JNMECON AND ImmuNr, Feb. 1975, p. 257-264
Vol. 11, No. 2 Printed in U.S.A.
Copyright 0) 1975 American Society for Microbiology
Isolation of Mitogenic and Adjuvant Active Fractions from Various Species of Nocardiae RITA CIORBARU, ARLETTE ADAM, JEAN-FRANCOIS PETIT, EDGAR LEDERER,* CONSTANTIN BONA, AND LOUIS CHEDID Institut de Biochimie, Universite Paris-Sud, 917405 Orsay, and Immunotherapie Experimentale, Institute Pasteur, 75015 Paris, France Received for publication 27 June 1974
Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N. rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.
We have previously shown that water-soluble lysozyme digests of Nocardia opaca possess an adjuvant activity (4) and are able to stimulate nonspecifically mouse and rabbit spleen lymphocytes (5a, 5b). We now show that digests of two other species of Nocardia, N. corallina and N. rubra (Corynebacterium rubrum), are also mitogenic and adjuvant. We present data on the fractionation of the digest of N. opaca which led to the isolation of a nonmitogenic adjuvant fraction and of a fraction which is endowed with both activities. In the course of these studies we have isolated from N. rubra a low-molecular-weight fraction which is adjuvant and does not contain any peptidoglycan fragment. MATERIALS AND METHODS
Preparation of the soluble extracts. (i) Delipidation of the cells. The cells were extracted in a Soxhlet apparatus with acetone, ethanol, ether, chloroform, methanol, and chloroform-methanol (87:13, vol/vol) and then suspended in acetone, filtered, and air-dried. (ii) Extraction of the delipidated cells with water. The delipidated cells were washed by suspension with a Potter homogenizer in water and centrifugation (1 h, 27,500 x g, 4 C). The water extract was filtered on a sintered glass, lyophilized, and fractionated on Sephadex G 75, giving five fractions; the first only was tested for adjuvant and mitogenic activities. (iii) Treatment of the cells with lysozyme. Prior to lysozyme treatment, the cells were extracted with 0.1 M ammonium acetate, pH 6.2. They were then suspended in 100 times their dry weight of 0.1 M ammonium acetate (pH 6.2) containing 0.01% eggwhite lysozyme (Industrie Biologique Fran,aise, 92230 Gennevilliers). The suspension was incubated for 18 h at 37 C in the presence of a few drops of toluene to prevent bacterial growth and centrifuged; the nonsolubilized material was reincubated with lysozyme and centrifuged. The two supernatants were pooled, lyophilized, resuspended in water, and lyophilized again several times to eliminate the ammonium acetate. The resulting material is called "crude lysozyme digest." Fractionation of the crude lysozyme digest. (i) Delipidation. The digest was delipidated by extraction at room temperature once with ether, three times with chloroform-methanol (2:1, vol/vol), and once again with ether, and dried. (ii) Fractionation on Sephadex. The delipidated
Growth conditions. The cells of the three species were grown in a 20-liter fermentor (Biolafitte, 78600 Maison-Lafitte). N. opaca (Pasteur Institute, Paris) was grown at 37 C for 2 days in the following medium: 0.4% meat extract (Difco, Detroit, Mich.), 0.2% yeast extract (Difco), 2% peptone (Difco), and 0.5% NaCl (pH adjusted to 7.2). N. corallina strain ATCC 999 was grown for 24 h at 34 C in 0.4% meat extract (Difco), 2% peptone (Difco), and 0.5% NaCl (pH adjusted to 7.2). N. rubra strain J. A. Crowle (ATCC 14898) was grown for 5 days at 25 C in heart infusion broth (Difco), 2.5%, glycerol, 10 ml/liter, and Na2HPO4 12 H20, 0.25 g/liter (pH adjusted to 7.5). 257
258
INFECT. IMMUN.
CIORBARU ET AL.
crude lysozyme digest was filtered on a Sephadex G 75 column (2.5 by 80 cm) equilibrated with 0.1 N acetic acid. Fractionation of fraction A of N. opaca. The first fraction of the Sephadex fractionation of N. opaca crude delipidated lysozyme digest was lyophilized, delipidated by the technique used for the crude lysozyme digest, resuspended in glacial acetic acid, and centrifuged at 48,000 x g for 1 h at 4 C. The pellet was resuspended and recentrifuged in the same conditions three times. The pellet and the pooled supernatants were lyophilized (Fig. 1). Analytical methods. (i) Amino acids. Total amino acids were measured after alkaline hydrolysis by the ninhydrin reaction (7). They were identified in hydrolysates (6 N HCl, 18 h, 110 C) either with a Technicon amino acid analyzer or by thin-layer chromatography on cellulose sheets (Eastman Kodak Co, Rochester, N.Y.), using butan-1-ol-acetic acid-pyridine-water (60:4:45:30, vol/vol/vol/vol) or butan-1-olacetic acid-water (62:15:25, vol/vol/vol), and detected with ninhydrin reagent (11). (ii) Amino sugars. Amino sugars were measured by the method of Elson-Morgan as modified by Belcher (5) after hydrolysis (4 h, 4 N HCl, 100 C). They were identified with the Technicon amino acid analyzer. (iii) Neutral sugars. Neutral sugars were measured by the orcinol method (15). They were identified by chromatography on Whatman no. 1 filter paper in the following solvent: ethyl acetate-pyridinewater (80:20:10, vol/vo/vol). The detection was made with the aniline oxalate reagent. Biological tests. (i) Adjuvant activity (17). Hartley female guinea pigs weighing 300 to 350 g were injected in both hind footpads with 0.1 ml of a water-in-oil emulsion made of equal parts of a solution of ovalbumin (50 mg/ml in saline) and either Freund complete adjuvant or Freund incomplete ajuvant in the case of the controls. Freund incomplete adjuvant was completed by various amounts of Nocardia fractions when injected into the test animals. To obtain the sera, the guinea pigs were killed 21 days after the injection. The adjuvant activity was estimated by determining the micrograms of antigen-antibody complex per milliliter at the equivalence point. The protein complex was estimated by the method of Lowry et al., with ovalbumin as the standard. Animals were challenged for delayed hypersensitivity 28 days after the injection. The resulting reactions were measured 48 h after the injection of ovalbumin. (ii) Mitogenic activity. Either 2- to 3-month-old AKR inbred mice (Pasteur Institute) or congenitally athymic nude mice were used. The latter were bred (C.N.R.S., Orleans) from an outbred wild strain homozygous for "nu" mutation originating from the Institute of Animal Genetics (Edinburgh). They were used at 4 to 8 weeks of age. Spleen or thymus lymphocytes were separated or cultivated according to a technique previously described (6; C. Bona, C. Damais, C. Galanos, and L.Chedid, In B. Urbaschek, ed., Pathophysiologie und Immunitat gramnegativer Infektionen sowie die Wir-
Delipidated cells of Nocardia opaca Washing with water, centrifugation o
Supernatant
Se diment
Washing with ammonium acetate, centrifugation
o: Supernatant
j
Sediment Di estion with lysozyme, centrifugation I
Sediment
Cr ude digest
Ilipidation
D4
Lipid fraction Crude delipidated digest Filtration
on
Sephadex G 75
I
Peak A (NWSM)
PeakB
Peak C
Delipidation
'I
Lipid fraction
I
Delipidated peak A Acetic
acid centrifugation
Pellet
bupernatant FIG. 1. Scheme for preparation of mitogenic fractions from N. opaca. NWSM, Nocardia water-soluble
mitogen. kungsweise der Endotoxine, in press). As controls to the various preparations previously described, the following mitogens were used: concanavalin A (Con A) (Miles) and lipopolysaccharide (LPS) of Salmonella enteritidis (16). The blast transformation of lymphocytes was determined by the incorporation of [3H]thymidine (specific activity, 1 Ci/mM; Saclay, France), which was measured by the scintillation method according to a procedure previously described (5b). The experiments were performed using four replicate tubes for each agent at a given dose.
RESULTS Chemical composition of the fractions obtained by Sephadex filtration of crude delipidated lysozyme digests. Filtration of crude delipidated lysozyme digests of N. opaca on
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MITOGENS AND ADJUVANTS FROM NOCARDIAE
Sephadex G 75 gave three fractions, A, B, and C (Fig. 2). Filtration of similar digests of N. corallina and N. rubra gave four fractions, A, B, C, and D. The composition of these fractions, which are surely not yet homogeneous, is reported in Table 1. With the exception of fraction D of N. rubra, all of them contain peptidoglycan, as shown by the presence of amino sugars and meso-1,6-diaminopimelic acid (DAP) as well as proteins (or glycoproteins) and polysaccharides. As described below, all of them have mitogenic and adjuvant activities except fraction D of N. rubra, which is only adjuvant (see Table 4). We attempted a separation of these activities on fraction A of N. opaca (called Nocardia water-soluble mitogen). Fractionation of fraction A of N. opaca. As described in Materials and Methods, 200 mg of this lyophilized delipidated fraction was separated by centrifugation in glacial acetic acid into a pellet (90 mg) and a supematant (80 mg, after lyophilization); their chemical compositions and detailed amino acid analyses are reported in Tables 2 and 3. Adjuvant activity. All the fractions are adjuvant (Table 4). It must be noted that fraction D of N. rubra does not contain peptidoglycan; its
2.
K
A
0 10
50
140 F,act ion N
100
FIG. 2. Chromatography of the crude delipidated lysozyme digest of N. opaca. The crude delipidated lysozyme digest (600 mg) dissolved in 0.1 N acetic acid (5 ml) was passed through a column (2.5 by 80 cm) of Sephadex G 75 equilibrated with 0.1 N acetic acid at a flow rate of 60 ml/h. Five-milliliter fractions were collected. Symbols:- - -, amino acids; . .. neutral sugars; ----, amino sugars. .,
TABLE 1. Composition of the fractions obtained by filtration on Sephadex G 75 of the delipidated lysozyme digestsa Neutral sugars
Amino sugars
Amino acids
F ractions
N. opaca A
25-30
B
12-15
C
5-10
N. ru bra A
40-45
B
28-35
C
13-20
Dt'
22-25
N. corallina A
52-55
Composition
S
Composition
Total %
(nmol/mg)
Gal, Glc Man traces Gal, Glc Man traces Gal, Glc Rib, Man traces
19-25
GlcNH2, Mur
32-35
175
21-25
GlcNH2, Mur
42-45
260
24-30
GlcNH2, Mur
50-55
599
Gal, Glc Man, Ara Gal, Glc Man, Ara Gal, Glc, Man Ara, Rib traces Gal, Rib traces
15-20
GlcNH2, Mur
15-20
241
7-12
GlcNH2, Mur
28-35
88
9-12
GlcNH2, Mur
34-40
346
c
10-15
GlcNH,, Mur
23-26
442
34-40 21-25 10-15
550 285 92
Gal, Glc Man, Ara Gal, Glc, Ara Glc, Ara traces Glc, Ara traces
1.8-5 10-15
GlcNH2, Mur GlcNH2, Mur GlcNH2, Mur a Lipids were not determined. Abbreviations: Gal, galactose; Glc, glucose; Man, GlcNH2, glucosamine; Mur, muramic acid. b This fraction is contaminated by salts. B C Do
c
Not identified.
18-20 13-20 17-22
20-25 7-15 4-10
mannose; Rib, ribose;
260
INFECT. IMMUN.
CIORBARU ET AL.
TABLE 2. Composition of the two fractions obtained after centrifugation in acetic acid ofpeak A of N. opacaa Neutral sugars
Amino sugars
Amino
Fractions
Composition
%
%
Composition
acids (%)b
Pellet
19-25
Gal, Glc
10-12
GlcNH2, Mur traces
45-48
Supematant
37-40
Gal, Glc, Rib traces
32-35
GlcNH2, Mur traces
18-20
See Table 1 for abbreviations. b The detailed amino acid composition is given in Table 3.
a
TABLE 3. Amino acid composition of the two fractions obtained after centrifugation in acetic acid of the delipidated fractions from N. opaca Pellet Composition Molar nmol/mg ratioa
Lysine Histidine Arginine Aspartic acid Threonine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine DAP a
221.5 52.8 183.75 519.54 499.34 477.96 72.24 431.76 283.4 295.26 209.32 311.98 56.91 108.36 50.0
0.78 0.18 0.64 1.83 1.76 1.68 0.25 1.52 1.00 1.04 0.73 1.10 0.20 0.38 0.17
Supernatant Molar nmol/mg ratioa 70.14 14.40 62.16 108.15 165.27 238.98
0.20 0.07 0.18 0.31 0.48 0.69
93.66 344.19 41.16 31.08
0.27 1.00 0.12 0.09
209.79
0.61
Molar ratio relative to alanine.
adjuvant activity cannot be related to this constituent. In these experiments we also tested the adjuvant activity of the first fraction obtained by Sephadex filtration of the water extract of delipidated cells of N. opaca and found it active. Mitogenic activity. (i) Dose-response relationship of AKR spleen lymphocytes stimulated by various Nocardia preparations. Various amounts of the Nocardia preparations were incubated with AKR mice spleen cells. The three fractions of N. opaca have a strong stimulatory effect, the peak of the activity being between 10 and 50 ,ug (Fig. 3). The three corresponding fractions isolated from N. rubra (fractions A, B, and C) were also active, whereas fraction D was very weakly active (Fig. 4). All four N. corallina fractions showed activity, although the response was lower than that obtained with the previous active preparations (Fig. 5). This weaker effect is equally demonstrated in Table 5. The first fraction obtained by Sephadex filtration of the water extract of delipidated cells, which is adjuvant, was also shown to be mito-
genic. The mitogenic activity of this product is low compared with the lysozyme digest of the same organism. (ii) Blastogenic activity of the Nocardia preparations, WSA, Con A, and LPS on mouse lymphocyte subpopulations. The mitogenic effect of the active Nocardia preparations was evaluated in comparison with the watersoluble adjuvant (WSA) of Mycobacterium smegmatis and Con A on spleen and thymus lymphocytes of AKR or nude mice. As previously reported, in contrast to the results with WSA extracted from M. smegmatis, the Nocardia preparations, like LPS, induced a marked stimulation of B-derived lymphocytes and had no effect on thymocytes. The results of a typical experiment are illustrated in Table 5. (iii) Mitogenic activity of the two fractions
obtained by fractionation of fraction A of N. opaca. In this study the supernatants and the pellet of fraction A of N. opaca were tested on spleen lymphocytes in comparison with the nonfractionated fraction A. The pellet was strongly active, and its activity has a dose-response relationship similar to the one of the original material (Fig. 6). In contrast, no mitogenic activity was found in the supernatant, although a strong adjuvant activity was found in both the supernatant and the pellet (Table 4). A comparison between the adjuvant and mitogenic activities of the various fractions tested is shown in Table 6. DISCUSSION The filtration of delipidated lysozyme digests from Nocardiae on Sephadex G 75 has given three distinct fractions in the case of N. opaca and four in the case of N. corallina and N. rubra. All these preparations possess an adjuvant activity demonstrated by the ability to increase the titer of circulating antibody and to induce delayed hypersensitivity to ovalbumin in guinea pigs. It is known (3) that bacterial peptidoglycans, either intact (12, 14) or enzymatically solubilized, are adjuvant (1, 2, 8-10, 13). The adju-
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261
TABLE 4. Adjuvant activity of Nocardia fractions
Humoral antibodies (gg/mI) Tested fractionSa
FIA M. butyricum (FCA) N. opaca A B C N. opaca Pellet
Supernatant Water extract of delipidated cells (fraction A) N. rubra A B C D N. corallina A B C D
Dose
(ug/animal)
No. of animals
Mean ± standard error
0 50
5 5
660 71 3,416 ± 373
50 50 50
4 4 4
5,205 4,272 3,200
2
5 10 50 50 50
5 5 5 4 4 4
1,728 i 230 285 2,816 106 3,235 ±521 4,980 ± 413 2,760 ± 281
50 50 50 50
5 5 5 5
4,656 4,476 4,788 3,272
50 50 50 50
5 5 5 5
Granuloma at injection site
Delayed hypersensitivity with ovalbumin challenge dose of:
o jg
100 "g
+
8b 13 I
12 I
+ + +
81 9I 8I
21/4 15/3 20/5
+ + + + + +
2I 4I 6I 8I 7I
740 459 533 232
+ + + +
12 I 8I 11I 9I
21/4
4,572 501 4,872 204 4,776 ±190 3,252 345
+ + + +
6I 7I 10 I 9I
18/7 12/5 17/6 16/5
789 336 310
2,336
-
3I
19/6c
8I 12 I
12 I 18/4 18/6 18/3
19/7 21/5 17/6
a FIA, Freund incomplete adjuvant; FCA, Freund complete adjuvant. b I, Induration. cThe first number is the diameter of erythema; the second is the diameter (mm) of necrosis.
vant activity of DAP-containing fractions (all except fraction D from N. rubra) could thus possibly be due to the peptidoglycan fragments they contain. This, however, does not hold for / -*pak A A--peak S fraction D from N. rubra (which does not --*peak C contain DAP), in which we postulate the presence of a different adjuvant active component. 150 As far as mitogenic activity is concerned, we have found that the three fractions of N. opaca induce a strong stimulation of mouse spleen lymphocytes. Three fractions of N. rubra (A, B, C) are mitogenic, whereas fraction D is not. In / 100 the case of N. corallina and its fractions, the mitogenic activity is lower than that found in N. opaca and N. rubra. As previously demonstrated (5a, 5b) for fraction A of N. opaca, all the mitogenic prepara50 tions of N. rubra and N. corallina stimulate selectively murine B lymphocytes. As WSA, a water-soluble cell wall fragment of M. smegmatis (3), is not mitogenic (5a, 5b), ,it is probable that the mitogenic activity of the ,__________________ .01 .1 1 10 50 lbOPg/1.5 x106 cells various Nocardiae fractions is due to a strucFIG. 3. Dose-response relationship of stimulation ture distinct from the peptidoglycan. In preliminary experiments undertaken to of mice spleen lymphocytes by the three fractions of isolate the molecule(s) responsible for mitogenic N. opaca. 200
-
262
INFECT. IMMUN.
CIORBARU ET AL.
activity, we performed a centrifugation of fraction A of N. opaca in glacial acetic acid. The resulting pellet is poor in DAP but has kept its full mitogenic activity. This fraction is also
#--
strongly adjuvant. The supernatant in which most of the DAP has been concentrated is highly adjuvant but not mitogenic (see Table 4 and Fig. 6). These data seem to exclude the possibility that the peptidoglycan is responsible for the mitogenic activity of this fraction. The
pea k A
pekl B
m_-- eaks D
I? 0E
Q
a
10
100)J9g
1.5 x-1
71.5x106cells
FIG. 4. Dose-response relationship of stimulation of mice spleen lymphocytes by the four fractions of N. rubra.
r
cells
FIG. 5. Dose-response relationship of stimulation of mice spleen lymphocytes by the four fractions of N. corallina.
TABLE 5. Blastogenic activity of the Nocardia preparations, WSA of M. smegmatis, Con A, and LPS on mouse lymphocytes subpopulations Substance added to culture
Thymocytes
Splenocytes
Splenocytes
9,749 ±910 286,198 + 13,414 213,513 4 42,800 128,301 ± 2, 830 13,303 4 1,645 615,915 ± 14,743 352,375 ± 45,969
9,796 ±1,874 40,398 ± 1,658 41,242 4 2,972 20,671 ± 6,892 17,494 + 992 4,437 + 1,477 91,905 ± 6,526
Nil
-
88 ± 360
Peak A (NWSMb) of N. opaca
10
99 ± 24
Peak A (NWSM) ofN. rubra
10
87 ±31
Peak A (NWSM) of N. corallina
10
143 ± 19
WSA of M. smegmatis
50
142 ± 52
5
1,239 ± 152
10
ND
Con A LPS of S. enteritidis
Counts per minute average of four tubes ± standard deviation. bNWSM, Nocardia water-soluble mitogen. c ND, Not done. a
Nude
AKR
Amt (Mg)
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MITOGENS AND ADJUVANTS FROM NOCARDIAE
263
purification of the molecule(s) inducing blast transformation is now in progress. ACKNOWLEDGMENTS We are grateful to Elise Zissman (Institut de Chimie des Substances Naturelles) for growing the various bacterial cultures and M.A. Yapo for amino acid analyses. This work was supported by grants from the Fondation pour la Recherche Medicale Fransaise and the New York Cancer Research Institute. LITERATURE CITED NW NWSM //u _
100
."eprnlnt.
of NWSM
1. Adam, A., C. Amar, R. Ciorbaru, E. Lederer, J. F. Petit, and E. Vilkas. 1974. Activite adjuvante des peptidogly-
de mycobacteries. C. R. Acad. Sci. Ser. D 278:799-801. Adam, A., R Ciorbaru, F. Ellouz, J. F. Petit, and E. Lederer. 1974. Adjuvant activitity of monomeric bacterial cell wall peptidoglycans. Biochem. Biophys. Res. Commun. 56:561-567. Adam, A., R. Ciorbaru, J. F. Petit, and E. Lederer. 1972. Isolation and properties of a macromolecular, water soluble, immuno-adjuvant, fraction from the cell wall of Mycobacterium smegmatis. Proc. Nat. Acad. Sci. U.S.A. 69:851-854. Adam, A., R. Ciorbaru, J. F. Petit, E. Lederer, L. Chedid, A. Lamensans, F. Parant, M. Parant, J. P. Rosselet, and F. M. Berger. 1973. Preparation and biological properties of water-soluble adjuvant fractions from delipidated cells of Mycobacterium smegmatis and Nocardia opaca. Infect. Immun. 7:855-861. Belcher, R., A. J. Nutten, and C. M. Sambrook. 1954. Determination of glucosamine. Analyst (London) canes
1 ---
p*1101
of NWSM
2.
3.
50
4.
5.
79:201-208.
.01
.1
10
loopg/1.5x106cells
FIG. 6. Separation of mitogenic activity in peak A of N. opaca by centrifugation.
TABLE 6. Comparison of the adjuvant and mitogenic activities of the various fractions testeda Fraction Fraction M. smegmatis WSA Water extract of N. opaca before
Adjuvant
~~activity
Mitogenic activity
++++
+++
++
++++
++++
Pellet
++++
+++++
Supernatant
++++
lysozyme treatment N. opaca fraction A (NWSMb) Fraction A Fraction B Fraction C N. corallina
++++ +++
+++
Fraction A (NWSM) Fraction B Fraction C Fraction D N. rubra Fraction A (NWSM) Fraction B Fraction C Fraction D
++++ ++++ ++++ +++
++
++++ ++++ ++++
++ + +
+ ++ +++ +++
+++ +
a The activity of the fractions has been rated from inactive (-) to very active (+ + + +). NWSM, Nocardia water-soluble mitogen.
5a. Bona, C., C. Dahmias, and L. Chedid. 1974. Blastic transformation of mice spleen lymphocytes by a water soluble mitogen extracted from Nocardia. Proc. Nat. Acad. Sci. U.S.A. 71:1602-1608. 5b. Bona, C.. C. Dahmias, A. Dimitriu, L. Chedid, R. Ciorbaru, A. Adam, J. F. Petit, E. Lederer, and J. P. Rosselet. 1974. Mitogenic effect of a water-soluble extract of Nocardia opaca: a comparative study with some bacterial adjuvants and peripheral lymphocytes of four mammalian species. J. Immunol. 112:20282035. 6. Bona, C., J. Tribiaciavsky, A. Anteunis, C. Heuclin, and R. Robineaux. 1972. Structure of the lymphocyte membrane. II-Evidence of the two classes of Ig. on the surface of rabbit lymphocytes during primary response to sheep red blood cells, using inhibition of rosetteforming cells and combined ultrastructural autoradiography and peroxidase methods. Eur. J. Immunol. 2:434-437. 7. Hirs, C. H. W., S. Moore, and W. H. Stein. 1956. Peptides obtained by tryptic hydrolysis of performic acid-oxidized ribonuclease. J. Biol. Chem. 219:623-624. 8. Hiu, I. J. 1972. Water soluble and lipid free fraction from B.C.G. with adjuvant and antitumor activity. Nature (London) New Biol. 238:241-242. 9. Migliore-Samour, D., and P. Jolles. 1972. Hydrosoluble
adjuvant-active mycobacterial polysaccharide peptidoglycan preparation by a simple extraction technique of the bacterial cells (strain peurois). FEBS Lett. 25:301-306. 10. Migliore-Samour, D., and P. Jolles. 1973. Hydrosoluble adjuvant-active mycobacterial fractions of low molecular weight. FEBS Lett. 35:317-321. 11. Moffat, E. D., and R. Lytle. 1959. Polychromatic technique for the identification of amino-acids on paper chromatograms. Anal. Chem. 31:926-928. 12. Nauciel, C., J. Fleck, J. P. Martin, and M. Mock. 1973. Activite adjuvante de peptidoglycanes de bacteries a
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Gram-negatives dans l'hypersensibilite de type retarde. C. R. Acad. Sci. Ser. D 276:3499-3500. 13. Nauciel, C., J. Fleck, M. Mock, and J. P. Martin. 1973. Activite adjuvante de fractions monomeriques de peptidoglycanes bacteriens dans l'hypersensibilite de type retarde. C. R. Acad. Sci. Ser. D 277:2841-2844. 14. Nguyen Dang, T., M. Hayat, E. Chenu, M. Mayer, and M. M. Janot. 1973. Proprietes immunologiques des parois cellulaires bacteriennes. Relation entre la structure reticulee et l'activite immunostimulante du pep-
INFECT. IMMUN.
tidoglycane. C. R. Acad. Sci. Ser. D 277:1717-1720. 15. Rimington, C. 1940. Seromucoid and the bound carbohydrate of the serum proteins. Biochem. J. 34:931-940. 16. Westphal, O., 0. Liideritz, and B. Bister. 1952. Ober die Extraction von Bakterien mit Phenol/Wasser. Z. Naturforsch. 7:148-155. 17. White, R. G., L. Bernstock, R. G. S. Johns, and E. Lederer. 1958. The influence of components of M. tuberculosis and other mycobacteria upon antibody production to ovalbumin. Immunology 1:54-66.
Vol. 11, No. 2 Printed in U.S.A.
Copyright 0) 1975 American Society for Microbiology
Isolation of Mitogenic and Adjuvant Active Fractions from Various Species of Nocardiae RITA CIORBARU, ARLETTE ADAM, JEAN-FRANCOIS PETIT, EDGAR LEDERER,* CONSTANTIN BONA, AND LOUIS CHEDID Institut de Biochimie, Universite Paris-Sud, 917405 Orsay, and Immunotherapie Experimentale, Institute Pasteur, 75015 Paris, France Received for publication 27 June 1974
Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N. rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.
We have previously shown that water-soluble lysozyme digests of Nocardia opaca possess an adjuvant activity (4) and are able to stimulate nonspecifically mouse and rabbit spleen lymphocytes (5a, 5b). We now show that digests of two other species of Nocardia, N. corallina and N. rubra (Corynebacterium rubrum), are also mitogenic and adjuvant. We present data on the fractionation of the digest of N. opaca which led to the isolation of a nonmitogenic adjuvant fraction and of a fraction which is endowed with both activities. In the course of these studies we have isolated from N. rubra a low-molecular-weight fraction which is adjuvant and does not contain any peptidoglycan fragment. MATERIALS AND METHODS
Preparation of the soluble extracts. (i) Delipidation of the cells. The cells were extracted in a Soxhlet apparatus with acetone, ethanol, ether, chloroform, methanol, and chloroform-methanol (87:13, vol/vol) and then suspended in acetone, filtered, and air-dried. (ii) Extraction of the delipidated cells with water. The delipidated cells were washed by suspension with a Potter homogenizer in water and centrifugation (1 h, 27,500 x g, 4 C). The water extract was filtered on a sintered glass, lyophilized, and fractionated on Sephadex G 75, giving five fractions; the first only was tested for adjuvant and mitogenic activities. (iii) Treatment of the cells with lysozyme. Prior to lysozyme treatment, the cells were extracted with 0.1 M ammonium acetate, pH 6.2. They were then suspended in 100 times their dry weight of 0.1 M ammonium acetate (pH 6.2) containing 0.01% eggwhite lysozyme (Industrie Biologique Fran,aise, 92230 Gennevilliers). The suspension was incubated for 18 h at 37 C in the presence of a few drops of toluene to prevent bacterial growth and centrifuged; the nonsolubilized material was reincubated with lysozyme and centrifuged. The two supernatants were pooled, lyophilized, resuspended in water, and lyophilized again several times to eliminate the ammonium acetate. The resulting material is called "crude lysozyme digest." Fractionation of the crude lysozyme digest. (i) Delipidation. The digest was delipidated by extraction at room temperature once with ether, three times with chloroform-methanol (2:1, vol/vol), and once again with ether, and dried. (ii) Fractionation on Sephadex. The delipidated
Growth conditions. The cells of the three species were grown in a 20-liter fermentor (Biolafitte, 78600 Maison-Lafitte). N. opaca (Pasteur Institute, Paris) was grown at 37 C for 2 days in the following medium: 0.4% meat extract (Difco, Detroit, Mich.), 0.2% yeast extract (Difco), 2% peptone (Difco), and 0.5% NaCl (pH adjusted to 7.2). N. corallina strain ATCC 999 was grown for 24 h at 34 C in 0.4% meat extract (Difco), 2% peptone (Difco), and 0.5% NaCl (pH adjusted to 7.2). N. rubra strain J. A. Crowle (ATCC 14898) was grown for 5 days at 25 C in heart infusion broth (Difco), 2.5%, glycerol, 10 ml/liter, and Na2HPO4 12 H20, 0.25 g/liter (pH adjusted to 7.5). 257
258
INFECT. IMMUN.
CIORBARU ET AL.
crude lysozyme digest was filtered on a Sephadex G 75 column (2.5 by 80 cm) equilibrated with 0.1 N acetic acid. Fractionation of fraction A of N. opaca. The first fraction of the Sephadex fractionation of N. opaca crude delipidated lysozyme digest was lyophilized, delipidated by the technique used for the crude lysozyme digest, resuspended in glacial acetic acid, and centrifuged at 48,000 x g for 1 h at 4 C. The pellet was resuspended and recentrifuged in the same conditions three times. The pellet and the pooled supernatants were lyophilized (Fig. 1). Analytical methods. (i) Amino acids. Total amino acids were measured after alkaline hydrolysis by the ninhydrin reaction (7). They were identified in hydrolysates (6 N HCl, 18 h, 110 C) either with a Technicon amino acid analyzer or by thin-layer chromatography on cellulose sheets (Eastman Kodak Co, Rochester, N.Y.), using butan-1-ol-acetic acid-pyridine-water (60:4:45:30, vol/vol/vol/vol) or butan-1-olacetic acid-water (62:15:25, vol/vol/vol), and detected with ninhydrin reagent (11). (ii) Amino sugars. Amino sugars were measured by the method of Elson-Morgan as modified by Belcher (5) after hydrolysis (4 h, 4 N HCl, 100 C). They were identified with the Technicon amino acid analyzer. (iii) Neutral sugars. Neutral sugars were measured by the orcinol method (15). They were identified by chromatography on Whatman no. 1 filter paper in the following solvent: ethyl acetate-pyridinewater (80:20:10, vol/vo/vol). The detection was made with the aniline oxalate reagent. Biological tests. (i) Adjuvant activity (17). Hartley female guinea pigs weighing 300 to 350 g were injected in both hind footpads with 0.1 ml of a water-in-oil emulsion made of equal parts of a solution of ovalbumin (50 mg/ml in saline) and either Freund complete adjuvant or Freund incomplete ajuvant in the case of the controls. Freund incomplete adjuvant was completed by various amounts of Nocardia fractions when injected into the test animals. To obtain the sera, the guinea pigs were killed 21 days after the injection. The adjuvant activity was estimated by determining the micrograms of antigen-antibody complex per milliliter at the equivalence point. The protein complex was estimated by the method of Lowry et al., with ovalbumin as the standard. Animals were challenged for delayed hypersensitivity 28 days after the injection. The resulting reactions were measured 48 h after the injection of ovalbumin. (ii) Mitogenic activity. Either 2- to 3-month-old AKR inbred mice (Pasteur Institute) or congenitally athymic nude mice were used. The latter were bred (C.N.R.S., Orleans) from an outbred wild strain homozygous for "nu" mutation originating from the Institute of Animal Genetics (Edinburgh). They were used at 4 to 8 weeks of age. Spleen or thymus lymphocytes were separated or cultivated according to a technique previously described (6; C. Bona, C. Damais, C. Galanos, and L.Chedid, In B. Urbaschek, ed., Pathophysiologie und Immunitat gramnegativer Infektionen sowie die Wir-
Delipidated cells of Nocardia opaca Washing with water, centrifugation o
Supernatant
Se diment
Washing with ammonium acetate, centrifugation
o: Supernatant
j
Sediment Di estion with lysozyme, centrifugation I
Sediment
Cr ude digest
Ilipidation
D4
Lipid fraction Crude delipidated digest Filtration
on
Sephadex G 75
I
Peak A (NWSM)
PeakB
Peak C
Delipidation
'I
Lipid fraction
I
Delipidated peak A Acetic
acid centrifugation
Pellet
bupernatant FIG. 1. Scheme for preparation of mitogenic fractions from N. opaca. NWSM, Nocardia water-soluble
mitogen. kungsweise der Endotoxine, in press). As controls to the various preparations previously described, the following mitogens were used: concanavalin A (Con A) (Miles) and lipopolysaccharide (LPS) of Salmonella enteritidis (16). The blast transformation of lymphocytes was determined by the incorporation of [3H]thymidine (specific activity, 1 Ci/mM; Saclay, France), which was measured by the scintillation method according to a procedure previously described (5b). The experiments were performed using four replicate tubes for each agent at a given dose.
RESULTS Chemical composition of the fractions obtained by Sephadex filtration of crude delipidated lysozyme digests. Filtration of crude delipidated lysozyme digests of N. opaca on
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259
MITOGENS AND ADJUVANTS FROM NOCARDIAE
Sephadex G 75 gave three fractions, A, B, and C (Fig. 2). Filtration of similar digests of N. corallina and N. rubra gave four fractions, A, B, C, and D. The composition of these fractions, which are surely not yet homogeneous, is reported in Table 1. With the exception of fraction D of N. rubra, all of them contain peptidoglycan, as shown by the presence of amino sugars and meso-1,6-diaminopimelic acid (DAP) as well as proteins (or glycoproteins) and polysaccharides. As described below, all of them have mitogenic and adjuvant activities except fraction D of N. rubra, which is only adjuvant (see Table 4). We attempted a separation of these activities on fraction A of N. opaca (called Nocardia water-soluble mitogen). Fractionation of fraction A of N. opaca. As described in Materials and Methods, 200 mg of this lyophilized delipidated fraction was separated by centrifugation in glacial acetic acid into a pellet (90 mg) and a supematant (80 mg, after lyophilization); their chemical compositions and detailed amino acid analyses are reported in Tables 2 and 3. Adjuvant activity. All the fractions are adjuvant (Table 4). It must be noted that fraction D of N. rubra does not contain peptidoglycan; its
2.
K
A
0 10
50
140 F,act ion N
100
FIG. 2. Chromatography of the crude delipidated lysozyme digest of N. opaca. The crude delipidated lysozyme digest (600 mg) dissolved in 0.1 N acetic acid (5 ml) was passed through a column (2.5 by 80 cm) of Sephadex G 75 equilibrated with 0.1 N acetic acid at a flow rate of 60 ml/h. Five-milliliter fractions were collected. Symbols:- - -, amino acids; . .. neutral sugars; ----, amino sugars. .,
TABLE 1. Composition of the fractions obtained by filtration on Sephadex G 75 of the delipidated lysozyme digestsa Neutral sugars
Amino sugars
Amino acids
F ractions
N. opaca A
25-30
B
12-15
C
5-10
N. ru bra A
40-45
B
28-35
C
13-20
Dt'
22-25
N. corallina A
52-55
Composition
S
Composition
Total %
(nmol/mg)
Gal, Glc Man traces Gal, Glc Man traces Gal, Glc Rib, Man traces
19-25
GlcNH2, Mur
32-35
175
21-25
GlcNH2, Mur
42-45
260
24-30
GlcNH2, Mur
50-55
599
Gal, Glc Man, Ara Gal, Glc Man, Ara Gal, Glc, Man Ara, Rib traces Gal, Rib traces
15-20
GlcNH2, Mur
15-20
241
7-12
GlcNH2, Mur
28-35
88
9-12
GlcNH2, Mur
34-40
346
c
10-15
GlcNH,, Mur
23-26
442
34-40 21-25 10-15
550 285 92
Gal, Glc Man, Ara Gal, Glc, Ara Glc, Ara traces Glc, Ara traces
1.8-5 10-15
GlcNH2, Mur GlcNH2, Mur GlcNH2, Mur a Lipids were not determined. Abbreviations: Gal, galactose; Glc, glucose; Man, GlcNH2, glucosamine; Mur, muramic acid. b This fraction is contaminated by salts. B C Do
c
Not identified.
18-20 13-20 17-22
20-25 7-15 4-10
mannose; Rib, ribose;
260
INFECT. IMMUN.
CIORBARU ET AL.
TABLE 2. Composition of the two fractions obtained after centrifugation in acetic acid ofpeak A of N. opacaa Neutral sugars
Amino sugars
Amino
Fractions
Composition
%
%
Composition
acids (%)b
Pellet
19-25
Gal, Glc
10-12
GlcNH2, Mur traces
45-48
Supematant
37-40
Gal, Glc, Rib traces
32-35
GlcNH2, Mur traces
18-20
See Table 1 for abbreviations. b The detailed amino acid composition is given in Table 3.
a
TABLE 3. Amino acid composition of the two fractions obtained after centrifugation in acetic acid of the delipidated fractions from N. opaca Pellet Composition Molar nmol/mg ratioa
Lysine Histidine Arginine Aspartic acid Threonine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine DAP a
221.5 52.8 183.75 519.54 499.34 477.96 72.24 431.76 283.4 295.26 209.32 311.98 56.91 108.36 50.0
0.78 0.18 0.64 1.83 1.76 1.68 0.25 1.52 1.00 1.04 0.73 1.10 0.20 0.38 0.17
Supernatant Molar nmol/mg ratioa 70.14 14.40 62.16 108.15 165.27 238.98
0.20 0.07 0.18 0.31 0.48 0.69
93.66 344.19 41.16 31.08
0.27 1.00 0.12 0.09
209.79
0.61
Molar ratio relative to alanine.
adjuvant activity cannot be related to this constituent. In these experiments we also tested the adjuvant activity of the first fraction obtained by Sephadex filtration of the water extract of delipidated cells of N. opaca and found it active. Mitogenic activity. (i) Dose-response relationship of AKR spleen lymphocytes stimulated by various Nocardia preparations. Various amounts of the Nocardia preparations were incubated with AKR mice spleen cells. The three fractions of N. opaca have a strong stimulatory effect, the peak of the activity being between 10 and 50 ,ug (Fig. 3). The three corresponding fractions isolated from N. rubra (fractions A, B, and C) were also active, whereas fraction D was very weakly active (Fig. 4). All four N. corallina fractions showed activity, although the response was lower than that obtained with the previous active preparations (Fig. 5). This weaker effect is equally demonstrated in Table 5. The first fraction obtained by Sephadex filtration of the water extract of delipidated cells, which is adjuvant, was also shown to be mito-
genic. The mitogenic activity of this product is low compared with the lysozyme digest of the same organism. (ii) Blastogenic activity of the Nocardia preparations, WSA, Con A, and LPS on mouse lymphocyte subpopulations. The mitogenic effect of the active Nocardia preparations was evaluated in comparison with the watersoluble adjuvant (WSA) of Mycobacterium smegmatis and Con A on spleen and thymus lymphocytes of AKR or nude mice. As previously reported, in contrast to the results with WSA extracted from M. smegmatis, the Nocardia preparations, like LPS, induced a marked stimulation of B-derived lymphocytes and had no effect on thymocytes. The results of a typical experiment are illustrated in Table 5. (iii) Mitogenic activity of the two fractions
obtained by fractionation of fraction A of N. opaca. In this study the supernatants and the pellet of fraction A of N. opaca were tested on spleen lymphocytes in comparison with the nonfractionated fraction A. The pellet was strongly active, and its activity has a dose-response relationship similar to the one of the original material (Fig. 6). In contrast, no mitogenic activity was found in the supernatant, although a strong adjuvant activity was found in both the supernatant and the pellet (Table 4). A comparison between the adjuvant and mitogenic activities of the various fractions tested is shown in Table 6. DISCUSSION The filtration of delipidated lysozyme digests from Nocardiae on Sephadex G 75 has given three distinct fractions in the case of N. opaca and four in the case of N. corallina and N. rubra. All these preparations possess an adjuvant activity demonstrated by the ability to increase the titer of circulating antibody and to induce delayed hypersensitivity to ovalbumin in guinea pigs. It is known (3) that bacterial peptidoglycans, either intact (12, 14) or enzymatically solubilized, are adjuvant (1, 2, 8-10, 13). The adju-
VOL. 11, 1975
MITOGENS AND ADJUVANTS FROM NOCARDIAE
261
TABLE 4. Adjuvant activity of Nocardia fractions
Humoral antibodies (gg/mI) Tested fractionSa
FIA M. butyricum (FCA) N. opaca A B C N. opaca Pellet
Supernatant Water extract of delipidated cells (fraction A) N. rubra A B C D N. corallina A B C D
Dose
(ug/animal)
No. of animals
Mean ± standard error
0 50
5 5
660 71 3,416 ± 373
50 50 50
4 4 4
5,205 4,272 3,200
2
5 10 50 50 50
5 5 5 4 4 4
1,728 i 230 285 2,816 106 3,235 ±521 4,980 ± 413 2,760 ± 281
50 50 50 50
5 5 5 5
4,656 4,476 4,788 3,272
50 50 50 50
5 5 5 5
Granuloma at injection site
Delayed hypersensitivity with ovalbumin challenge dose of:
o jg
100 "g
+
8b 13 I
12 I
+ + +
81 9I 8I
21/4 15/3 20/5
+ + + + + +
2I 4I 6I 8I 7I
740 459 533 232
+ + + +
12 I 8I 11I 9I
21/4
4,572 501 4,872 204 4,776 ±190 3,252 345
+ + + +
6I 7I 10 I 9I
18/7 12/5 17/6 16/5
789 336 310
2,336
-
3I
19/6c
8I 12 I
12 I 18/4 18/6 18/3
19/7 21/5 17/6
a FIA, Freund incomplete adjuvant; FCA, Freund complete adjuvant. b I, Induration. cThe first number is the diameter of erythema; the second is the diameter (mm) of necrosis.
vant activity of DAP-containing fractions (all except fraction D from N. rubra) could thus possibly be due to the peptidoglycan fragments they contain. This, however, does not hold for / -*pak A A--peak S fraction D from N. rubra (which does not --*peak C contain DAP), in which we postulate the presence of a different adjuvant active component. 150 As far as mitogenic activity is concerned, we have found that the three fractions of N. opaca induce a strong stimulation of mouse spleen lymphocytes. Three fractions of N. rubra (A, B, C) are mitogenic, whereas fraction D is not. In / 100 the case of N. corallina and its fractions, the mitogenic activity is lower than that found in N. opaca and N. rubra. As previously demonstrated (5a, 5b) for fraction A of N. opaca, all the mitogenic prepara50 tions of N. rubra and N. corallina stimulate selectively murine B lymphocytes. As WSA, a water-soluble cell wall fragment of M. smegmatis (3), is not mitogenic (5a, 5b), ,it is probable that the mitogenic activity of the ,__________________ .01 .1 1 10 50 lbOPg/1.5 x106 cells various Nocardiae fractions is due to a strucFIG. 3. Dose-response relationship of stimulation ture distinct from the peptidoglycan. In preliminary experiments undertaken to of mice spleen lymphocytes by the three fractions of isolate the molecule(s) responsible for mitogenic N. opaca. 200
-
262
INFECT. IMMUN.
CIORBARU ET AL.
activity, we performed a centrifugation of fraction A of N. opaca in glacial acetic acid. The resulting pellet is poor in DAP but has kept its full mitogenic activity. This fraction is also
#--
strongly adjuvant. The supernatant in which most of the DAP has been concentrated is highly adjuvant but not mitogenic (see Table 4 and Fig. 6). These data seem to exclude the possibility that the peptidoglycan is responsible for the mitogenic activity of this fraction. The
pea k A
pekl B
m_-- eaks D
I? 0E
Q
a
10
100)J9g
1.5 x-1
71.5x106cells
FIG. 4. Dose-response relationship of stimulation of mice spleen lymphocytes by the four fractions of N. rubra.
r
cells
FIG. 5. Dose-response relationship of stimulation of mice spleen lymphocytes by the four fractions of N. corallina.
TABLE 5. Blastogenic activity of the Nocardia preparations, WSA of M. smegmatis, Con A, and LPS on mouse lymphocytes subpopulations Substance added to culture
Thymocytes
Splenocytes
Splenocytes
9,749 ±910 286,198 + 13,414 213,513 4 42,800 128,301 ± 2, 830 13,303 4 1,645 615,915 ± 14,743 352,375 ± 45,969
9,796 ±1,874 40,398 ± 1,658 41,242 4 2,972 20,671 ± 6,892 17,494 + 992 4,437 + 1,477 91,905 ± 6,526
Nil
-
88 ± 360
Peak A (NWSMb) of N. opaca
10
99 ± 24
Peak A (NWSM) ofN. rubra
10
87 ±31
Peak A (NWSM) of N. corallina
10
143 ± 19
WSA of M. smegmatis
50
142 ± 52
5
1,239 ± 152
10
ND
Con A LPS of S. enteritidis
Counts per minute average of four tubes ± standard deviation. bNWSM, Nocardia water-soluble mitogen. c ND, Not done. a
Nude
AKR
Amt (Mg)
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MITOGENS AND ADJUVANTS FROM NOCARDIAE
263
purification of the molecule(s) inducing blast transformation is now in progress. ACKNOWLEDGMENTS We are grateful to Elise Zissman (Institut de Chimie des Substances Naturelles) for growing the various bacterial cultures and M.A. Yapo for amino acid analyses. This work was supported by grants from the Fondation pour la Recherche Medicale Fransaise and the New York Cancer Research Institute. LITERATURE CITED NW NWSM //u _
100
."eprnlnt.
of NWSM
1. Adam, A., C. Amar, R. Ciorbaru, E. Lederer, J. F. Petit, and E. Vilkas. 1974. Activite adjuvante des peptidogly-
de mycobacteries. C. R. Acad. Sci. Ser. D 278:799-801. Adam, A., R Ciorbaru, F. Ellouz, J. F. Petit, and E. Lederer. 1974. Adjuvant activitity of monomeric bacterial cell wall peptidoglycans. Biochem. Biophys. Res. Commun. 56:561-567. Adam, A., R. Ciorbaru, J. F. Petit, and E. Lederer. 1972. Isolation and properties of a macromolecular, water soluble, immuno-adjuvant, fraction from the cell wall of Mycobacterium smegmatis. Proc. Nat. Acad. Sci. U.S.A. 69:851-854. Adam, A., R. Ciorbaru, J. F. Petit, E. Lederer, L. Chedid, A. Lamensans, F. Parant, M. Parant, J. P. Rosselet, and F. M. Berger. 1973. Preparation and biological properties of water-soluble adjuvant fractions from delipidated cells of Mycobacterium smegmatis and Nocardia opaca. Infect. Immun. 7:855-861. Belcher, R., A. J. Nutten, and C. M. Sambrook. 1954. Determination of glucosamine. Analyst (London) canes
1 ---
p*1101
of NWSM
2.
3.
50
4.
5.
79:201-208.
.01
.1
10
loopg/1.5x106cells
FIG. 6. Separation of mitogenic activity in peak A of N. opaca by centrifugation.
TABLE 6. Comparison of the adjuvant and mitogenic activities of the various fractions testeda Fraction Fraction M. smegmatis WSA Water extract of N. opaca before
Adjuvant
~~activity
Mitogenic activity
++++
+++
++
++++
++++
Pellet
++++
+++++
Supernatant
++++
lysozyme treatment N. opaca fraction A (NWSMb) Fraction A Fraction B Fraction C N. corallina
++++ +++
+++
Fraction A (NWSM) Fraction B Fraction C Fraction D N. rubra Fraction A (NWSM) Fraction B Fraction C Fraction D
++++ ++++ ++++ +++
++
++++ ++++ ++++
++ + +
+ ++ +++ +++
+++ +
a The activity of the fractions has been rated from inactive (-) to very active (+ + + +). NWSM, Nocardia water-soluble mitogen.
5a. Bona, C., C. Dahmias, and L. Chedid. 1974. Blastic transformation of mice spleen lymphocytes by a water soluble mitogen extracted from Nocardia. Proc. Nat. Acad. Sci. U.S.A. 71:1602-1608. 5b. Bona, C.. C. Dahmias, A. Dimitriu, L. Chedid, R. Ciorbaru, A. Adam, J. F. Petit, E. Lederer, and J. P. Rosselet. 1974. Mitogenic effect of a water-soluble extract of Nocardia opaca: a comparative study with some bacterial adjuvants and peripheral lymphocytes of four mammalian species. J. Immunol. 112:20282035. 6. Bona, C., J. Tribiaciavsky, A. Anteunis, C. Heuclin, and R. Robineaux. 1972. Structure of the lymphocyte membrane. II-Evidence of the two classes of Ig. on the surface of rabbit lymphocytes during primary response to sheep red blood cells, using inhibition of rosetteforming cells and combined ultrastructural autoradiography and peroxidase methods. Eur. J. Immunol. 2:434-437. 7. Hirs, C. H. W., S. Moore, and W. H. Stein. 1956. Peptides obtained by tryptic hydrolysis of performic acid-oxidized ribonuclease. J. Biol. Chem. 219:623-624. 8. Hiu, I. J. 1972. Water soluble and lipid free fraction from B.C.G. with adjuvant and antitumor activity. Nature (London) New Biol. 238:241-242. 9. Migliore-Samour, D., and P. Jolles. 1972. Hydrosoluble
adjuvant-active mycobacterial polysaccharide peptidoglycan preparation by a simple extraction technique of the bacterial cells (strain peurois). FEBS Lett. 25:301-306. 10. Migliore-Samour, D., and P. Jolles. 1973. Hydrosoluble adjuvant-active mycobacterial fractions of low molecular weight. FEBS Lett. 35:317-321. 11. Moffat, E. D., and R. Lytle. 1959. Polychromatic technique for the identification of amino-acids on paper chromatograms. Anal. Chem. 31:926-928. 12. Nauciel, C., J. Fleck, J. P. Martin, and M. Mock. 1973. Activite adjuvante de peptidoglycanes de bacteries a
264
CIORBARU ET AL.
Gram-negatives dans l'hypersensibilite de type retarde. C. R. Acad. Sci. Ser. D 276:3499-3500. 13. Nauciel, C., J. Fleck, M. Mock, and J. P. Martin. 1973. Activite adjuvante de fractions monomeriques de peptidoglycanes bacteriens dans l'hypersensibilite de type retarde. C. R. Acad. Sci. Ser. D 277:2841-2844. 14. Nguyen Dang, T., M. Hayat, E. Chenu, M. Mayer, and M. M. Janot. 1973. Proprietes immunologiques des parois cellulaires bacteriennes. Relation entre la structure reticulee et l'activite immunostimulante du pep-
INFECT. IMMUN.
tidoglycane. C. R. Acad. Sci. Ser. D 277:1717-1720. 15. Rimington, C. 1940. Seromucoid and the bound carbohydrate of the serum proteins. Biochem. J. 34:931-940. 16. Westphal, O., 0. Liideritz, and B. Bister. 1952. Ober die Extraction von Bakterien mit Phenol/Wasser. Z. Naturforsch. 7:148-155. 17. White, R. G., L. Bernstock, R. G. S. Johns, and E. Lederer. 1958. The influence of components of M. tuberculosis and other mycobacteria upon antibody production to ovalbumin. Immunology 1:54-66.
