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Colchicine and phytohaemagglutinin-stimulation of human lymphocytes
REFERENCES 1.
I. and BEKGQVIST, H. ?L, ./. Cell Uiol. 15, 60-1 (1962). ,J. and FICQ, A., Ezpfl Cell Res. 38, 153 (1965). BKACHET, .J. and QUARTIER, J., Expfl Cell Res. 32, 110 (1963). E:ar~su~~ssos, H., Acla Physiol. Scund. 52, 195 (1961). ~-~ Expl/ Cell Res. 42, 537 (1966). L.rsc, \\‘. and R~AUIIEH, XV., Expt/ Cell Res. 39, 1 (1965). I..\YG, C. A. and 311~s~ Jn, F., Proc. St//l Accctl. Sri. 55, 152.5 (1966).
AGKEI,L,
2. BRACHET, 3.
1. 5. 6. i.
COLCHICINE
AND PHYTOHAEMAGGLUTININ-STIMULATION OF HUMAN G. ASTALDI,
The Blood
Research
LYMPHOCYTES1 M.
Foundation
GOCIU2
Center,3
and
Municipal
Received November
R.
AIR6
Hospital
of Tortona,
Italy
9, 1966
SIPiCE 19-17, our group has adopted the use of colchicine to evaluate the proliferative activity of blood-forming cells in tissue culture from different physiopathologic conditions [2]. Contrary to expectations, it was possible to ascertain with this method that the proliferative activity of the bone marrow cells in human leukaemias is rather low [ 1,3,4]. Later investigations by many others using different approaches and methods have confirmed this. Recently, we initiated an investigation as to the effect of colchitine on the blastic development of human peripheral blood lymphocytes stimulated with phytohaemagglutinin (PHA) in cell cultures. This research project has a twofold purpose: (1) To test the effect of colchicine on the blastic development process occurring in consequence of the intense RNA- and DNA-synthesis presented by human lymphocytes in the PHA-culture system. (2) To evaluate the extent of proliferation of the PHA-transformed lymphocytes, because the divisions which occur in these cells could definitely contribute in increasing their number. In fact, the number of blast-like cells obtained after 3-4 days of cell culture with PHA is a function not only of the number of lymphocytes which undergo the blastic development, but also of the number of the same blast-like cells which undergo mitotic division. Another factor which could influence the number of blasts occurring in a given interval of a lymphocyte-culture stimulated with PHA, is the possible loss of different types of cells in a different proportion during the incubation period. However, 1 This investigation has been supported in part by a grant from Comm. L. curone, Italy. 2 Institutul de Medicina Interna al Acad. R.S.R., Bucharest, Rumania. 3 The Blood Research Foundation Center, Tortona, Italy, is supported by the Foundation, Washington, D.C., U.S.A. Experimenfal
Cell Research
46
I’agano, Blood
PonteResearch
Colclzicine mtl ~~hytohrremrrgglotir1in-stinllrlrrtiorl
I;ig. l.--Blast-like culture with PHA of the lymphocytes,
cells developed from human peripheral and colcemid-U’hile colchicine does not it blocks the proliferative activity of the
blood prevent blasts.
lymphocytes the blastic
‘229
in a 72 hr cell transformation
the lymphocyte aggregation which occurs in these cultures causes serious difficulties in making absolute counts, and consequently, in evaluating the above-mentioned loss. In these experiments, colchicine (colcemid) has been added to the PHA-culture system at the very beginning of the culture, and th.e drug concentration was 0.25 y/ml. However, colcemid was not added to the control cultures. Periodically, every 12 hr for a total of 96 hr, samples of cells were taken from these cultures, and then smeared and stained with May Gruenwald-Giemsa. Both the percentages of the blast-like cells among all the lymphatic cell population, and the percentages of mitoses among the same blasts wcrc determined on these smears. Fig. 1 shows blast-like cells obtained from normal peripheral blood lymphocytes cultivated in the above-mentioned experimental conditions with colcemid. So far, it is apparent that colchicine did not prevent the hlastic development of the cultured lymphocytes, and simultaneously, all the blasts entering mitosis were blocked at the pre-metaphase stage. On the other hand, it has also been previously shown that colchicine does not prevent the maturation of bone-marrow erythroblasts in tissue culture [5]. Fig. 2 graphically shows the behaviour during culture of the percentages of blasts among all the lymphatic cell population. There is no apparent significant difference between the extent of blasts developed in the control cultures, and the extent of blasts developed in the colchicine-cultures during the first 24-36 hr of cultivation, i.e., during the period of the RNA-synthesis in the PHA-stimulated lymphocytes. On Experimentul
Cell
Research
46
230
G. Astaldi, M. Gociu and R. Airi,
the contrary, from the period of 48 hr on, a significant difference is evident, due to the fact that in the control-cultures the blast-like cells underwent divisions, whereas in the colchicine-cultures blast divisions did not occur. These results would seem to show that: (1) Colchicine (which was sufficient to block in premetaphase all the blasts entering mitosis at the experimented dose) does not interfere with the capacity of normal human lymphocytes to undergo the blastic development in the PHA-culture system.
Fig. 2.-Percentages
of blast-like cells obtained in PHA-cultures with and without Colcemid. The difference approximately expresses the extent of blasts derived through the transformation process of the lymphocytes, and the extent of those blasts derived through mitotic division. O- 0, Cultures with PHA; 0 - 0, cultures with PHA and colchicine.
(2) This experimental procedure may provide some information on the number of blast-like cells obtained in a 3-day culture. In other words, there exists the possibility that this method may help to judge the extent of blasts derived through the transformation process of lymphocytes, and of those blasts derived through mitotic divisions. The effect of colchicine on the blastic development of human peripheral blood lymphocytes stimulated in cell culture with Phytohaemagglutinin (PHA) has been investigated. The results showed that: (I) Colchicine (Cokemid, in dose of 0.25 y/ml, which was sufficient to block in premetaphase all the blasts entering mitosis) does not interfere with the capacity of normal human lymphocytes to undergo the blastic transformation in the PHA-culture system; (2) The experimented method may Experimental
Cell Research
46
Mammalian
bursa Fabricii
231
provide some information for explaining the number of the blast-like cells obtained in a 3-day culture. In other words, this method may help in judging the extent of blasts derived through the transformation process of lymphocytes and, on the other hand, the extent of those blasts derived through mitotic division.
REFERENCES
1. ASTALDI, G., in Ciba Foundation Symposium on Haemopoiesis, p. 99. London, 1960. 2. ASTALDI, G., ALLEGRI, A. and MAURI, C., Experientia 3, 499 (1947). 3. ASTALDI, G. and MAURI, C., Le Sang 21, 378 (1950). 4. __ Rev. Belg Path. MCd. Expptl 23, 69 (1953). 5. ASTALDI, G., MAURI, C. and TOLENTINO, P., ibid. 20, 101 (1948).
THE
MAMMALIAN
EQUIVALENT
TO BURSA
J.
& A.
Churchill,
FABRIC11
OF BIRDS K. Histological
E.
FICHTELIUS
Institute, Uppsala, Received
University Sweden
November
of Uppsala, 11, 1966
A
theory is here presented about the mammalian equivalent to bursa Fabricii of birds. The theory is based on data about the phylogenesis of the lymphatic tissue presented mainly by Good and collaborators, and on recent findings on the DNAsynthesis of lymphocytes within epithelium obtained at our laboratory. From some points of view the birds have the most highly developed immune system. The work of Cooper, Peterson and Good [2,3] has shown that there is a dissociation of immunological functions in the chicken. The bursa of Fabricius controls the development of immunoglobulin production, and the thymus is largely responsible for the ontogeny of the cellular immunity. Cooper et al. [2, 31 added sublethal irradiation to thymectomy or bursectomy in the immediate post-hatching period of chicken. In the bursectomized and irradiated chicken there were no germinal centres or plasma cells, no gammaM-, or gammaG-immunoglobulins detected by immunoelectrophoresis and no detectable antibody production to protein or bacterial antigens even after repeated stimulation. The number of small lymphocytes in the splenic white pulp and in the blood were normal as was the capacity for graft-versus-host reactions. There was definite, though diminished, ability to reject skin homografts. The immunological defect in thymectomized-irradiated chickens resembled that of neonatally thymectomized mice, rats and hamsters. There were impaired delayed hypersensitivity reactions and graft-versus-host capabilities, absent or slow, abnormal, homograft rejection, and a moderately reduced antibody response to both protein and bacterial antigens. As in mammals, the cell type most affected by thymectomy was the small Experimental
Cell Research
46
I. and BEKGQVIST, H. ?L, ./. Cell Uiol. 15, 60-1 (1962). ,J. and FICQ, A., Ezpfl Cell Res. 38, 153 (1965). BKACHET, .J. and QUARTIER, J., Expfl Cell Res. 32, 110 (1963). E:ar~su~~ssos, H., Acla Physiol. Scund. 52, 195 (1961). ~-~ Expl/ Cell Res. 42, 537 (1966). L.rsc, \\‘. and R~AUIIEH, XV., Expt/ Cell Res. 39, 1 (1965). I..\YG, C. A. and 311~s~ Jn, F., Proc. St//l Accctl. Sri. 55, 152.5 (1966).
AGKEI,L,
2. BRACHET, 3.
1. 5. 6. i.
COLCHICINE
AND PHYTOHAEMAGGLUTININ-STIMULATION OF HUMAN G. ASTALDI,
The Blood
Research
LYMPHOCYTES1 M.
Foundation
GOCIU2
Center,3
and
Municipal
Received November
R.
AIR6
Hospital
of Tortona,
Italy
9, 1966
SIPiCE 19-17, our group has adopted the use of colchicine to evaluate the proliferative activity of blood-forming cells in tissue culture from different physiopathologic conditions [2]. Contrary to expectations, it was possible to ascertain with this method that the proliferative activity of the bone marrow cells in human leukaemias is rather low [ 1,3,4]. Later investigations by many others using different approaches and methods have confirmed this. Recently, we initiated an investigation as to the effect of colchitine on the blastic development of human peripheral blood lymphocytes stimulated with phytohaemagglutinin (PHA) in cell cultures. This research project has a twofold purpose: (1) To test the effect of colchicine on the blastic development process occurring in consequence of the intense RNA- and DNA-synthesis presented by human lymphocytes in the PHA-culture system. (2) To evaluate the extent of proliferation of the PHA-transformed lymphocytes, because the divisions which occur in these cells could definitely contribute in increasing their number. In fact, the number of blast-like cells obtained after 3-4 days of cell culture with PHA is a function not only of the number of lymphocytes which undergo the blastic development, but also of the number of the same blast-like cells which undergo mitotic division. Another factor which could influence the number of blasts occurring in a given interval of a lymphocyte-culture stimulated with PHA, is the possible loss of different types of cells in a different proportion during the incubation period. However, 1 This investigation has been supported in part by a grant from Comm. L. curone, Italy. 2 Institutul de Medicina Interna al Acad. R.S.R., Bucharest, Rumania. 3 The Blood Research Foundation Center, Tortona, Italy, is supported by the Foundation, Washington, D.C., U.S.A. Experimenfal
Cell Research
46
I’agano, Blood
PonteResearch
Colclzicine mtl ~~hytohrremrrgglotir1in-stinllrlrrtiorl
I;ig. l.--Blast-like culture with PHA of the lymphocytes,
cells developed from human peripheral and colcemid-U’hile colchicine does not it blocks the proliferative activity of the
blood prevent blasts.
lymphocytes the blastic
‘229
in a 72 hr cell transformation
the lymphocyte aggregation which occurs in these cultures causes serious difficulties in making absolute counts, and consequently, in evaluating the above-mentioned loss. In these experiments, colchicine (colcemid) has been added to the PHA-culture system at the very beginning of the culture, and th.e drug concentration was 0.25 y/ml. However, colcemid was not added to the control cultures. Periodically, every 12 hr for a total of 96 hr, samples of cells were taken from these cultures, and then smeared and stained with May Gruenwald-Giemsa. Both the percentages of the blast-like cells among all the lymphatic cell population, and the percentages of mitoses among the same blasts wcrc determined on these smears. Fig. 1 shows blast-like cells obtained from normal peripheral blood lymphocytes cultivated in the above-mentioned experimental conditions with colcemid. So far, it is apparent that colchicine did not prevent the hlastic development of the cultured lymphocytes, and simultaneously, all the blasts entering mitosis were blocked at the pre-metaphase stage. On the other hand, it has also been previously shown that colchicine does not prevent the maturation of bone-marrow erythroblasts in tissue culture [5]. Fig. 2 graphically shows the behaviour during culture of the percentages of blasts among all the lymphatic cell population. There is no apparent significant difference between the extent of blasts developed in the control cultures, and the extent of blasts developed in the colchicine-cultures during the first 24-36 hr of cultivation, i.e., during the period of the RNA-synthesis in the PHA-stimulated lymphocytes. On Experimentul
Cell
Research
46
230
G. Astaldi, M. Gociu and R. Airi,
the contrary, from the period of 48 hr on, a significant difference is evident, due to the fact that in the control-cultures the blast-like cells underwent divisions, whereas in the colchicine-cultures blast divisions did not occur. These results would seem to show that: (1) Colchicine (which was sufficient to block in premetaphase all the blasts entering mitosis at the experimented dose) does not interfere with the capacity of normal human lymphocytes to undergo the blastic development in the PHA-culture system.
Fig. 2.-Percentages
of blast-like cells obtained in PHA-cultures with and without Colcemid. The difference approximately expresses the extent of blasts derived through the transformation process of the lymphocytes, and the extent of those blasts derived through mitotic division. O- 0, Cultures with PHA; 0 - 0, cultures with PHA and colchicine.
(2) This experimental procedure may provide some information on the number of blast-like cells obtained in a 3-day culture. In other words, there exists the possibility that this method may help to judge the extent of blasts derived through the transformation process of lymphocytes, and of those blasts derived through mitotic divisions. The effect of colchicine on the blastic development of human peripheral blood lymphocytes stimulated in cell culture with Phytohaemagglutinin (PHA) has been investigated. The results showed that: (I) Colchicine (Cokemid, in dose of 0.25 y/ml, which was sufficient to block in premetaphase all the blasts entering mitosis) does not interfere with the capacity of normal human lymphocytes to undergo the blastic transformation in the PHA-culture system; (2) The experimented method may Experimental
Cell Research
46
Mammalian
bursa Fabricii
231
provide some information for explaining the number of the blast-like cells obtained in a 3-day culture. In other words, this method may help in judging the extent of blasts derived through the transformation process of lymphocytes and, on the other hand, the extent of those blasts derived through mitotic division.
REFERENCES
1. ASTALDI, G., in Ciba Foundation Symposium on Haemopoiesis, p. 99. London, 1960. 2. ASTALDI, G., ALLEGRI, A. and MAURI, C., Experientia 3, 499 (1947). 3. ASTALDI, G. and MAURI, C., Le Sang 21, 378 (1950). 4. __ Rev. Belg Path. MCd. Expptl 23, 69 (1953). 5. ASTALDI, G., MAURI, C. and TOLENTINO, P., ibid. 20, 101 (1948).
THE
MAMMALIAN
EQUIVALENT
TO BURSA
J.
& A.
Churchill,
FABRIC11
OF BIRDS K. Histological
E.
FICHTELIUS
Institute, Uppsala, Received
University Sweden
November
of Uppsala, 11, 1966
A
theory is here presented about the mammalian equivalent to bursa Fabricii of birds. The theory is based on data about the phylogenesis of the lymphatic tissue presented mainly by Good and collaborators, and on recent findings on the DNAsynthesis of lymphocytes within epithelium obtained at our laboratory. From some points of view the birds have the most highly developed immune system. The work of Cooper, Peterson and Good [2,3] has shown that there is a dissociation of immunological functions in the chicken. The bursa of Fabricius controls the development of immunoglobulin production, and the thymus is largely responsible for the ontogeny of the cellular immunity. Cooper et al. [2, 31 added sublethal irradiation to thymectomy or bursectomy in the immediate post-hatching period of chicken. In the bursectomized and irradiated chicken there were no germinal centres or plasma cells, no gammaM-, or gammaG-immunoglobulins detected by immunoelectrophoresis and no detectable antibody production to protein or bacterial antigens even after repeated stimulation. The number of small lymphocytes in the splenic white pulp and in the blood were normal as was the capacity for graft-versus-host reactions. There was definite, though diminished, ability to reject skin homografts. The immunological defect in thymectomized-irradiated chickens resembled that of neonatally thymectomized mice, rats and hamsters. There were impaired delayed hypersensitivity reactions and graft-versus-host capabilities, absent or slow, abnormal, homograft rejection, and a moderately reduced antibody response to both protein and bacterial antigens. As in mammals, the cell type most affected by thymectomy was the small Experimental
Cell Research
46