Relationship between ANS fluorescence and electrokinetic potential of activated lymphocytes

Life Sciences, Vol . 24, pp . 1183-1194 Printed in the U .S .A .

RELATIONSHIP BETWEEN ANS FLUORESCENCE POTENTIAL OF

Pergamon Press

AND ELECTROKINETIC

ACTIVATED LYMPHOCYTES

Jacques Vaillier, Jean Lematre, Dominique Vaillier and Mireille Donner INSERM - Unit of Experimental Cancerology and Radiobiology Plateau de Brabois, 54500 Vandoeuvre-lès-Nancy, France . (Received in final form February 15, 1979) SUMMARY An immune response was induced in vivo on C3H/He d'mouse strain with Bovine Serum Albumin (BSA), or Sheep Red Blood Cells (SRBC) . The membrane fluorescence changes of activated splenic lymphocytes were studied two weeks after the injection of antigens . Experiments were performed with the hydrophobic fluorescent probe : 1-anilino-4-naphthalene sulphonate (ANS) . The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on antigens . Their fluorescence intensities were lower than control values . A maximum decrease of fluorescence was recorded on days 1 , 6 and 9 after immunization with BSA-stimulated lymphocytes . SRBC-stimulated lymphocytes exhibited a maximum ANS fluorescence decrease on days 4 and 9 after immunization . These fluorescence phenomena would be in an inverse relationship with the electrokinetic surface potential of activated lymphocytes, as assessed by the electrophoretic mobility analysis (EPM) . Some parameters affecting the ANS fluorescence in T and B cells are discussed . Quantification of hydrophobic sites in splenic cells would indicate that forces other than the hydrophobic ones may also be involved in the dyebinding changes following immune activation . The major functions of biological membranes are known, and the structural framework for various processes are essential for living cells . Also, the conformation of the membranes, and conformational changes in biological systems are of great interest with respect to their functions . Many fluorescent dyes have been used as probes of structural changes in proteins and membranes (1,2,3,4,5,6,7,$) . The validity of the use of fluorescent probes in the study of membrane changes has been discussed by Radda and Vanderkooi (5) . These aromatic compounds are not fluorescent in water, they become highly fluorescent when non-covalently bound to some proteins or membranes . One of the chromophores which presented this effect was 1-anilino-d-naphthalene sulphonate (ANS) . The increase in quantum yield of fluorescence seems to occur due to the changes in the micropolarity in the vicinity of the ANS molecule (6) . Numerous studies suggest that ANS can be used in a study of conformational changes (as well on free membranes as on proteins) (3,4,5,6,7, 4,9,11) . However, until recently, only a few experiments have been performed on living cells (9,10,12) . I t was of great interest to consider an immune response at the subcellular level of lymphocytes, according to their ANS-fluorescence changes after an antigenic stimulation . This study of the kinetics of such changes perhaps could help in the characterization of another parameter of the immune response . Using another technique, it was possible to measure the electrokinetic potential of lymphocytes . This electrokinetic potential is an estimating value of the surface electrostatic potential : this is an important functional property of biological membranes . It determines the partition ions between the aqueous phase and the membrane surface, and thus affects their binding, and it undoubtedly plays a main role in processes such a membrane aggregation, fusion, 0024-3205/79/131183-10$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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and cellular interactions . These phenomena are important, particularly in the course of the immune process . These investigations showed that there exists an inverse relationship between intensity of ANS bound to activated lymphocytes and their electrokinetic surface potential . MATERIALS AND METHODS Animals Male mice of inbred strain C3H/He were used (Centre de Sélection et d'Elevage des Animaux de Laboratoire, Orléans-la-Source, France) . They were about two months old, and weighted 25 _+ 2 g . Food and water were given ad libitum . The animals were caged in groups of 10, temperature was maintained at 20 + 1°C . Immunization procedure Antigens were given by a single intravenous injection . The first group received 0 .5 mg BSA (Miles Laboratories Inc ., Fraction V) in 0 .4 ml PBS pH 7 .2 . SRBC (Institut Pasteur Production, Paris) were washed three times in PBS solution before injection . 10 8 SRBC/0 .4 ml were injected to a second group of mice . Control mice received only 0 .4 rnl PBS . _ANS

This was used as an ammonium salt, purchased from Sigma Laboratories . The compound was used in chrometographically pure form . In a previous study (10), it was found that with the concentration of ANS used (25 .10 -5 M), there was no cell lysis : the number of viable cel Is was always greater than 90 °lo at the end of experiments . Abreover, ANS in low concentration produces minor effects on the structure of the membrane, as assessed by Lesslauer et al . (13) and Layton et al . (14) . Cell preparation s Three spleens of : control, BSA- and SRBC-immunized mice were removed . After a gentle homogenization with a glass Potter, splenic cells were washed once in RPMI 1640 medium . Purified splenic lymphocyte suspensions were obtained by separation on Ficoll-Isopaque of 1 .09 g/ml density . A ratio of 2 : 3 splenic cell suspension : Ficoll-Isopaque was used . The siliconized tubes of 15 mi were centrifuged for 30 mn at 20 ° C with an interface strength of 400 g . The cells of the interface were collected with a Pasteur pipette ; the suspension was diluted 5 times, centrifuged and washed 3 times in PBS pH 7 .2 . Viability was 95-97~ . Fluorescence spectrophotometr y Every day, after immunization, 10 7 splenic, stimulated and control lymphocytes were incubated with ANS at room temperature, in a final volume of 3 ml PBS (pH 7 .2) . The fluorescence intensity was measured when the rate of increase became negligible (10) . The small contribution of fluorescence from ANS alone in the buffer was deducted . Fluorescence emission was measured in a 1 cm quartz cuvette at 90° with respect to the incident beam, using an Optica model 115 fluospectrophotometer . Excitation was at 396 nm . Excitation slit of 35 nm and emission slit of 20 nm . Emission was at 480 nm with ANS-cells ; at 51d nm with ANS solution alone . Arbitrary units used to measure fluorescence intensity were the same for all experiments . All measurements were carried out at room temperature (20°C) . Cell electrophoresis (electrokinetic potential ) The electrophoresic mobility (EPM) was determined by the use of an electrophoretic chamber of a circular cross section and a small volume (0 .8 ml) equipped with reversible silver-silver chloride-potassium electrodes (1 .0 M) at 25°C . All measurements were carried out in NaC) 0 .145 M adjusted to pH 7 .2 with sodium hydrogenocarbonate and the EPM was expressed in um sec -l V -l cm .

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It is established that mouse spleen cells exhibit a bimodal electrophoretic distribution, corresponding to B and T lymphocytes (24,25) . So, we have calculated the total mobility of B lymphocytes, the total mobility of T lymphocytes . The sum of both results divided by the number of all counted cells gave the general mean EPM (M) . (Thus, the comparison between fluorescence and the whole mean EPM is homogeneous) . The mobility of human washed erythrocytes was determined before and after each experiment to monitor the reliable performance of the apparatus and was found to be : - 1 .0$ _+ 0 .03 y~m . sec - lV - lcm . In each experiment, at least 140 cells were scored . The relation between ~ (zeta potential) and M is thus given as follows (15) : 4nq

M

E

12 .85 M

in conditions NaCI 0 .145 M, pH 7 .2, and 25°C, we obtain : 1 "vl

Number of ANS binding sites Binding studies of ANS were performed by measuring the concentration of the dye remaining in the supernatant fluid after centrifugation of lymphocytes and substracting it from the concentration of added dye . Free ANS, was measured with a Jobin 6 Yvon Duospac 203 spectrophotometer at 219 .6 nm . RESULTS Fluorescence changes in BSA-stimulated lymphocytes Figure lA illustrates the fluorescence changes in BSA-stimulated lymphocytes (measured as a plateau value) during a period of 14 days after inoculation of BSA . The curves are polyphasic, with maxima and minima values . On days 1, 6 and 9 after immunization, there was a decrease in mean fluorescence plateau intensity of spleen cel Is, reaching a maximum value on day 11 . On day 12, the fluorescence values approached the control values again . Each point represents the average + S .E . of 4 separate experiments in the both cases of the control and BSA-stimulated lymphocytes .

Fig . 1 Kinetics of ANS fluorescence and EPM ( ~, ) changes after BSA immunization (splenic lymphocytes ) Curve (A) : changes in fluorescence intensity (arbitrary units)( " ") BSA-stimulated cells,(- ) control cells Curve (B) : changes in mean EPM ( m .sec -l V - lcm) or zeta potential 12 .85 mV) . (*--~*) BSAstimulated cells, (*-~-~) control cells . \

-+.os!

161 ~\\

/

I

1 1

i

-o.es-oszo o ~ v

e

s

~o

~

m

na oys

118 6

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ANS-Bound Lymphocytes

Fluorescence changes in SRBC-stimulated lymphocytes Figure 2A shows the fluorescence intensity changes of splenic SRBC-stimulated lymphocytes . The curves are clearly polyphasic . On days 4 and 9 after immunization, there was a minimum value in fluorescence intensity of splenic lymphocytes, reaching a maximum value on day 12 . On day 13, the fluorescence values approached the control values . The fluorescence intensity was always lower than control values up to day 10 . The decrease or increase of fluorescence could not be attributed to the stress due to injection alone . Indeed, splenic control lymphocytes from mice injected with 0 .4 ml PBS presented the same fluorescence intensity during a period of 14 days . Each point on the Figure represents the average of 4 determinations .

-o.~e1 0

2

4

6

8

10

12

14

0"h

Fig . 2 fluorescence and EPM ( ~ ) changes, after SRBC-immunization (splenic Kinetics of ANS l ymphocytes ) " ) SRBC-stirtuCurve (A) : changes in fluorescence intensity (arbitrary units) . (" lated cells ; (~--) control cells . Curve (B) : changes in mean EPM m .sec -l V -l cm) or ~, potential (x 12 .85 m V) . (*---~) SRBC-stimulated cells ; ~*-~-~-) control cells .

Electrokinetic potential changes ; correlation between fluorescence and zeta potentia l Figure 1B shows the variations of zeta potential of splenic lymphocytes, over 14 days after immunization with BSA . Each point is the mean of 4 separate experiments . Three waves appear, indicating a possible regulatory mechanism . The mean EPM (or ~ ) increases by 1096 on day 1 , falls on the 4th, and rises again on the 6th and 9th days . It returns to the control level on the 12th day . In comparison with the daily fluorescence changes (Figure lA), it should be noted that :

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a weak fluorescence corresponds to a high zeta potential ; a strong fluorescence corresponds to a low ~ . With respect to an imaginary horizontal axis, the curves lA and 1B present an inverse shape . An inverse relationship appears between fluorescence intensity and zeta potential . The zeta potential evolution of SRBC-stimulated lymphocytes is presented in Figure 2B . Each point is mean of 4 separate experiments . ~ increases until the 4th day . There is then a plateau between the 4th and the 9th days . I t returns to the control value on day 10 . An inverse relationship appears also, again between fluorescence intensity and zeta potential of SRBC-stimulated lyrr.phocytes . The linear regression (straight line), shown in Figure 3 confirms the correlation between fluorescence intensity and mean EPM or ; . These data give the correlation factor of - 0 .96 .

30 ~

a

20 -

-0 .92

-0 .5

_7

-1A5

-7~1

( " ~ttilittl

Fig . 3 Correlation between electrophoretic mobility and fluorescence intensity of stimulated splenic lymphocytes . Correlation coefficient : r = - 0 .96 .

The electrophoretic distribution profile of activated lymphocytes The observed correlation between mean EPM and apparent ANS binding may be incidental to a shift in the distribution of the lymphocyte population, because the EPM is bimodally distributed . Table I shows the mobility of lymphocytes according to the number of days after immunization with BSA . Cells with a mobility greater than - 1 .00}~m .sec-lV-lcm (in our conditions) were classified as "fast" (T cells) and those with a lower mobility as "slow" (B cells) . The results evide~5ced that there is a shift in the distribution of the lymphocyte subpopulations, but it is not associated with fluorescence . The mean EPM of the slow lymphocytes changed after immunization . The mean EPM of the fast cells showed less variations . Moreover, the proportion of B cells rapidly decreased to a value of 3$% on the 1st day, and regularly increased for the following days . Neighbour results were observed on SRBC-activated lymphocytes (Table I I) . However, the mean EPM of the T cells showed weak variations with respect to B cells . The proportion of B cells decreased to reach a value of 36°k on the 6th day, and regularly increased for the following days .

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Table I Electrophoretic mobility* (y.~m .sec . -l V -1 cm) of splenic lymphocytes from C3H/He mice immunized on day 0 with BMA antigen ; compared to cells from normal C3H/He mice (day 0)

days after immunization

general mean EPM _+ SE

0

- 0 .96 ± 0 .02

1

Splenic Slow EPM + SE

lymphocytes

Fast EPM + SE

Percentage** slow

-0 .78+0 .02 (316)

-1 .19+0 .03 (244)

~±3

- 1 .06 + 0 .02

-0 .81 +0 .02 (228)

- 1 .21 +0 .03 (372)

38 + 4

2

- 1 .04 + 0 .02

- 0 .79 + 0 .03 (224)

- 1 .20 _+ 0 .02 (364)

38 .5 + 4

3

- 1 .O1 + 0 .02

- 0 .73 + 0 .02 (236)

- 1 .20 + 0 .03 (376)

38 .5 + 4

4

- 0 .93 + 0 .03

5

- 0 .98 + 0 .02

-0 .64+0 .03 (248) -0 .72+0 .02 (252)

-1 .10+0 .03 (372) -1 .20+0 .02 (340)

6

- 1 .02 + 0 .01

-0 .78+0 .01 (260)

-1 .20_+0 .01 (320)

45 ± 3

7

- 1 .O1 ± 0 .02

-0 .77+0 .01 (276)

-1 .23+0 .01 (312)

47 ± 4

8

- 1 .02 + 0 .02

-0 .81 +0 .01 (3~4)

-1 .24+0 .02 (2t34)

51 .5 + 3

9

- 1 .05 + 0 .02

-0 .85+0 .02 (324)

-1 .28+0 .03 (2134)

53 .5 + 4

10

- 0 .95 + 0 .03

-0 .77_+0 .03 (344)

-1 .17+0 .02 (2130)

55 + 3

11

- 0 .92 + 0 .02

-0 .74+0 .02 (3~8)

-1 .15+0 .01 (256)

55 + 4

12

- 0 .95 ± 0 .02

-0 .77+0 .02 (352)

-1 .18+0 .02 (276)

~ ±3

13

- 0 .96 ± 0 .02

-0 .78+0 .02 (32d)

-1 .19_+0 .02 (260)

~ ±3

14

- 0 .96 ± 0 .02

-0 .77+0 .03 (320)

-1 .19+0 .02 (252)

56 ± 3

40 + 5 42 .5 + 3

* Number of cells examined between brackets . ** The percentage of fast lymphocytes is the complement to 100 .

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Table I I Electrophoretic mobility*~y m .sec -l V -l cm) of splenic lymphocytes from C3H/He mice immunized on day 0 with SRBC antigen ; compared to cells from normal C3H/He mice (day 0)

days after immunization

mean EPM _+ SE

0

-0 .96+0 - .02

1

Splenic lymphocytes Slow EPM + SE

Fast EPM + SE

Percentage** slow

-0 .7d+0 .02 (316)

-1 .19+0 .02 (244)

56+3 -

X1 .01+0 .03 -

-0 .82+0 .03 (292)

-1 .20+0 .02 (300)

4g+4 -

2

-1 .03+0 - .02

-0 .83+0 .02 (268)

-1 .21+0 .02 (312)

46+4 -

3

- 1 .05 ± 0 .02

-0 .84+0 .02 (260)

-1 .21 +0 .02 (344)

43 + 3

4

-1 .10+0 .02 -

- 0 " 90+0 .02 (248)

-1 .24+0 .02 (348)

41 +4 -

5

- 1 .09 ± 0 .03

-0 .86+0 .02 (220)

-1 .22+0 .03 (392)

36 ± 4

6

-1 .09+0 .03 -

-0 .87+0 .02 (228)

-1 .22+0 .03 (362)

3g+3 -

7

-1 .08+0 .02 -

-0 " 87_+0 .02 (234)

-1 .21+0 .02 (35d)

40+3 -

8

- 1 .08 + 0 .03

-0 .89+0 .02 (232)

-1 .21+0 .03 (348)

40 ± 4

9

-1 .10+0 .02 -

-0 .92+0 .02 (264)

-1 .24+0 .02 (348)

43+2 -

10

-0 .98+0 - .02

-0 " 77+0 .02 (2t34)

-1 .18+0 .02 (308)

48+4 -

11

-0 .95+0 .02 -

-0 " 76+0 .02 (304)

-1 .17+0 .02 (272)

52 .5+3 -

12

-0 .95+0 .03 -

-0 .76+0 .03 (300)

-1 .16+0 .02 (268)

53+4 -

13

-0 .94+0 - .03

-0 .75+0 .02 (312)

-1 .16_+0 .03 (280)

53+3 -

14

-0 .94 .+0 .02 -

-0 " 75+0 .02 (312)

-1 .16_+0 .02 (264)

~+3 -

* Number of cells examined between brackets . ** The percentage of fast lymphocytes is the complement to 100 .

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ANS-Bound Lymphocytes

Number of ANS binding site s In order to confirm or to exclude the possibility of several ANS binding mechanisms, estimates of the total number of ANS molecules per cells were performed . The results are presented in Figure 4 . They express the variation of ANS amount bound to 10~ cells, after BSA- and SRBC-stimulation (the results are pooled) . If we draw the respective curves from the values given, they closely follow the curves of fluorescence changes . This relationship is not linear . The curve is a parabolic segment . The number of ANS molecules passing into the cells is not in proportional relationship with the fluorescence intensity .

30-

é

20-

0

50

700

7b0

200

A11A ~nrlrerlrr x'ot9

Fig . 4 Relation between ANS fluorescence intensity and ANS molecule contents bound to_10~ stimulated splenic lymphocytes .

DISCUSSION As seen in a previous study (12), mice stimulated with 0 .5 mg BSA or 10 8 SRBC exhibited an immune response : the splenic lymphocytes are suitably stimulated . It is well known that during induction of humoral antibody formation, only a limited number of the available lymphocytes present signs of specific activation (16, 17) . However, there are also signs of nonspecific activation (cellular cooperation and memory cell productior~ involving a much larger proportion of the cells that will display detectable specificity in their reactions to the administered antigens (18,19,20) . Thus, the fluorescence changes observed in Figures lA and 2A are the reflect of the whole activated lymphocytes . The fluorescence changes are induced by the action of antigens because the control lymphocytes show insignificant fluorescence changes during the 14 successive days (Figs . lA and 2A) . These curves clearly show a difference between BSA- and SRBC-activated lymphocytes in fluorescence intensity . In a previous work (10), we have shown that ANS penetrates into the cells ; ANS is bound to hydrophobic sites of intracellular membranes . Our results now suggest that some intracellular phenomena, have different kinetics according to the antigen used . However, the intracellular phenomena (metabolism) which are produced during a primary response generally follow similar kinetics whatever antigen is used .

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Haynes et al . (21) have claimed that ANS is a fluorescent indicator of electrostatic potential on lecithin membranes . Its approach is given by the electrokinetic potential ~ . Fig,~res 1B and 2B show the mean EPM or mean electrokinetic potential changes of BSA- and SRBC-stimulated lymphocytes, respectively . The results seem to demonstrate that fluorescence intensity and mean electrophoretic mobility are in an inverse relationship . In our experimental living system, there is a good correlation between electrophoretic mobility and fluorescence intensity, as shown in Figure 3 . When the antigen-stimulated lymphocytes have an electrokinetic surface potential more negative than control lymphocytes, they exhibit a low fluorescence ; and vice-versa . Dallner and Azzi (22) have provided evidence that bound ANS in smooth membranes is less fluorescent than in rough membranes . Flanagan and Hesketh (23) concluded that a more negative surface charge inhibits binding . These hypothesis formulated from experimental models, can be now extrapolated to living membranes . The inverse relationship between fluorescence and ~ can be explained by a greater affinity of the membranes for ANS, when ~ is less negative than that of control lymphocytes . An electrokinetic surface potential more negative inhibits the binding by electrostatic repulsion of ANS molecules . It can be suggested that : these changes of are caused by antigenic stimulation and could be a consequence of the lymphocytes metabolic activity . However, there is still some controversy with regard to the exact nature of the surface potential existing on a living membrane . The hypothesis formulated from the inverse relationship between EPM and fluorescence now explains the results on Figure 4 . When the EPM of lymphocytes increases, the number of ANS molecules bound to the cells decreases and becomes proportional to the fluorescence (i .e . between 50 and 70 .10 13 /107 cells) . Thus, it seems that the hydrophobic sites are always saturated . Then, it appears that the electrostatic effects on ANS molecules at hydrophobic level sites are negligible . As the number of ANS molecules passing into the cells decreases when the EPM of lymphocytes increases, there is a partial inhibition of ANS molecules diffusion . In Figure 4, the shape of the curve could be interpreted as a quenching effect induced by the reabsorption of fluorescence intensity by the high number of ANS molecules passing into the cells . Several authors (24,25) have shown that mouse spleen cells exhibit a bimodal electrophoretic distribution . Cells with the lowest mobility have been reported to correspond mainly to B lymphocytes, while cells with the highest mobility include mainly T lymphocytes . Thus, the changes in the EPM of the whole spleen cell population may be involved (and, in turn, observed fluorescence) by alterations in the frequency of B and T cells, or an electrophoretic shift of one, the other, or both of these subpopulations . The data deducted from histogram analysis, evidenced that both parameters are connected, as shown in Tables I and I I . Such EPM changes might be the results of maturation and differentiation events following antigenic stimulation . Indeed, surface charge is known to primarily reflect the molecular architecture of the cell periphery (26) and that this architecture is modified during the immune response of both B and T cells has been suggested by a lot of studies (27,28) . Whether these changes occur on the same cells or on different B or T cell subpopulations is not clear . The binding of A.NS to cells will also be affected by other cell parameters : cell volume, water, lipid and protein content . Miller et al . (29) have shown that there was no relation between B and T cells and their surface to volume ratio . As assessed by other authors (29,30), the primary response in our experiments is not characterized by cellular proliferation, or a significant difference of size of immune cells : the ANS fluorescence is not affected by this parameter . Several authors (31,32,33) have shown a stricking difference in surface membranes of T and B cells ( surface proteins and biochemical groups) . Neither the B nor the T cell protein appears to be identical . But ANS is not a marker for B or T cells, as shown in a previous study (34) . Moreover, ANS fluorescence of activated T cells is weak (12) . Consequently, it seems that the fluorescence observed is mainly induced by the activated B cells . The biological meaning of apparent by large response would be caused by the metabolic activation of lymphocytes before and during secretion of immunoglobulins . However,

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there is no direct relation between ANS fluorescence and lipid, protein, nucleic acid contents of immune cells (Chronobiologia, in. press) . According to Miller et al . (29) there is a sequence for B and T cell differentiations in the spleen following antigenic stimulation . These results could explain the changes in EPM of B and T subpopulations and the fluorescence changes of both subpopulations . Thus, it cannot be excluded that forces other than the hydrophobic ones may also be involved in the dye-binding changes in activated lymphocytes . It seems that conformational changes of intracellular membranes accompany electrokinetic surface potential changes of splenic lymphocytes in the course of immune response . However, suggestions about alterations in the conformation of the ANS binding site appear to be premature . Work is in progress to isolate each cell type, but in this case, an inevitable loss of immune cell subpopulations will occur . The Nude mouse strain appears to be rmre adequate in this study . Preliminary results show that there are conformational changes but more data are required before this possibility can be fully evaluated . ACKNOWLEDGEMENTS We would like to express our appreciation to Mrs Suzanne Droesch and Miss Maryse Girrfortheir valuable technical assistance, and Miss Josiane Bara for her excellent secretarial assistance . This investigation was supported by a grant from the Institut National de la Santé et de la Recherche Médicale (C .R .L . 76-5-194-1) . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .

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