Clin. exp. Immunol. (1977) 29, 311-315.
The effects of azathioprine and levamisole on rosette-forming cells of healthy subjects and cancer patients H. VERHAEGEN, W. DE COCK & J. DE CREE Clinical Research LUnit, St Bartholomeus, Merksem, Antwerp, Belgium (Received 13 December 1976)
SUMMARY
The in vitro effects of azathioprine and levamisole on the capacity of peripheral lymphocytes to form rosettes with sheep erythrocytes were studied in fifteen healthy subjects and forty-one cancer patients. Azathioprine significantly inhibited rosette formation of T lymphocytes in healthy subjects and cancer patients in remission. In contrast, azathioprine could not inhibit rosette formation of T lymphocytes in cancer patients with advanced disease. Patients with advanced tumour had also fewer rosette-forming cells (RFC) than healthy subjects. Levamisole, an antianergic chemotherapeutic agent, restored azathioprine-inhibited RFC in healthy subjects and in fifteen out of twenty-two cancer patients. Levamisole alone significantly enhanced low RFC in cancer patients.
INTRODUCTION Azathioprine is an immunosuppressive agent of both humoral and cellular immune responses, widely used in clinical organ transplantation. Bach, Dardenne & Fournier (1969) and Bach & Dardenne (1971) reported that azathioprine in vitro inhibits the capacity of T cells to form rosettes with sheep red blood cells and suggested that this inhibition is due to blocking of receptor sites. Poulter, Bradley & Turk (1974) showed that azathioprine in vivo depletes certain T populations and decreases the 0-antigenicity of others. Recently, we reported (Verhaegen et al., 1975) that levamisole restored E-rosette formation of peripheral lymphocytes of healthy subjects after in vitro inhibition with azathioprine. Here, we studied the in vitro effects of levamisole and azathioprine on E-rosette formation of peripheral lymphocytes in healthy subjects and cancer patients. MATERIALS AND METHODS Peripheral blood was obtained from fifteen adult healthy subjects and from forty-one cancer patients and collected on heparin. The forty-one cancer patients were known to have rectum- or colon-carcinoma (seventeen patients), carcinoma of the stomach (four patients), mamma carcinoma (three patients), lung carcinoma (nine patients), ovarium carcinoma (two patients), and carcinoma of the uterus, bladder, prostate, kidney and parotis (one patient each). Twenty-two were males, and nineteen females; their ages ranged from 48-82 (median: 65) years. None of the patients were receiving cytostatic therapy. Twenty patients were in remission after complete surgery and tests were performed at least 4 months after surgery. The other twenty-one patients had an extensive localized (five patients) or metastatic tumour (sixteen patients). Separation of lymphocytes. A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml blood and allowing the erythrocytes to sediment at room temperature for about 1 hr. The leucocyte-rich plasma was then layered onto a Ficoll-Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1500 rev/min for 30 min. The lymphocytes at the interface were aspirated and washed with Hanks' balanced salt solution (HBSS, pH 7-7) and the concentration adjusted to 2 x 106 cells/ml in HBSS. The viability of the lymphocytes was tested with I% trypan blue and the Correspondence: Dr H. Verhaegen, Clinical Research Unit, St Bartholomeus, Elf Novemberstraat 43, B-2060 Merksem, Antwerp, Belgium.
311
312
H. Verhaegen, W. De Cock & J. De Cree
purity was evaluated on a smear stained by the May-Grunwald-Giemsa method. Experiments were only performed when lymphocyte viability and purity was above 90%. Sheep red blood cells (SRBC). SRBC were always obtained from the same sheep under sterile conditions in Alsever's solution (v/v, 1/1). The cells were stored at 4VC and washed three times with HBSS before use. A 1% solution of SRBC in HBSS was made. Experimental design. Lymphocyte suspension, 0 2 ml, was mixed with: (a) 0 4 ml HBSS; (b) 0-2 ml HBSS and 0-2 ml azathioprine (Wellcome, Kent, 150 ,ug/ml); (c) 0-2 ml azathioprine and 0-2 ml levamisole (0-24 pg/ml, 10-6 M) and (d) 0-2 ml HBSS and 0-2 ml levamisole (0.24 pg/ml). These four mixtures were incubated for 1 hr at 370C. SRBC suspension, 0-25 ml, was added to 0-25 ml of these lymphocyte suspensions, incubated for 5 min at 370C, centrifuged at 700 rev/min for 5 min and then incubated for 1 hr at 0C. After gentle resuspension, RFC were counted in a haemocytometer. Two hundred cells were counted, and lymphocytes binding three or more SRBC were considered positive. Statistical analysis. Intergroup differences were analysed by the Mann-Whitney U-test (two-tailed) (Siegel, 1956). Intragroup differences due to azathioprine and/or levamisole treatment were analysed by the Wilcoxon matched-pairs signed-ranks test (two-tailed) (Siegel, 1956).
RESULTS As shown in Fig. 1 the capacity of T lymphocytes to form rosettes with SRBC was significantly inhibited by azathioprine (50 pg/ml*) treatment in healthy subjects (P = 0.00064) and cancer patients in remission (P = 0.0012). In contrast, RFC of cancer patients with advanced disease could not be significantly inhibited by azathioprine. -a
80
0
00
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o
_
0
a
0
70
000
0
00
A0 -'6o 60
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0
0
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0
30 0
20 0
10 A C Healthy
subjects
C
A
Cancer potients in remission
A C Cancer patients with advanced disease
FIG. 1. The in vitro effect of azathioprine (A) on control RFC (C) of healthy subjects and cancer patients. (-) Median values. *
End concentration.
313
Levamisole and azathioprine-sensitive T cells
Intergroup statistical analysis showed that cancer patients with advanced disease differ significantly from healthy subjects (P = 00011) and patients in remission (P = 00012) as regards the azathioprine sensitivity of their T lymphocytes. In Fig. 2 the effect of levamisole on azathioprine-treated T lymphocytes of healthy subjects and cancer patients is represented. The azathioprine-inhibited RFC of healthy subjects could be restored (P = 00006) in all subjects by levamisole (3V10-7 M*). The effects of azathioprine and levamisole on RFC a
80
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FIG. 2. In vitro effects of azathioprine (A) and levamisole (L) on control RFC (C) in fifteen healthy subjects and forty-one cancer patients, divided into three groups of different response. (i) Healthy subjects; (a) cancer patients in remission; (A) cancer patients with local tumour; (e) cancer patients with metastatic tumour. (b)
Ca)
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Conwd Levamisole Cotr patients with advanced remission disease FIG. 3. (a) RFC in peripheral blood of healthy subjects, cancer patients in remission and cancer patients with advanced disease; (b) in vitro effect of levamisole on low RFC of healthy subjects (c), cancer patients in remission (a) and cancer patients with advanced disease (e). camer subjects patients in ah
*
End concentration.
1Oa-6,m
314
H. Verhaegen, W. De Cock & 7. De Cree
of cancer patients led to three different responses. In fifteen patients azathioprine inhibited RFC and this inhibition could be offset by levamisole, in seven patients the azathioprine-inhibited RFC could not be restored by levamisole and the other nineteen patients had no azathioprine-sensitive RFC. The distribution of patients over the three different responses and arranged according to the stage ofthe disease is also shown in the figure. In Fig. 3 the percentages of RFC in healthy subjects and cancer patients is represented. RFC of healthy subjects and cancer patients in remission did not differ significantly, but RFC of cancer patients with advanced disease were significantly lower than RFC of healthy subjects (P = 0.025). Levamisole in vitro (310'- M) significantly (P = 0 03) enhanced low RFC*.
DISCUSSION The SRBC-rosette formation is a convenient technique for detecting T-lymphocytes. These rosettes are formed by light centrifugation of a mixture of the two types of cells after a short incubation at 370C, followed by incubation at 40C. Using this method the number of E rosettes is significantly lower in patients with advanced cancer than in healthy subjects (Anthony et al., 1975; Dellon, Potvin & Chretien, 1975; Gross et al., 1975; Potvin, Tarpley & Chretien, 1975), and we have been able to confirm this finding. Our results show that azathioprine inhibited RFC of healthy subjects and cancer patients in remission, but not of cancer patients with advanced disease. The effects of azathioprine on RFC of mice have been described by Bach, Dardenne & Fournier (1969) and Poulter, Bradley & Turk (1974). They showed that the less T cells are differentiated the more sensitive they are to inhibition by azathioprine. With this in mind our results suggest that peripheral T-lymphocytes of cancer patients with advanced disease may be more differentiated than T lymphocytes of healthy subjects and cancer patients in remission. This is substantiated by the results of Gross et al. (1975) who showed that peripheral T lymphocytes of patients with lung carcinoma are more resistant to anti-lymphocyte serum, which is a measure of fully immunocompetent lymphocytes. Levamisole in vitro restored azathioprine-inhibited peripheral RFC of healthy subjects and of some cancer patients. These results are in agreement with the observation of Van Ginckel & Hoebeke (1976), who showed that levamisole in vivo could depress the azathioprine sensitivity of spleen RFC of normal mice. Similar effects have been reported for thymic extracts (Dardenne & Bach, 1973) and drugs interfering with cyclic nucleotide metabolism (Bach & Bach, 1973). Since levamisole has been shown to interact with the cyclic nucleotide metabolism (Hadden et al., 1975), levamisole may well act by inducing T-cell changes, which in turn may be prerequisite for T-cell maturation. Lastly we should mention that levamisole in vitro significantly enhanced low RFC of cancer patients. The enhancing effects of levamisole in vitro (Biniaminov & Ramot, 1975) and in vivo on low RFC have already been reported in patients with Hodgkin's disease (Ramot et al., 1976) and other immunological disorders (Verhaegen et al., 1977). We are indebted to Dr A. de Beukelaar and Dr F. Krug, St Bartholomeus Hospital, Merksem, Antwerp, and to Dr Ceulemans, H. Familie Hospital, Antwerp, for sending us the patients; to 0. Vogels, and L. Van der Veken for technical assistance; to J. Dony for statistical analysis; and to H. Vanhove for reviewing the manuscript. The work was supported by a grant from the 'Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw' (IWONL). * The criterium for low RFC is less than 50%O, being lower than 2 s.d. of the mean of eighty healthy subjects tested in our laboratory. REFERENCES ANTHONY, H.M., KIRK, J.A., MADSEN, K.E., MASON, M.K. & TEMPLEMAN, G.H. (1975) E- and EAC-rosetting lymphocytes in patients with carcinoma of bronchus. Clin. exp. Immunol. 20, 29.
on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette forming cells. Interactions with thymic factor. Europ. J. Immunol. 3, 778.
BACH, M.-A. & BACH, J.-F. (1973) Studies
Levamisole and azathioprine-sensitive T cells BACH, J.-F. & DARDENNE, M. (1971) Activities of immunosuppressive agents in vitro. I. Rosette inhibition by azathioprine. Rev. Fr. Etud. Clin. Biol. 16, 770. BACH, J.-F., DARDENNE, M. & FOURNIER, C. (1969) In vitro evaluation of immunosuppressive drugs. Nature (Lond.), 222, 998. BINIAMINOV, M. & RAMOT, B. (1975) In vitro restoration by levamisole of thymus-derived lymphocyte function in Hodgkin's disease. Lancet, i, 464. DARDENNE, M. & BACH, J.-F. (1973) Studies on thymus products. I. Modification of rosette-forming cells by thymic extracts. Determination of the target RFC subpopulation. Immunology, 25, 343. DELLON, A.C., POTVIN, C. & CHRETIEN, P.B. (1975) Thymus-dependent lymphocyte levels in bronchogenic carcinoma: correlations with histology, clinical stage and clinical course after surgical treatment. Cancer, 35, 687. GROSS, R.L., LATTY, A., WILLIAMS, E.A. & NEWBERNE, P.M. (1975) Abnormal spontaneous rosette formation and rosette inhibition in lung carcinoma. New Engl. J. Med.
292, 439. HADDEN, J.W., COFFEY, R.G., HADDEN, E.M., LOPEZCORRALES, E. & SUNSHINE, G.H. (1975) Effects of levamisole and imidazole on lymphocyte proliferation and cyclic nucleotide levels. Cell. Immunol. 20, 98. POTVIN, C., TARPLEY, J.L. & CHRETIEN, B. (1975) Thymus-
I
315
derived lymphocytes in patients with solid malignancies. Clin. Immunol. Immunopathol. 3, 476. POULTER, L.W., BRADLEY, N.J. & TuRK, J.L. (1974) Differential effect of azathioprine on 0-antigenicity of mouse lymphocytes. Immunology, 26, 777. RAMOT, B., BINIAMINOV, M. SHOHAM, CH. & ROSENTHAL, E. (1976) Effect of levamisole on E-rosette forming cells in vivo and in vitro in Hodgkin's disease. New Engl. J. Med. 294, 809. SIEGEL, S. (1956) Mann-Whitney U test. Nonparametric Statistics for the Behavioral Sciences, p. 116. McGrawHill Book Co., Inc., New York. SIEGEL, S. (1956) Wilcoxon matched-pairs signed-ranks test. Nonparametric Statistics for the Behavioral Sciences, p. 75. McGraw-Hill Book Co., Inc., New York. VAN GINCKEL, R.F. & HOEBEKE, J. (1976) Effects of levamisole on spontaneous rosette forming cells in murine spleen. Europ. 7. Immunol. 6, 305. VERHAEGEN, H., DE COCK, W., DE CREE, J., VERBRUGGEN, F., VERHAEGEN-DECLERCQ, M. & BRUGMANS, J. (1975) In vitro restoration by levamisole of E-rosette forming cells after inhibition with azathioprine. Lancet, i, 978.
VERHAEGEN, H., DE CREE, J., DE COCK, W. & VERBRUGGEN,
F. (1977) Restoration by levamisole of low E-rosette forming cells in patients suffering from various diseases. Clin. exp. Immunol. 27, 313.
The effects of azathioprine and levamisole on rosette-forming cells of healthy subjects and cancer patients H. VERHAEGEN, W. DE COCK & J. DE CREE Clinical Research LUnit, St Bartholomeus, Merksem, Antwerp, Belgium (Received 13 December 1976)
SUMMARY
The in vitro effects of azathioprine and levamisole on the capacity of peripheral lymphocytes to form rosettes with sheep erythrocytes were studied in fifteen healthy subjects and forty-one cancer patients. Azathioprine significantly inhibited rosette formation of T lymphocytes in healthy subjects and cancer patients in remission. In contrast, azathioprine could not inhibit rosette formation of T lymphocytes in cancer patients with advanced disease. Patients with advanced tumour had also fewer rosette-forming cells (RFC) than healthy subjects. Levamisole, an antianergic chemotherapeutic agent, restored azathioprine-inhibited RFC in healthy subjects and in fifteen out of twenty-two cancer patients. Levamisole alone significantly enhanced low RFC in cancer patients.
INTRODUCTION Azathioprine is an immunosuppressive agent of both humoral and cellular immune responses, widely used in clinical organ transplantation. Bach, Dardenne & Fournier (1969) and Bach & Dardenne (1971) reported that azathioprine in vitro inhibits the capacity of T cells to form rosettes with sheep red blood cells and suggested that this inhibition is due to blocking of receptor sites. Poulter, Bradley & Turk (1974) showed that azathioprine in vivo depletes certain T populations and decreases the 0-antigenicity of others. Recently, we reported (Verhaegen et al., 1975) that levamisole restored E-rosette formation of peripheral lymphocytes of healthy subjects after in vitro inhibition with azathioprine. Here, we studied the in vitro effects of levamisole and azathioprine on E-rosette formation of peripheral lymphocytes in healthy subjects and cancer patients. MATERIALS AND METHODS Peripheral blood was obtained from fifteen adult healthy subjects and from forty-one cancer patients and collected on heparin. The forty-one cancer patients were known to have rectum- or colon-carcinoma (seventeen patients), carcinoma of the stomach (four patients), mamma carcinoma (three patients), lung carcinoma (nine patients), ovarium carcinoma (two patients), and carcinoma of the uterus, bladder, prostate, kidney and parotis (one patient each). Twenty-two were males, and nineteen females; their ages ranged from 48-82 (median: 65) years. None of the patients were receiving cytostatic therapy. Twenty patients were in remission after complete surgery and tests were performed at least 4 months after surgery. The other twenty-one patients had an extensive localized (five patients) or metastatic tumour (sixteen patients). Separation of lymphocytes. A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml blood and allowing the erythrocytes to sediment at room temperature for about 1 hr. The leucocyte-rich plasma was then layered onto a Ficoll-Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1500 rev/min for 30 min. The lymphocytes at the interface were aspirated and washed with Hanks' balanced salt solution (HBSS, pH 7-7) and the concentration adjusted to 2 x 106 cells/ml in HBSS. The viability of the lymphocytes was tested with I% trypan blue and the Correspondence: Dr H. Verhaegen, Clinical Research Unit, St Bartholomeus, Elf Novemberstraat 43, B-2060 Merksem, Antwerp, Belgium.
311
312
H. Verhaegen, W. De Cock & J. De Cree
purity was evaluated on a smear stained by the May-Grunwald-Giemsa method. Experiments were only performed when lymphocyte viability and purity was above 90%. Sheep red blood cells (SRBC). SRBC were always obtained from the same sheep under sterile conditions in Alsever's solution (v/v, 1/1). The cells were stored at 4VC and washed three times with HBSS before use. A 1% solution of SRBC in HBSS was made. Experimental design. Lymphocyte suspension, 0 2 ml, was mixed with: (a) 0 4 ml HBSS; (b) 0-2 ml HBSS and 0-2 ml azathioprine (Wellcome, Kent, 150 ,ug/ml); (c) 0-2 ml azathioprine and 0-2 ml levamisole (0-24 pg/ml, 10-6 M) and (d) 0-2 ml HBSS and 0-2 ml levamisole (0.24 pg/ml). These four mixtures were incubated for 1 hr at 370C. SRBC suspension, 0-25 ml, was added to 0-25 ml of these lymphocyte suspensions, incubated for 5 min at 370C, centrifuged at 700 rev/min for 5 min and then incubated for 1 hr at 0C. After gentle resuspension, RFC were counted in a haemocytometer. Two hundred cells were counted, and lymphocytes binding three or more SRBC were considered positive. Statistical analysis. Intergroup differences were analysed by the Mann-Whitney U-test (two-tailed) (Siegel, 1956). Intragroup differences due to azathioprine and/or levamisole treatment were analysed by the Wilcoxon matched-pairs signed-ranks test (two-tailed) (Siegel, 1956).
RESULTS As shown in Fig. 1 the capacity of T lymphocytes to form rosettes with SRBC was significantly inhibited by azathioprine (50 pg/ml*) treatment in healthy subjects (P = 0.00064) and cancer patients in remission (P = 0.0012). In contrast, RFC of cancer patients with advanced disease could not be significantly inhibited by azathioprine. -a
80
0
00
-0 -
o
_
0
a
0
70
000
0
00
A0 -'6o 60
0
t.0
09
0 0
0
0 00 0 0 oo
0
00
0 0
00
8
0 0
00 00
00
0 00 0 0
0
50
0
uu
000
40
0 O
-0
0
00 0@ 0 0 0
0
0
0
0 S 0
0
30 0
20 0
10 A C Healthy
subjects
C
A
Cancer potients in remission
A C Cancer patients with advanced disease
FIG. 1. The in vitro effect of azathioprine (A) on control RFC (C) of healthy subjects and cancer patients. (-) Median values. *
End concentration.
313
Levamisole and azathioprine-sensitive T cells
Intergroup statistical analysis showed that cancer patients with advanced disease differ significantly from healthy subjects (P = 00011) and patients in remission (P = 00012) as regards the azathioprine sensitivity of their T lymphocytes. In Fig. 2 the effect of levamisole on azathioprine-treated T lymphocytes of healthy subjects and cancer patients is represented. The azathioprine-inhibited RFC of healthy subjects could be restored (P = 00006) in all subjects by levamisole (3V10-7 M*). The effects of azathioprine and levamisole on RFC a
80
_
-~~o o0
0~~~
*
A
°
70 0O
~a
o+&
a:
a i
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.
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0
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LIJ.1 C A A+L
I
:I LJI C A A+L
I
-
C A A+L
FIG. 2. In vitro effects of azathioprine (A) and levamisole (L) on control RFC (C) in fifteen healthy subjects and forty-one cancer patients, divided into three groups of different response. (i) Healthy subjects; (a) cancer patients in remission; (A) cancer patients with local tumour; (e) cancer patients with metastatic tumour. (b)
Ca)
90r a
80F oa
70 -
00
o
8
AP.
ono
8o
a
60
-se-.
00
0"
0 0
0
o Ir
50 F
S
o 8
40
*
*
A
t 0
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30
2oL
9
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I
Conwd Levamisole Cotr patients with advanced remission disease FIG. 3. (a) RFC in peripheral blood of healthy subjects, cancer patients in remission and cancer patients with advanced disease; (b) in vitro effect of levamisole on low RFC of healthy subjects (c), cancer patients in remission (a) and cancer patients with advanced disease (e). camer subjects patients in ah
*
End concentration.
1Oa-6,m
314
H. Verhaegen, W. De Cock & 7. De Cree
of cancer patients led to three different responses. In fifteen patients azathioprine inhibited RFC and this inhibition could be offset by levamisole, in seven patients the azathioprine-inhibited RFC could not be restored by levamisole and the other nineteen patients had no azathioprine-sensitive RFC. The distribution of patients over the three different responses and arranged according to the stage ofthe disease is also shown in the figure. In Fig. 3 the percentages of RFC in healthy subjects and cancer patients is represented. RFC of healthy subjects and cancer patients in remission did not differ significantly, but RFC of cancer patients with advanced disease were significantly lower than RFC of healthy subjects (P = 0.025). Levamisole in vitro (310'- M) significantly (P = 0 03) enhanced low RFC*.
DISCUSSION The SRBC-rosette formation is a convenient technique for detecting T-lymphocytes. These rosettes are formed by light centrifugation of a mixture of the two types of cells after a short incubation at 370C, followed by incubation at 40C. Using this method the number of E rosettes is significantly lower in patients with advanced cancer than in healthy subjects (Anthony et al., 1975; Dellon, Potvin & Chretien, 1975; Gross et al., 1975; Potvin, Tarpley & Chretien, 1975), and we have been able to confirm this finding. Our results show that azathioprine inhibited RFC of healthy subjects and cancer patients in remission, but not of cancer patients with advanced disease. The effects of azathioprine on RFC of mice have been described by Bach, Dardenne & Fournier (1969) and Poulter, Bradley & Turk (1974). They showed that the less T cells are differentiated the more sensitive they are to inhibition by azathioprine. With this in mind our results suggest that peripheral T-lymphocytes of cancer patients with advanced disease may be more differentiated than T lymphocytes of healthy subjects and cancer patients in remission. This is substantiated by the results of Gross et al. (1975) who showed that peripheral T lymphocytes of patients with lung carcinoma are more resistant to anti-lymphocyte serum, which is a measure of fully immunocompetent lymphocytes. Levamisole in vitro restored azathioprine-inhibited peripheral RFC of healthy subjects and of some cancer patients. These results are in agreement with the observation of Van Ginckel & Hoebeke (1976), who showed that levamisole in vivo could depress the azathioprine sensitivity of spleen RFC of normal mice. Similar effects have been reported for thymic extracts (Dardenne & Bach, 1973) and drugs interfering with cyclic nucleotide metabolism (Bach & Bach, 1973). Since levamisole has been shown to interact with the cyclic nucleotide metabolism (Hadden et al., 1975), levamisole may well act by inducing T-cell changes, which in turn may be prerequisite for T-cell maturation. Lastly we should mention that levamisole in vitro significantly enhanced low RFC of cancer patients. The enhancing effects of levamisole in vitro (Biniaminov & Ramot, 1975) and in vivo on low RFC have already been reported in patients with Hodgkin's disease (Ramot et al., 1976) and other immunological disorders (Verhaegen et al., 1977). We are indebted to Dr A. de Beukelaar and Dr F. Krug, St Bartholomeus Hospital, Merksem, Antwerp, and to Dr Ceulemans, H. Familie Hospital, Antwerp, for sending us the patients; to 0. Vogels, and L. Van der Veken for technical assistance; to J. Dony for statistical analysis; and to H. Vanhove for reviewing the manuscript. The work was supported by a grant from the 'Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw' (IWONL). * The criterium for low RFC is less than 50%O, being lower than 2 s.d. of the mean of eighty healthy subjects tested in our laboratory. REFERENCES ANTHONY, H.M., KIRK, J.A., MADSEN, K.E., MASON, M.K. & TEMPLEMAN, G.H. (1975) E- and EAC-rosetting lymphocytes in patients with carcinoma of bronchus. Clin. exp. Immunol. 20, 29.
on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette forming cells. Interactions with thymic factor. Europ. J. Immunol. 3, 778.
BACH, M.-A. & BACH, J.-F. (1973) Studies
Levamisole and azathioprine-sensitive T cells BACH, J.-F. & DARDENNE, M. (1971) Activities of immunosuppressive agents in vitro. I. Rosette inhibition by azathioprine. Rev. Fr. Etud. Clin. Biol. 16, 770. BACH, J.-F., DARDENNE, M. & FOURNIER, C. (1969) In vitro evaluation of immunosuppressive drugs. Nature (Lond.), 222, 998. BINIAMINOV, M. & RAMOT, B. (1975) In vitro restoration by levamisole of thymus-derived lymphocyte function in Hodgkin's disease. Lancet, i, 464. DARDENNE, M. & BACH, J.-F. (1973) Studies on thymus products. I. Modification of rosette-forming cells by thymic extracts. Determination of the target RFC subpopulation. Immunology, 25, 343. DELLON, A.C., POTVIN, C. & CHRETIEN, P.B. (1975) Thymus-dependent lymphocyte levels in bronchogenic carcinoma: correlations with histology, clinical stage and clinical course after surgical treatment. Cancer, 35, 687. GROSS, R.L., LATTY, A., WILLIAMS, E.A. & NEWBERNE, P.M. (1975) Abnormal spontaneous rosette formation and rosette inhibition in lung carcinoma. New Engl. J. Med.
292, 439. HADDEN, J.W., COFFEY, R.G., HADDEN, E.M., LOPEZCORRALES, E. & SUNSHINE, G.H. (1975) Effects of levamisole and imidazole on lymphocyte proliferation and cyclic nucleotide levels. Cell. Immunol. 20, 98. POTVIN, C., TARPLEY, J.L. & CHRETIEN, B. (1975) Thymus-
I
315
derived lymphocytes in patients with solid malignancies. Clin. Immunol. Immunopathol. 3, 476. POULTER, L.W., BRADLEY, N.J. & TuRK, J.L. (1974) Differential effect of azathioprine on 0-antigenicity of mouse lymphocytes. Immunology, 26, 777. RAMOT, B., BINIAMINOV, M. SHOHAM, CH. & ROSENTHAL, E. (1976) Effect of levamisole on E-rosette forming cells in vivo and in vitro in Hodgkin's disease. New Engl. J. Med. 294, 809. SIEGEL, S. (1956) Mann-Whitney U test. Nonparametric Statistics for the Behavioral Sciences, p. 116. McGrawHill Book Co., Inc., New York. SIEGEL, S. (1956) Wilcoxon matched-pairs signed-ranks test. Nonparametric Statistics for the Behavioral Sciences, p. 75. McGraw-Hill Book Co., Inc., New York. VAN GINCKEL, R.F. & HOEBEKE, J. (1976) Effects of levamisole on spontaneous rosette forming cells in murine spleen. Europ. 7. Immunol. 6, 305. VERHAEGEN, H., DE COCK, W., DE CREE, J., VERBRUGGEN, F., VERHAEGEN-DECLERCQ, M. & BRUGMANS, J. (1975) In vitro restoration by levamisole of E-rosette forming cells after inhibition with azathioprine. Lancet, i, 978.
VERHAEGEN, H., DE CREE, J., DE COCK, W. & VERBRUGGEN,
F. (1977) Restoration by levamisole of low E-rosette forming cells in patients suffering from various diseases. Clin. exp. Immunol. 27, 313.
