Journal o f Immunological Methods, 14 (1977) 19--24 © Elsevier/North-Holland Biomedical Press
19
P H A G O C Y T O S I S M E A S U R E D A S INHIBITION OF U R I D I N E U P T A K E BY C A N D I D A A L B I C A N S
M. YAMAMURA, JILL BOLER and H. VALDIMARSSON Department o f Immunology, St. Mary's Hospital Medical School, London, W2 1PG, U.K. (Received 9 July 1976, accepted 11 August 1976)
Inhibition of 3H-uridine incorporation into Candida albicans can be used as a sensitive index of phagocytic function because: 1) there is a linear correlation between uridine incorporation and yeast number; 2) phagocytic cells do not incorporate significant amounts of uridine in short term cultures; and 3) C. albicans replicating inside phagocytic cells does not take up uridine from culture medium. Appropriate conditions for measuring phagocytic capacity of human polymorphonuclear cells (PMNs) were 5 x l 0 s C. albicans and 5 x 104 PMNs in 0.5 ml of medium containing 2.5% AB serum. This mixture was incubated for 30 min at 37°C. Aliquots were then transferred into microtiter wells and incubated for a further 60 min in the presence of 3H-uridine. Under these conditions PMN leucocytes from 25 healthy individuals caused suppression of uridine incorporation ranging from 33 to 75% (50 ± 12).
INTRODUCTION F r o m t h e d a y s o f M e t c h n i k o f f ( 1 9 0 5 ) t e c h n i q u e s f o r assessing p h a g o c y t o s i s have m o s t l y b e e n b a s e d on m i c r o s c o p i c a l c o u n t i n g o f i n t r a - a n d e x t r a c e l l u l a r p a r t i c l e s . A n o b j e c t i v e t e c h n i q u e has b e e n d e s c r i b e d b y S t o s s e l ( 1 9 7 3 ) , b u t t h e r e q u i r e m e n t f o r a large n u m b e r o f PMNs (10 X 106 p e r t e s t ) is a m a j o r d r a w b a c k t o a p p l y i n g t h i s m e t h o d in c l i n i c a l s t u d i e s . S e n s i t i s e d r e d cells l a b e l l e d w i t h S l C h r o m i u m have also b e e n u s e d ( K l a u s , 1 9 7 3 ) b u t t h i s t e c h n i q u e m a y n o t be s u i t a b l e f o r m e a s u r i n g o p s o n i z i n g activi t y in s e r u m . In t h i s p a p e r we d e s c r i b e a n e w a s s a y f o r m e a s u r i n g p h a g o c y t o s i s . T h e t e c h n i q u e is o b j e c t i v e a n d e a s y t o p e r f o r m , r e q u i r e s less t h a n 1 m l o f b l o o d a n d c a n be a p p l i e d f o r m e a s u r i n g o p s o n i z i n g a c t i v i t y in s e r u m . MATERIALS AND METHODS 3H-uridine ( s p e c i f i c a c t i v i t y 5 C i / m m o l ) was o b t a i n e d f r o m t h e R a d i o c h e m i c a l C e n t r e , A m e r s h a m , E n g l a n d . LP3 p l a s t i c t u b e s { L u c h a m ) w e r e u s e d f o r m i x i n g C. a l b i c a n s a n d PMNs d u r i n g p h a g o c y t o s i s , w h i l e a l i q u o t s f o r measuring uridine uptake were transferred into microtiter plates (Cooke).
20 RPMI 1640 medium was used t h r o u g h o u t in all experiments. C. Albicans, strain UC 820 was maintained on 2% agar slope containing 1% p e p t o n e (Difco), and 2% glucose. Subcultures were made in 1% p e p t o n e and 2% glucose broth, for 3--4 days at r o o m temperature. The broth cultures gave a h o m o g e n e o u s yeast form of C. albicans which maintained more than 98% viability when kept at 4°C for up to t w o weeks.
Leucocyte preparation PMN leucocytes were prepared from blood, containing 20 units/ml of preservative free heparin, by sedimenting the red cells with Dextran 110 (6% w/v in 0.9% NaC1). The leucocytes were washed five times in medium containing 10 units heparin per ml and finally resuspended to 2.5 X 10 S PMNs per ml. Standard AB serum was kept in liquid nitrogen.
Assay procedure Unless otherwise stated the standard assay procedure was as follows: C. albicans and PMNs were mixed in duplicates in LP3 tubes, each t ube containing 5 X 10 s Candida organisms and 5 X 104 PMNs suspended in 0.5 ml of medium with 2.5% AB serum. Duplicates of 5 X l 0 s C. albicans only in 2.5% serum were always included and ot her controls as appropriate. The tubes were incubated u n d er constant r ot a t i on for 30 min at 37°C. Aliquots of 0.1 ml were then transferred to microtiter wells 5 gl of 3H-uridine (10 pCi/ml) added, and the mic r ot i t er plates incubated for a furt her 60 min at 37°C. Cell associated radioactivity was t hen collected o n t o glass fibre discs with Skatron harvester, placed into toluene--PPO scintillation fluid and c o u n t e d in a Packard spectrometer, model B2450.
Presentation o f data Replicate variability was 7%. Results were expressed as per cent inhibition of uridine in co r p or at i on by the following formula: 100 -- cpm (C. albicans + PMNs) X 100 cpm (C. albicans only) RESULTS
Can phagocytosed C. albicans incorporate 3H-uridine from culture medium For answering this question we used patients with chronic granulomatous disease {CGD), whose PMNs are know n to have normal capacity to phagocytose but are not capable of killing or preventing germination of ingested C.
21 TABLE 1 Effects of PMN leucocytes from CGD patients on uridine incorporation by C. albicans. PMN leucocyte donor
CGD patient (R.Y.) CGD patient (P.Y.) Mother and CGD patients Healthy control Normal ranges
Per cent Candida killing Methylene blue
s 1Cr release *
Per cent inhibition of uridine incorporation
7 0 46 45 ND
1 2 15 60 35--71
45 44 44 46 33--75
* Yamamura et al., 1976.
albicans ( L e h r e r , 1 9 7 0 ) . It was f o u n d t h a t PMNs f r o m 2 such p a t i e n t s , while u n a b l e to release s lCr f r o m C. albicans o r g a n i s m s , h a d an i n h i b i t o r y e f f e c t on t h e i r uridine i n c o r p o r a t i o n to the s a m e e x t e n t as did PMNs f r o m h e a l t h y c o n t r o l subjects (table 1). I t was c o n c l u d e d t h e r e f o r e , t h a t p h a g o c y t o s e d C. albicans c o u l d n o t i n c o r p o r a t e 3H-uridine f r o m t h e c u l t u r e m e d i u m . Incorporation of 3H-uridine by C. albicans Fig. 1 s h o w s t h e i n c o r p o r a t i o n o f 3H-uridine b y v a r y i n g n u m b e r s o f C. albicans i n c u b a t e d at 3 7 ° C f o r 1 h. T h e r e was a linear rise in uridine u p t a k e w i t h increasing n u m b e r s o f C. albicans o r g a n i s m s f r o m 1 X 104 t o 1 X 106 p e r well. T h e n u m b e r s o f e x t r a - a n d intra-cellular C. albicans c o u l d t h e r e f o r e be c a l c u l a t e d f r o m such a s t a n d a r d c u r v e p r o v i d e d t h a t b e t w e e n 1 X 104 a n d 1 X 106 o r g a n i s m s r e m a i n e d u n p h a g o c y t o s e d . A m i x t u r e c o n t a i n i n g 1 X l 0 s C. albicans and 1 X 104 PMNs p e r m i c r o t i t e r - w e l l i n c o r p o r a t e d uridine corres p o n d i n g to a p p r o x i m a t e l y 5 X 104 u n c o n s u m e d y e a s t o r g a n i s m s (50%). It s h o u l d be n o t e d t h a t 2.5% s e r u m significantly increased t h e uridine i n c o r p o r a t i o n c o m p a r e d w i t h s e r u m free m e d i u m (fig. 1).
Serum requirements C. albicans was o p t i m a l l y o p s o n i z e d in 2.5% s e r u m (fig. 2), and this c o n c e n t r a t i o n was t h e r e f o r e used in s u b s e q u e n t p h a g o c y t o s i s assays. T h e r e was no significant d i f f e r e n c e b e t w e e n t h e o p s o n i z i n g a c t i v i t y o f s e r u m and h e p a rinized p l a s m a at a n y c o n c e n t r a t i o n . P h a g o c y t o s i s , while a b o l i s h e d in 0.01 M E D T A , was p r e s e n t in s e r u m h e a t e d at 56°C f o r 30 m i n , b u t m i n i m a l p h a g o c y t i c killing was o b s e r v e d u n d e r t h e s e c o n d i t i o n s (table 2). Effects o f varying PMN/C. albicans ratio Fig. 3 s h o w s curves f o r uridine i n c o r p o r a t i o n in 2 e x p e r i m e n t s w h e r e increasing n u m b e r s o f P M N l e u c o c y t e s (4 X 103 t o 4 X l 0 s) w e r e i n c u b a t e d
22
o o
o 2.5% o Serum
Serum Absent
CPM.
10/.
103
102j, 104
105
106
NO, of C. albicans/well
Fig. 1. Incorporation o f 3H-uridine b y varying numbers o f C. albicans incubated at 37°C for 1 h. [] El, Serum free m e d i u m , o o, 2.5% serum present.
100 c-CZ)
c-¢..-
,~ 50 (39 (_...)
0
0,3125
0,625 '
1,25 '
2,5 '
5
1'0
Log 2 Concentration of Plasma ( % ) Fig. 2. Effects o f different plasma concentrations on the incorporation o f ]H/uridine by C. albicans in 3 different experiments. The PMN/C. albicans ratio was 1/5.
23 TABLE 2 Serum requirements for phagocytosis and killing of C. albicans.
Untreated serum Heated serum (56°C for 30 min) Serum + 0.01 M EDTA Medium without serum
Per cent inhibition of uridine incorporation
Per cent chromium release *
Exp. 1
Exp. 2
Exp. 1
Exp. 2
55 46 2 3
67 56 1 7
60 1 2 2
69 2 3 1
* Yamamura et al., 1976. w i t h 4 X 1 0 S C. albicans. T h e r e was a p p r o x i m a t e l y 5 0 % i n h i b i t i o n o f u r i d i n e u p t a k e a t a P M N / C . albicans r a t i o o f 1 / 1 0 a n d t h i s r a t i o was t h e r e f o r e s u b s e q u e n t l y used in the s t a n d a r d assay for p h a g o c y t i c c a p a c i t y of PMN leucocytes.
N o r m a l ranges PMN leucocytes from 25 healthy individuals were tested for phagocytic c a p a c i t y i n t h e p r e s e n c e o f 2 . 5 % s t a n d a r d A B s e r u m . A t a P M N / C . albicans
Fixed No. of C.albicans
4xi05
I00
O
Z
1
•
,
. . . .
,
,
.
.
i
.
.
104
.
.
I
105
.
.
.
,
. . . .
L
106
NO, of PINs
Fig. 3. Two experiments in which PMN/C. albicans ratios were varied from 1/100 to 1/1. There was approximately 50% inhibition of uridine incorporation at a PMN/C. albicans ratio of 1/10.
24 r a t i o o f 1/5, a range o f 74 t o 99% i n h i b i t i o n was o b s e r v e d w i t h a m e d i a n o f 92%. A r a t i o o f 1 / 1 0 gave a Poisson d i s t r i b u t i o n ranging f r o m 33 to 75% w i t h a m e a n o f 50%. DISCUSSION This p a p e r describes a n e w m e t h o d f o r o b j e c t i v e a s s e s s m e n t o f p h a g o c y t o sis. It is b a s e d on the o b s e r v a t i o n t h a t C. albicans replicating inside phagoc y t i c cells c a n n o t i n c o r p o r a t e e x t r a c e l l u l a r uridine, while t h e r e is a linear rel a t i o n s h i p b e t w e e n t h e n u m b e r o f n o n - p h a g o c y t o s e d y e a s t a n d uridine upt a k e . It is t h e r e f o r e possible t o use a s t a n d a r d curve f o r e s t i m a t i n g the n u m b e r o f C. albicans p h a g o c y t o s e d a n d t h e r e b y t o calculate t h e average n u m ber o f yeasts ingested b y each PMN l e u c o c y t e . A t a PMN/C. albicans ratio o f 1 / 1 0 a n d a s e r u m c o n c e n t r a t i o n o f 2.5%, a p p r o x i m a t e l y 50% o f t h e y e a s t is ingested, c o r r e s p o n d i n g to an average i n t a k e o f 5 o r g a n i s m s p e r p h a g o c y t e . This assay p r i n c i p l e can p r o b a b l y be used f o r m e a s u r i n g p h a g o c y t o s i s o f o t h e r m i c r o - o r g a n i s m s . It has several d i s t i n c t a d v a n t a g e s such as sensitivity and o b j e c t i v e end p o i n t . T h e small a m o u n t s o f b l o o d r e q u i r e d m a k e s the m e t h o d p a r t i c u l a r l y suitable for clinical use. We are c u r r e n t l y using this t e c h n i q u e t o g e t h e r with a p r e v i o u s l y r e p o r t e d m e t h o d f o r m e a s u r i n g c a n d i d a c i d a l a c t i v i t y ( Y a m a m u r a et al., 1 9 7 6 ) in o r d e r to analyse t h e various f a c t o r s involved in o p s o n i z a t i o n and p h a g o c y t i c killing o f C. albicans. REFERENCES Klaus, G.G., 1973, Exp. Cell. Res. 79, 73 Lehrer, R.I., 1970, Infect. Immunol. 2, 42 Metchnikoff, E., 1905, Immunity in Infectious Disease Cambridge University Press (Translated by F.G. Binnie) Stossel, T.P., 1973, Blood 42, 121 Yamamura, M., J. Boler and H. Valdimarsson, 1976, J. Immunol. Methods (in press)
19
P H A G O C Y T O S I S M E A S U R E D A S INHIBITION OF U R I D I N E U P T A K E BY C A N D I D A A L B I C A N S
M. YAMAMURA, JILL BOLER and H. VALDIMARSSON Department o f Immunology, St. Mary's Hospital Medical School, London, W2 1PG, U.K. (Received 9 July 1976, accepted 11 August 1976)
Inhibition of 3H-uridine incorporation into Candida albicans can be used as a sensitive index of phagocytic function because: 1) there is a linear correlation between uridine incorporation and yeast number; 2) phagocytic cells do not incorporate significant amounts of uridine in short term cultures; and 3) C. albicans replicating inside phagocytic cells does not take up uridine from culture medium. Appropriate conditions for measuring phagocytic capacity of human polymorphonuclear cells (PMNs) were 5 x l 0 s C. albicans and 5 x 104 PMNs in 0.5 ml of medium containing 2.5% AB serum. This mixture was incubated for 30 min at 37°C. Aliquots were then transferred into microtiter wells and incubated for a further 60 min in the presence of 3H-uridine. Under these conditions PMN leucocytes from 25 healthy individuals caused suppression of uridine incorporation ranging from 33 to 75% (50 ± 12).
INTRODUCTION F r o m t h e d a y s o f M e t c h n i k o f f ( 1 9 0 5 ) t e c h n i q u e s f o r assessing p h a g o c y t o s i s have m o s t l y b e e n b a s e d on m i c r o s c o p i c a l c o u n t i n g o f i n t r a - a n d e x t r a c e l l u l a r p a r t i c l e s . A n o b j e c t i v e t e c h n i q u e has b e e n d e s c r i b e d b y S t o s s e l ( 1 9 7 3 ) , b u t t h e r e q u i r e m e n t f o r a large n u m b e r o f PMNs (10 X 106 p e r t e s t ) is a m a j o r d r a w b a c k t o a p p l y i n g t h i s m e t h o d in c l i n i c a l s t u d i e s . S e n s i t i s e d r e d cells l a b e l l e d w i t h S l C h r o m i u m have also b e e n u s e d ( K l a u s , 1 9 7 3 ) b u t t h i s t e c h n i q u e m a y n o t be s u i t a b l e f o r m e a s u r i n g o p s o n i z i n g activi t y in s e r u m . In t h i s p a p e r we d e s c r i b e a n e w a s s a y f o r m e a s u r i n g p h a g o c y t o s i s . T h e t e c h n i q u e is o b j e c t i v e a n d e a s y t o p e r f o r m , r e q u i r e s less t h a n 1 m l o f b l o o d a n d c a n be a p p l i e d f o r m e a s u r i n g o p s o n i z i n g a c t i v i t y in s e r u m . MATERIALS AND METHODS 3H-uridine ( s p e c i f i c a c t i v i t y 5 C i / m m o l ) was o b t a i n e d f r o m t h e R a d i o c h e m i c a l C e n t r e , A m e r s h a m , E n g l a n d . LP3 p l a s t i c t u b e s { L u c h a m ) w e r e u s e d f o r m i x i n g C. a l b i c a n s a n d PMNs d u r i n g p h a g o c y t o s i s , w h i l e a l i q u o t s f o r measuring uridine uptake were transferred into microtiter plates (Cooke).
20 RPMI 1640 medium was used t h r o u g h o u t in all experiments. C. Albicans, strain UC 820 was maintained on 2% agar slope containing 1% p e p t o n e (Difco), and 2% glucose. Subcultures were made in 1% p e p t o n e and 2% glucose broth, for 3--4 days at r o o m temperature. The broth cultures gave a h o m o g e n e o u s yeast form of C. albicans which maintained more than 98% viability when kept at 4°C for up to t w o weeks.
Leucocyte preparation PMN leucocytes were prepared from blood, containing 20 units/ml of preservative free heparin, by sedimenting the red cells with Dextran 110 (6% w/v in 0.9% NaC1). The leucocytes were washed five times in medium containing 10 units heparin per ml and finally resuspended to 2.5 X 10 S PMNs per ml. Standard AB serum was kept in liquid nitrogen.
Assay procedure Unless otherwise stated the standard assay procedure was as follows: C. albicans and PMNs were mixed in duplicates in LP3 tubes, each t ube containing 5 X 10 s Candida organisms and 5 X 104 PMNs suspended in 0.5 ml of medium with 2.5% AB serum. Duplicates of 5 X l 0 s C. albicans only in 2.5% serum were always included and ot her controls as appropriate. The tubes were incubated u n d er constant r ot a t i on for 30 min at 37°C. Aliquots of 0.1 ml were then transferred to microtiter wells 5 gl of 3H-uridine (10 pCi/ml) added, and the mic r ot i t er plates incubated for a furt her 60 min at 37°C. Cell associated radioactivity was t hen collected o n t o glass fibre discs with Skatron harvester, placed into toluene--PPO scintillation fluid and c o u n t e d in a Packard spectrometer, model B2450.
Presentation o f data Replicate variability was 7%. Results were expressed as per cent inhibition of uridine in co r p or at i on by the following formula: 100 -- cpm (C. albicans + PMNs) X 100 cpm (C. albicans only) RESULTS
Can phagocytosed C. albicans incorporate 3H-uridine from culture medium For answering this question we used patients with chronic granulomatous disease {CGD), whose PMNs are know n to have normal capacity to phagocytose but are not capable of killing or preventing germination of ingested C.
21 TABLE 1 Effects of PMN leucocytes from CGD patients on uridine incorporation by C. albicans. PMN leucocyte donor
CGD patient (R.Y.) CGD patient (P.Y.) Mother and CGD patients Healthy control Normal ranges
Per cent Candida killing Methylene blue
s 1Cr release *
Per cent inhibition of uridine incorporation
7 0 46 45 ND
1 2 15 60 35--71
45 44 44 46 33--75
* Yamamura et al., 1976.
albicans ( L e h r e r , 1 9 7 0 ) . It was f o u n d t h a t PMNs f r o m 2 such p a t i e n t s , while u n a b l e to release s lCr f r o m C. albicans o r g a n i s m s , h a d an i n h i b i t o r y e f f e c t on t h e i r uridine i n c o r p o r a t i o n to the s a m e e x t e n t as did PMNs f r o m h e a l t h y c o n t r o l subjects (table 1). I t was c o n c l u d e d t h e r e f o r e , t h a t p h a g o c y t o s e d C. albicans c o u l d n o t i n c o r p o r a t e 3H-uridine f r o m t h e c u l t u r e m e d i u m . Incorporation of 3H-uridine by C. albicans Fig. 1 s h o w s t h e i n c o r p o r a t i o n o f 3H-uridine b y v a r y i n g n u m b e r s o f C. albicans i n c u b a t e d at 3 7 ° C f o r 1 h. T h e r e was a linear rise in uridine u p t a k e w i t h increasing n u m b e r s o f C. albicans o r g a n i s m s f r o m 1 X 104 t o 1 X 106 p e r well. T h e n u m b e r s o f e x t r a - a n d intra-cellular C. albicans c o u l d t h e r e f o r e be c a l c u l a t e d f r o m such a s t a n d a r d c u r v e p r o v i d e d t h a t b e t w e e n 1 X 104 a n d 1 X 106 o r g a n i s m s r e m a i n e d u n p h a g o c y t o s e d . A m i x t u r e c o n t a i n i n g 1 X l 0 s C. albicans and 1 X 104 PMNs p e r m i c r o t i t e r - w e l l i n c o r p o r a t e d uridine corres p o n d i n g to a p p r o x i m a t e l y 5 X 104 u n c o n s u m e d y e a s t o r g a n i s m s (50%). It s h o u l d be n o t e d t h a t 2.5% s e r u m significantly increased t h e uridine i n c o r p o r a t i o n c o m p a r e d w i t h s e r u m free m e d i u m (fig. 1).
Serum requirements C. albicans was o p t i m a l l y o p s o n i z e d in 2.5% s e r u m (fig. 2), and this c o n c e n t r a t i o n was t h e r e f o r e used in s u b s e q u e n t p h a g o c y t o s i s assays. T h e r e was no significant d i f f e r e n c e b e t w e e n t h e o p s o n i z i n g a c t i v i t y o f s e r u m and h e p a rinized p l a s m a at a n y c o n c e n t r a t i o n . P h a g o c y t o s i s , while a b o l i s h e d in 0.01 M E D T A , was p r e s e n t in s e r u m h e a t e d at 56°C f o r 30 m i n , b u t m i n i m a l p h a g o c y t i c killing was o b s e r v e d u n d e r t h e s e c o n d i t i o n s (table 2). Effects o f varying PMN/C. albicans ratio Fig. 3 s h o w s curves f o r uridine i n c o r p o r a t i o n in 2 e x p e r i m e n t s w h e r e increasing n u m b e r s o f P M N l e u c o c y t e s (4 X 103 t o 4 X l 0 s) w e r e i n c u b a t e d
22
o o
o 2.5% o Serum
Serum Absent
CPM.
10/.
103
102j, 104
105
106
NO, of C. albicans/well
Fig. 1. Incorporation o f 3H-uridine b y varying numbers o f C. albicans incubated at 37°C for 1 h. [] El, Serum free m e d i u m , o o, 2.5% serum present.
100 c-CZ)
c-¢..-
,~ 50 (39 (_...)
0
0,3125
0,625 '
1,25 '
2,5 '
5
1'0
Log 2 Concentration of Plasma ( % ) Fig. 2. Effects o f different plasma concentrations on the incorporation o f ]H/uridine by C. albicans in 3 different experiments. The PMN/C. albicans ratio was 1/5.
23 TABLE 2 Serum requirements for phagocytosis and killing of C. albicans.
Untreated serum Heated serum (56°C for 30 min) Serum + 0.01 M EDTA Medium without serum
Per cent inhibition of uridine incorporation
Per cent chromium release *
Exp. 1
Exp. 2
Exp. 1
Exp. 2
55 46 2 3
67 56 1 7
60 1 2 2
69 2 3 1
* Yamamura et al., 1976. w i t h 4 X 1 0 S C. albicans. T h e r e was a p p r o x i m a t e l y 5 0 % i n h i b i t i o n o f u r i d i n e u p t a k e a t a P M N / C . albicans r a t i o o f 1 / 1 0 a n d t h i s r a t i o was t h e r e f o r e s u b s e q u e n t l y used in the s t a n d a r d assay for p h a g o c y t i c c a p a c i t y of PMN leucocytes.
N o r m a l ranges PMN leucocytes from 25 healthy individuals were tested for phagocytic c a p a c i t y i n t h e p r e s e n c e o f 2 . 5 % s t a n d a r d A B s e r u m . A t a P M N / C . albicans
Fixed No. of C.albicans
4xi05
I00
O
Z
1
•
,
. . . .
,
,
.
.
i
.
.
104
.
.
I
105
.
.
.
,
. . . .
L
106
NO, of PINs
Fig. 3. Two experiments in which PMN/C. albicans ratios were varied from 1/100 to 1/1. There was approximately 50% inhibition of uridine incorporation at a PMN/C. albicans ratio of 1/10.
24 r a t i o o f 1/5, a range o f 74 t o 99% i n h i b i t i o n was o b s e r v e d w i t h a m e d i a n o f 92%. A r a t i o o f 1 / 1 0 gave a Poisson d i s t r i b u t i o n ranging f r o m 33 to 75% w i t h a m e a n o f 50%. DISCUSSION This p a p e r describes a n e w m e t h o d f o r o b j e c t i v e a s s e s s m e n t o f p h a g o c y t o sis. It is b a s e d on the o b s e r v a t i o n t h a t C. albicans replicating inside phagoc y t i c cells c a n n o t i n c o r p o r a t e e x t r a c e l l u l a r uridine, while t h e r e is a linear rel a t i o n s h i p b e t w e e n t h e n u m b e r o f n o n - p h a g o c y t o s e d y e a s t a n d uridine upt a k e . It is t h e r e f o r e possible t o use a s t a n d a r d curve f o r e s t i m a t i n g the n u m b e r o f C. albicans p h a g o c y t o s e d a n d t h e r e b y t o calculate t h e average n u m ber o f yeasts ingested b y each PMN l e u c o c y t e . A t a PMN/C. albicans ratio o f 1 / 1 0 a n d a s e r u m c o n c e n t r a t i o n o f 2.5%, a p p r o x i m a t e l y 50% o f t h e y e a s t is ingested, c o r r e s p o n d i n g to an average i n t a k e o f 5 o r g a n i s m s p e r p h a g o c y t e . This assay p r i n c i p l e can p r o b a b l y be used f o r m e a s u r i n g p h a g o c y t o s i s o f o t h e r m i c r o - o r g a n i s m s . It has several d i s t i n c t a d v a n t a g e s such as sensitivity and o b j e c t i v e end p o i n t . T h e small a m o u n t s o f b l o o d r e q u i r e d m a k e s the m e t h o d p a r t i c u l a r l y suitable for clinical use. We are c u r r e n t l y using this t e c h n i q u e t o g e t h e r with a p r e v i o u s l y r e p o r t e d m e t h o d f o r m e a s u r i n g c a n d i d a c i d a l a c t i v i t y ( Y a m a m u r a et al., 1 9 7 6 ) in o r d e r to analyse t h e various f a c t o r s involved in o p s o n i z a t i o n and p h a g o c y t i c killing o f C. albicans. REFERENCES Klaus, G.G., 1973, Exp. Cell. Res. 79, 73 Lehrer, R.I., 1970, Infect. Immunol. 2, 42 Metchnikoff, E., 1905, Immunity in Infectious Disease Cambridge University Press (Translated by F.G. Binnie) Stossel, T.P., 1973, Blood 42, 121 Yamamura, M., J. Boler and H. Valdimarsson, 1976, J. Immunol. Methods (in press)
