1247 response between levamisole treated and untreated
lymphocyte interpreted as a positive effect by levamisole. In the accompanying table an augmentating effect by levamisole is shown in relation to the P.H.A. responsiveness state of lymphocytes from subjects in the 4 groups, and in all 63 subjects. It can be seen that in 5 (14%) of 37 subjects whose lymphocytes showed a normal response levamisole augmented the response. Of the 26 subjects showing P.H.A. hyporesponsiveness, lymphocyte response was augmented in 18 (69%). This difference was Levamisole statistically significant (x2=20.5; p< 0-001). treatment restored P.H.A. responsiveness to within the normal range in 11 (61%) of these 18 P.H.A. hyporesponsive subjects. An inhibitory effect was observed in 3 (5%) of the 63 subjects. All 3 (2 P.T.B., 1 N.P.C.) were normoresponsive to P.H.A. cultures
.
in-vitro action of levamisole which are hyporesponsive to P.H.A. are used as target cells. This culture system might be suitable as an in-vitro assay of the lymphoThe culture cyte augmenting effect of levamisole. system may also be useful in elucidating the mechanism of P.H.A. hyporesponsiveness in that an augmenting effect indicates that r.H.A.-responsive cells are present and that their responsiveness can be at least partly restored.
is
-
was
The results indicate that more pronounced when
an
lymphocytes
Department of Pathology,
University
of
Singapore.
S. H. CHAN.
W.H.O. Immunology Research and
Training Centre, University of Singapore, McAlister Road,
Singapore
3.
M. J. SIMONS.
NORMAL RECIRCULATION OF T LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKÆMIA
per hour in the
peripheral lymph was calculated by multiplying lymphocyte concentration by the hourly lymph flow. Spontaneous rosette formation with sheep erythro° its
cytes
used
as a
T-cell marker. 4,6
hourly output of peripheral lymph was within the normal range in the c.L.L. patients. The normal migration of T lymphocytes from blood to lymph suggests a normal recirculation of these cells in patients with C.L.L. This finding indicates that the T cells in c.L.L. represent a normally functioning lymphocyte population. This interpretation is further supported by the normal cell-mediated immunity as assessed in vivo by positive skin-test reactivity to bacterial and fungal antigens in the patients studied. The almost complete absence of B cells in the peripheral lymph confirms the data of Engeset et a1. and our previous findings of impaired recirculation of B-cell-like c.L.L. lymphocytes 9,10 and is in agreement with recent animal studies showing a slower and reduced recirculation of B cells compared with T cells.11,12 This work was supported by the Deutsche Forschungsgemeinschaft, the Alfred and Clare-Pott-Stiftung, and the Ministerium fur Wissenschaft und
Forschung, Nordrhein-Westfalen. K. BREMER
SIR,-In chronic lymphocytic leukxmia (C.L.L.) monoclonal B cells proliferate and accumulate, 1, leaving only small percentages of T cells.3,4 However, absolute reveals normal or even increased numbers of T cells in the blood of patients with C.L.L., and the findings by Dr Catovsky and his colleagues (Sept. 28, p. 751) suggest that increased numbers of circulating T cells are associated with a more stable C.L.L. disease process. To further elucidate the in-vivo functional capacity of T cells in c.L.L., we studied their ability to migrate from blood to
was
As shown in the accompanying table, the percentage of T cells was diminished in the blood of the C.L.L. patients compared with that in the hasmatologically normal controls, whereas the majority (82-98%) of the c.L.L. cells had B-cell surface charac.teristics-e.g., membrane-bound immunoglobulins. In contrast, the peripheral lymph of the C.L.L. patients and the controls contained 72-89 % T cells and no or only few B cells (0-2%). 2 of the c.L.L. patients had increased absolute numbers of T cells both in blood and peripheral lymph. Furthermore, the ratio between the numbers of T cells per ml. blood and those in the
Division of Hæmatology, Department of Medicine, University of Essen,
Essen, Germany.
G. COHNEN W. AUGENER G. BRITTINGER.
measurement
lymph. hsmatologically normal patients (blood-lymphocyte 1500-2000 per c.mm.) and 3 patients with C.L.L. (blood-lymphocyte counts 20,000-130,000 per c.mm.) a peripheral lymph vessel in the lower leg was cannulated to collect prenodal afferent lymph.5 The lymphocyte output In 4
counts
PHAGOCYTOSIS IN CHRONIC GRANULOMATOUS DISEASE SIR,--We should like to comment on the phagocytosis experiments in chronic granulomatous disease reported by Dr Biggar (May 3, p. 991), which has led to the conclusion of an increased phagocytosis of C.G.D. leucocytes. The differences found between the number of viable bacteria in c.G.D. leucocytes and control leucocytes in his 6. 7.
Preud’homme, J. L., Seligmann, M. Blood, 1972, 40, 777. Aisenberg, A. C., Bloch, K. J. New Engl. J. Med. 1972, 287, 272. 3. Brown, G., Greaves, M. F., Lister, T. A., Rapson, N., Papamichail, M. Lancet, 1974, ii, 753. 4. Cohnen, G., Augener, W., Buka, A., Brittinger, G. Acta hœmat. Basel, 1974, 51, 65. 5. Engeset, A., Hager, B., Nesheim, A., Kolbenstvedt, A. Lymphology, 1973, 6, 1.
1. 2.
Jondal, M., Holm, G., Wigzell, H. J. exp. Med. 1972, 136, 207. Augener, W., Cohnen, G., Brittinger, G. Biomed. Express, 1974, 21,
6. 8. Engeset, A., Fröland, S. S., Bremer, K., Scand. J. Hœmat. 1974, 13, 93. 9. Bremer, K., Wack, O., Schick, P. Biomedicine, 1973, 18, 393. 10. Flad, H. D., Huber, Ch., Bremer, K., Menne, H. D., Huber, H. Eur. J. Immunol. 1973, 3, 688. 11. Sprent, J., Miller, J. F. A. ibid. 1972, 2, 384. 12. Howard, J. C. J. exp. Med. 1972, 135, 185.
T LYMPHOCYTES IN BLOOD AND PERIPHERAL LYMPH OF PATIENTS WITH C.L.L. AND HÆMATOLOGICALLY NORMAL PATIENTS
(CONTROLS)
1248 cellular bacteria of 60-75% occurred in the 20-minute phago-
cytosis samples. The reduction was not so striking in the 5-minute phagocytosis samples, probably because killing starts about 15 minutes after phagocytosis. Why the phagocytosis of the control leucocytes in Dr Biggar’s experiments was so slow remains unclear. Our results indicate that the interpretation of increased
phagocytosis of C.G.D. leucocytes is not sound. impaired bactericidal ability of C.G.D. leucocytes may
The well be the cause of the differences in the number of viable intracellular bacteria between C.G.D. and control leucocytes in his experiments. Department of Internal Medicine, Laboratory for Medical Microbiology, University Hospital, Groningen, Netherlands.
G. C. DE GAST WIETSKE S. YDEMA MARGRIET E. WIERSMA J. DANKERT.
ACTION OF HEPATITIS-B ANTIGEN ON NICOTIANA SYLVESTRIS AND VIGNA SINENSIS
SIR,-Banatvala and Paynereported that
Comparison of number of viable intracellular Staph. aureus after 5 and 20 minutes’ phagocytosis in 2 control leucocyte suspensions when incubated in penicillinistreptomycin without phenylbutazone ( •,[]) and when incubated in penicillin/ streptomycin with phenylbutazone (0, []). Bacteria control (X). A striking reduction of viable intracellular Staph. aureus occurred when intracellular killing of granulocytes was not inhibited
by phenylbutazone.
phagocytosis studies can be explained by the well-known impaired bactericidal ability of C.G.D. leucocytes.1 During incubation with lysostaphin (20 minutes) and trypsin (10 minutes) control leucocytes may kill a large percentage of the phagocytised bacteria, whereas c.G.D. leucocytes are unable to kill such bacteria as staphylococci. This possibility at least was not excluded by Dr Biggar and was rejected on account of morphological studies only briefly mentioned. We were prompted to test this possibility as follows: Phagocytosis of control leucocytes was studied in a bacterialeucocyte suspension similar to that used by Dr Biggar (106 Staph. aureus and 5 x 106 leucocytes per ml. with 10% pooled normal human serum). After 5 and 20 minutes aliquots were removed, put into saline with penicillin 1000 u per ml. and streptomycin 500 µg. per ml. and into saline with penicillin/ streptomycin supplemented with phenylbutazone (2 mg. per ml.), and incubated at 370C for 20 minutes to kill extracellular bacteria. In the solution with phenylbutazone intracellular killing will be inhibited immediately,2 whereas in the solution without phenylbutazone intracellular killing is not inhibited. The leucocytes were then washed twice, lysed with sterile distilled water, and the number of viable intracellular bacteria measured by standard dilution and pour-plate techniques. As shown in the accompanying figure, leucocytes of 2 controls phagocytised nearly the same number of Staph. aureus as did the c.G.D. leucocytes described by Dr Biggar, if the samples were incubated in penicillin/streptomycin with phenylbutazone. When the samples were incubated in penicillin/streptomycin without phenylbutazone, however, a reduction of viable intra1. 2.
Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A. Lancet, 1966, i, 1225. Strauss, R. R., Paul, B. B., Sbarra, A. J. J. Bact. 1968, 96, 1982.
sera
con-
taining hepatitis-B antigen (HBAg) induced necrosis in the leaves of Nicotiana sylvestris. Beasley and Su 2 subsequently reported negative results and suggested that their failure might be due to a different HBAg subtype prevalent in Taiwan (ad). Howard and McCarthy3 also reported negative results and indicated that young plants inoculated mechanically can be readily damaged in an irreversible manner, resulting in large areas of collapse which may be mistaken for necrosis and which could lead retardation of growth. We report here negative results in attempting to induce necrosis with HBAg positive sera in young plants of Nicotiana sylvestris and Vigna sinensis grown under different environmental conditions. Seeds of tobacco plants were germinated in sterilised vermiculite at 29 °C. When the seedlings just emerged to
-
HBAg-
TABLE I-EFFECT OF POOLED SERA FROM PATIENTS WITH POSITIVE ACUTE HEPATITIS ON NICOTIANA SYLVESTRIS AND VIGNA SINENSIS
they were transplanted in sterilised sandy loam soil rich in organic matter and were grown in a controlled environmental growth chamber where a 28 °C temperature and a 78,’relative humidity were maintained. The plants were with 14 hours of 500 ft-c. light intensity and 10 hours of dark in the 24-hour day and night cycle.
supplied
Plants were watered as needed. Plants grown under the above-mentioned conditions are highly susceptible to infection by plant viruses. The first series of experiments were conducted using the leaf inoculation technique described by Banatvala and Payne,l simply dusting about 2-month-old (5-10 cm. in 1. 2. 3.
Banatvala, J. E., Payne, C. Lancet, 1973, i, 1359. Beasley, R. P., Su, H. J. ibid. 1973, ii, 1203. Howard, C. R., McCarthy, D. A. ibid. 1974, ii, 219.
lymphocyte interpreted as a positive effect by levamisole. In the accompanying table an augmentating effect by levamisole is shown in relation to the P.H.A. responsiveness state of lymphocytes from subjects in the 4 groups, and in all 63 subjects. It can be seen that in 5 (14%) of 37 subjects whose lymphocytes showed a normal response levamisole augmented the response. Of the 26 subjects showing P.H.A. hyporesponsiveness, lymphocyte response was augmented in 18 (69%). This difference was Levamisole statistically significant (x2=20.5; p< 0-001). treatment restored P.H.A. responsiveness to within the normal range in 11 (61%) of these 18 P.H.A. hyporesponsive subjects. An inhibitory effect was observed in 3 (5%) of the 63 subjects. All 3 (2 P.T.B., 1 N.P.C.) were normoresponsive to P.H.A. cultures
.
in-vitro action of levamisole which are hyporesponsive to P.H.A. are used as target cells. This culture system might be suitable as an in-vitro assay of the lymphoThe culture cyte augmenting effect of levamisole. system may also be useful in elucidating the mechanism of P.H.A. hyporesponsiveness in that an augmenting effect indicates that r.H.A.-responsive cells are present and that their responsiveness can be at least partly restored.
is
-
was
The results indicate that more pronounced when
an
lymphocytes
Department of Pathology,
University
of
Singapore.
S. H. CHAN.
W.H.O. Immunology Research and
Training Centre, University of Singapore, McAlister Road,
Singapore
3.
M. J. SIMONS.
NORMAL RECIRCULATION OF T LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKÆMIA
per hour in the
peripheral lymph was calculated by multiplying lymphocyte concentration by the hourly lymph flow. Spontaneous rosette formation with sheep erythro° its
cytes
used
as a
T-cell marker. 4,6
hourly output of peripheral lymph was within the normal range in the c.L.L. patients. The normal migration of T lymphocytes from blood to lymph suggests a normal recirculation of these cells in patients with C.L.L. This finding indicates that the T cells in c.L.L. represent a normally functioning lymphocyte population. This interpretation is further supported by the normal cell-mediated immunity as assessed in vivo by positive skin-test reactivity to bacterial and fungal antigens in the patients studied. The almost complete absence of B cells in the peripheral lymph confirms the data of Engeset et a1. and our previous findings of impaired recirculation of B-cell-like c.L.L. lymphocytes 9,10 and is in agreement with recent animal studies showing a slower and reduced recirculation of B cells compared with T cells.11,12 This work was supported by the Deutsche Forschungsgemeinschaft, the Alfred and Clare-Pott-Stiftung, and the Ministerium fur Wissenschaft und
Forschung, Nordrhein-Westfalen. K. BREMER
SIR,-In chronic lymphocytic leukxmia (C.L.L.) monoclonal B cells proliferate and accumulate, 1, leaving only small percentages of T cells.3,4 However, absolute reveals normal or even increased numbers of T cells in the blood of patients with C.L.L., and the findings by Dr Catovsky and his colleagues (Sept. 28, p. 751) suggest that increased numbers of circulating T cells are associated with a more stable C.L.L. disease process. To further elucidate the in-vivo functional capacity of T cells in c.L.L., we studied their ability to migrate from blood to
was
As shown in the accompanying table, the percentage of T cells was diminished in the blood of the C.L.L. patients compared with that in the hasmatologically normal controls, whereas the majority (82-98%) of the c.L.L. cells had B-cell surface charac.teristics-e.g., membrane-bound immunoglobulins. In contrast, the peripheral lymph of the C.L.L. patients and the controls contained 72-89 % T cells and no or only few B cells (0-2%). 2 of the c.L.L. patients had increased absolute numbers of T cells both in blood and peripheral lymph. Furthermore, the ratio between the numbers of T cells per ml. blood and those in the
Division of Hæmatology, Department of Medicine, University of Essen,
Essen, Germany.
G. COHNEN W. AUGENER G. BRITTINGER.
measurement
lymph. hsmatologically normal patients (blood-lymphocyte 1500-2000 per c.mm.) and 3 patients with C.L.L. (blood-lymphocyte counts 20,000-130,000 per c.mm.) a peripheral lymph vessel in the lower leg was cannulated to collect prenodal afferent lymph.5 The lymphocyte output In 4
counts
PHAGOCYTOSIS IN CHRONIC GRANULOMATOUS DISEASE SIR,--We should like to comment on the phagocytosis experiments in chronic granulomatous disease reported by Dr Biggar (May 3, p. 991), which has led to the conclusion of an increased phagocytosis of C.G.D. leucocytes. The differences found between the number of viable bacteria in c.G.D. leucocytes and control leucocytes in his 6. 7.
Preud’homme, J. L., Seligmann, M. Blood, 1972, 40, 777. Aisenberg, A. C., Bloch, K. J. New Engl. J. Med. 1972, 287, 272. 3. Brown, G., Greaves, M. F., Lister, T. A., Rapson, N., Papamichail, M. Lancet, 1974, ii, 753. 4. Cohnen, G., Augener, W., Buka, A., Brittinger, G. Acta hœmat. Basel, 1974, 51, 65. 5. Engeset, A., Hager, B., Nesheim, A., Kolbenstvedt, A. Lymphology, 1973, 6, 1.
1. 2.
Jondal, M., Holm, G., Wigzell, H. J. exp. Med. 1972, 136, 207. Augener, W., Cohnen, G., Brittinger, G. Biomed. Express, 1974, 21,
6. 8. Engeset, A., Fröland, S. S., Bremer, K., Scand. J. Hœmat. 1974, 13, 93. 9. Bremer, K., Wack, O., Schick, P. Biomedicine, 1973, 18, 393. 10. Flad, H. D., Huber, Ch., Bremer, K., Menne, H. D., Huber, H. Eur. J. Immunol. 1973, 3, 688. 11. Sprent, J., Miller, J. F. A. ibid. 1972, 2, 384. 12. Howard, J. C. J. exp. Med. 1972, 135, 185.
T LYMPHOCYTES IN BLOOD AND PERIPHERAL LYMPH OF PATIENTS WITH C.L.L. AND HÆMATOLOGICALLY NORMAL PATIENTS
(CONTROLS)
1248 cellular bacteria of 60-75% occurred in the 20-minute phago-
cytosis samples. The reduction was not so striking in the 5-minute phagocytosis samples, probably because killing starts about 15 minutes after phagocytosis. Why the phagocytosis of the control leucocytes in Dr Biggar’s experiments was so slow remains unclear. Our results indicate that the interpretation of increased
phagocytosis of C.G.D. leucocytes is not sound. impaired bactericidal ability of C.G.D. leucocytes may
The well be the cause of the differences in the number of viable intracellular bacteria between C.G.D. and control leucocytes in his experiments. Department of Internal Medicine, Laboratory for Medical Microbiology, University Hospital, Groningen, Netherlands.
G. C. DE GAST WIETSKE S. YDEMA MARGRIET E. WIERSMA J. DANKERT.
ACTION OF HEPATITIS-B ANTIGEN ON NICOTIANA SYLVESTRIS AND VIGNA SINENSIS
SIR,-Banatvala and Paynereported that
Comparison of number of viable intracellular Staph. aureus after 5 and 20 minutes’ phagocytosis in 2 control leucocyte suspensions when incubated in penicillinistreptomycin without phenylbutazone ( •,[]) and when incubated in penicillin/ streptomycin with phenylbutazone (0, []). Bacteria control (X). A striking reduction of viable intracellular Staph. aureus occurred when intracellular killing of granulocytes was not inhibited
by phenylbutazone.
phagocytosis studies can be explained by the well-known impaired bactericidal ability of C.G.D. leucocytes.1 During incubation with lysostaphin (20 minutes) and trypsin (10 minutes) control leucocytes may kill a large percentage of the phagocytised bacteria, whereas c.G.D. leucocytes are unable to kill such bacteria as staphylococci. This possibility at least was not excluded by Dr Biggar and was rejected on account of morphological studies only briefly mentioned. We were prompted to test this possibility as follows: Phagocytosis of control leucocytes was studied in a bacterialeucocyte suspension similar to that used by Dr Biggar (106 Staph. aureus and 5 x 106 leucocytes per ml. with 10% pooled normal human serum). After 5 and 20 minutes aliquots were removed, put into saline with penicillin 1000 u per ml. and streptomycin 500 µg. per ml. and into saline with penicillin/ streptomycin supplemented with phenylbutazone (2 mg. per ml.), and incubated at 370C for 20 minutes to kill extracellular bacteria. In the solution with phenylbutazone intracellular killing will be inhibited immediately,2 whereas in the solution without phenylbutazone intracellular killing is not inhibited. The leucocytes were then washed twice, lysed with sterile distilled water, and the number of viable intracellular bacteria measured by standard dilution and pour-plate techniques. As shown in the accompanying figure, leucocytes of 2 controls phagocytised nearly the same number of Staph. aureus as did the c.G.D. leucocytes described by Dr Biggar, if the samples were incubated in penicillin/streptomycin with phenylbutazone. When the samples were incubated in penicillin/streptomycin without phenylbutazone, however, a reduction of viable intra1. 2.
Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A. Lancet, 1966, i, 1225. Strauss, R. R., Paul, B. B., Sbarra, A. J. J. Bact. 1968, 96, 1982.
sera
con-
taining hepatitis-B antigen (HBAg) induced necrosis in the leaves of Nicotiana sylvestris. Beasley and Su 2 subsequently reported negative results and suggested that their failure might be due to a different HBAg subtype prevalent in Taiwan (ad). Howard and McCarthy3 also reported negative results and indicated that young plants inoculated mechanically can be readily damaged in an irreversible manner, resulting in large areas of collapse which may be mistaken for necrosis and which could lead retardation of growth. We report here negative results in attempting to induce necrosis with HBAg positive sera in young plants of Nicotiana sylvestris and Vigna sinensis grown under different environmental conditions. Seeds of tobacco plants were germinated in sterilised vermiculite at 29 °C. When the seedlings just emerged to
-
HBAg-
TABLE I-EFFECT OF POOLED SERA FROM PATIENTS WITH POSITIVE ACUTE HEPATITIS ON NICOTIANA SYLVESTRIS AND VIGNA SINENSIS
they were transplanted in sterilised sandy loam soil rich in organic matter and were grown in a controlled environmental growth chamber where a 28 °C temperature and a 78,’relative humidity were maintained. The plants were with 14 hours of 500 ft-c. light intensity and 10 hours of dark in the 24-hour day and night cycle.
supplied
Plants were watered as needed. Plants grown under the above-mentioned conditions are highly susceptible to infection by plant viruses. The first series of experiments were conducted using the leaf inoculation technique described by Banatvala and Payne,l simply dusting about 2-month-old (5-10 cm. in 1. 2. 3.
Banatvala, J. E., Payne, C. Lancet, 1973, i, 1359. Beasley, R. P., Su, H. J. ibid. 1973, ii, 1203. Howard, C. R., McCarthy, D. A. ibid. 1974, ii, 219.
