -2 ND --

PU, Ponmcea urceus; PC, Pomacea canaliculata; PO, Pile oveta; RDE, neuraminidase (Behring Werke Marburg) ; ND, not determined.