370 MITOGENIC STIMULATION BY LEVAMISOLE ON NORMAL HUMAN LYMPHOCYTES AND LEUKÆMIC
TABLE II-CHROMOSOME POPULATIONS IN BONE-MARROW AND PERIPHERAL BLOOD FROM BABY WITH A.L.L.
LYMPHOBLASTS
SiR,—Several letters have discussed the immunomodulatory effects of levamisole on lymphocytes in vitrol-3 and its enhancement of the immune response in man 4-6 To elucidate cytogenetically a possible effect by levamisole on leuksemic lymphocytes, 2 x 106 lymphocytes from 10 normal controls and 11 patients with leukaemia were cultured for 72 hours at 37C in 4 ml Eagle’s modified medium supplemented with 10% fetal-calf serum and antibiotics, in the presence of phytohaemagglutinin (P.H.A.) (3[ig/ml), levamisole (30flglml) (Janssen Pharmaceutica, Belgium), or both. "’he mitotic index per 1000 cells and chromosome abnormalities per 100 metaphases were estimated in all the cultures. In the normal controls, G-banding showed no differences in the frequency of chromosome abnormalities between levamisoletreated and untreated cultures, but mitotic indices were high when levamisole and P.H.A. were added together (table I). A deTABLE I-RESPONSES FROM NORMAL AND LEUKAEMIC LYMPHOCYTES
STIMULATED IN VITRO BY P.H.A., LEVAMISOLE, OR P.H.A. + I
,
LEVAMISOLE*
1
by P.H.A.+levamisole on normal and lymphoblastic-leukæmic lymphocyte populations. Possibly, mitogenic stimulation of leuktemic lymphoblasts
might be useful as an in-vitro assay to study the chromosome characteristics and behaviour of A.L.L. cells in peripheralblood cultures. In vivo, levamisole has been used as a stimulator of the immune response.’-9 Could immunomodulators also generate direct modifications on neoplastic lymphocytes and in tumours if they were administered in leukaemias and lymphomas in vivo? Service of Pathology, Juan Canalejo Hospital, La Coruña, Spain.
V. GOYANES-VILLAESCUSA
ENTEROCHROMAFFIN CELLS AND CŒLIAC DISEASE
*
After 3 hours’ culture with colchicine.
tectable increase in mitotic index and chromosome abnormalities was seen in 4 of 8 cultures from acute lymphoblastic leukaemic (A.L.L.) cells stimulated by P.H.A.+levamisole, compared with P.H.A.-treated cultures from the same patients (table i). However, no significant changes were observed between cultures from chronic lymphocytic leukaemia (c.L.L.) treated with P.H.A. or P.H.A.+levamisole. In a baby with A.L.L. with 101 000 blasts/mm3 in peripheral blood, direct study of bone-marrow metaphases (75%) showed a cell-line with an unfamiliar marker C chromosome. The same chromosome was identified in 32% of the metaphases from peripheral-blood lymphocytes grown in the presence of P.H.A.+levamisole, but not when P.H.A. was used alone (table n). In the P.H.A.+levamisole stimulated cultures, the levels of labelled lymphocytes following incubation with 3H-thymidine (3Ci/mM, lµCi/ml) for the last 2 h of culture were 38% for the controls, 6% for c.L.L., and 40% for A.L.L. cells. These results suggest effective mitogenic stimulation in vitro Biniaminov, M., Ramot, B. Lancet, 1975, i, 464. De Cock, W., De Cree, J., Verbruggen, F., Verhaegen-Declercq, M., Brugmans, J. ibid. p. 978. 3. Chan, S., Simons, M. ibid. p. 1246. 4. De Cree, J., Verhaegen, H., De Cock, W., Vanheule, R., Brugmans, J., Schuermans, V. ibid. 1974, ii, 294. 5. Schuermans, Y. ibid. 1975, i, 111. 6. Dinai, Y., Pras, M. ibid. 1975, ii, 556. 1. 2.
Verhaegen, H.,
SIR Increased urinary excretion of 5-hydroxyindoleacetic acid (5-H.I.A.A.), a metabolite of 5-hydroxytryptamine (5-H.T.), has been reported in both adults and children with untreated cceliac disease.10-13Clinical recovery following the introduction of a gluten-free diet is accompanied by a fall in urinary 5-H.I.A.A.35 Raised blood-levels of 5-H.T. in coeliac disease also return to normal after gluten withdrawal from the diet." Since 5-H.T. is synthesised by enterochromaffin (E.c.) cells in the small intestine, hyperplasia and/or hyperactivity of these cells could explain the raised levels of both blood-5-H.T. and urinary 5-H.I.A.A. in coeliac disease. To examine this hypothesis we have used a morphometric method 16 to count E.c. cells in samples of duodenal mucosa obtained by peroral biopsy from a standard site between the third and fourth part of the duodenum in three groups of children. 17 E.c.-cell counts were com pared in 10 children with normal biopsy findings on light mic roscopy and 10 children with coeliac disease whose biopsy material showed flattened villi. A third group of 4 children with a clinical history suggestive of cceliac disease was also studied. The duodenal mucosa in these patients initially showed equivocal changes, but after 10 days on an oral gluten challenge (10 g gluten 3 times a day) a second duodenal-biopsy specimen showed more severe epithelial damage. These children subsequently responded well to gluten withdrawal. E.c. cells were stained with alkaline diazonium" and counted by inserting a grid into the eyepiece of a light microscope. Tlus gave a final image magification of x 250. The 1 cm square gnd contained 100 uniformly distributed points, and the section of duodenal mucosa was systematically covered by the grid unui the E.C. cells within tissue bounded by 1000 points had been 7. Tripodi, D., Parks, L., Brugmans, J. New. Engl. J. Med. 1973, 289, 354 8. Amery, W. Lancet, 1975, i, 389. 9. Webster, D., Hughes, L. ibid. 10. Haverback, B. J., Davidson, J. D. Gastroenterology, 1958, 35, 570. 11. Kowlessar, O. D., Williams, R. C., Law, D. H., Sleisenger, M. H. New Eng J. Med. 1958, 259, 340. 12. Challacombe, D. N., Brown, G. A., Black, S. C., Storris, M. H. Archs Dis. Childh. 1972, 47, 442. 13. Challacombe, D. N., Goodall, M., Gaze, H., Brown, G. A. ibid. 1935, 50, 779. 14. Sleisenger, M. H. New Engl. J. Med. 1961, 265, 49. 15. Pimparker, B. D., Senesky, D., Kaiser, M. H. Gastroenterology, 1961. 46 504. 16. Piris, J., Whitehead, R. J. clin. Path. 1975, 28, 636. 17. Challacombe, D. N., Robertson, K. Unpublished. 18. Pearse, A. G. E. Histochemistry, Theoretical and Applied. London. 1965
TABLE II-CHROMOSOME POPULATIONS IN BONE-MARROW AND PERIPHERAL BLOOD FROM BABY WITH A.L.L.
LYMPHOBLASTS
SiR,—Several letters have discussed the immunomodulatory effects of levamisole on lymphocytes in vitrol-3 and its enhancement of the immune response in man 4-6 To elucidate cytogenetically a possible effect by levamisole on leuksemic lymphocytes, 2 x 106 lymphocytes from 10 normal controls and 11 patients with leukaemia were cultured for 72 hours at 37C in 4 ml Eagle’s modified medium supplemented with 10% fetal-calf serum and antibiotics, in the presence of phytohaemagglutinin (P.H.A.) (3[ig/ml), levamisole (30flglml) (Janssen Pharmaceutica, Belgium), or both. "’he mitotic index per 1000 cells and chromosome abnormalities per 100 metaphases were estimated in all the cultures. In the normal controls, G-banding showed no differences in the frequency of chromosome abnormalities between levamisoletreated and untreated cultures, but mitotic indices were high when levamisole and P.H.A. were added together (table I). A deTABLE I-RESPONSES FROM NORMAL AND LEUKAEMIC LYMPHOCYTES
STIMULATED IN VITRO BY P.H.A., LEVAMISOLE, OR P.H.A. + I
,
LEVAMISOLE*
1
by P.H.A.+levamisole on normal and lymphoblastic-leukæmic lymphocyte populations. Possibly, mitogenic stimulation of leuktemic lymphoblasts
might be useful as an in-vitro assay to study the chromosome characteristics and behaviour of A.L.L. cells in peripheralblood cultures. In vivo, levamisole has been used as a stimulator of the immune response.’-9 Could immunomodulators also generate direct modifications on neoplastic lymphocytes and in tumours if they were administered in leukaemias and lymphomas in vivo? Service of Pathology, Juan Canalejo Hospital, La Coruña, Spain.
V. GOYANES-VILLAESCUSA
ENTEROCHROMAFFIN CELLS AND CŒLIAC DISEASE
*
After 3 hours’ culture with colchicine.
tectable increase in mitotic index and chromosome abnormalities was seen in 4 of 8 cultures from acute lymphoblastic leukaemic (A.L.L.) cells stimulated by P.H.A.+levamisole, compared with P.H.A.-treated cultures from the same patients (table i). However, no significant changes were observed between cultures from chronic lymphocytic leukaemia (c.L.L.) treated with P.H.A. or P.H.A.+levamisole. In a baby with A.L.L. with 101 000 blasts/mm3 in peripheral blood, direct study of bone-marrow metaphases (75%) showed a cell-line with an unfamiliar marker C chromosome. The same chromosome was identified in 32% of the metaphases from peripheral-blood lymphocytes grown in the presence of P.H.A.+levamisole, but not when P.H.A. was used alone (table n). In the P.H.A.+levamisole stimulated cultures, the levels of labelled lymphocytes following incubation with 3H-thymidine (3Ci/mM, lµCi/ml) for the last 2 h of culture were 38% for the controls, 6% for c.L.L., and 40% for A.L.L. cells. These results suggest effective mitogenic stimulation in vitro Biniaminov, M., Ramot, B. Lancet, 1975, i, 464. De Cock, W., De Cree, J., Verbruggen, F., Verhaegen-Declercq, M., Brugmans, J. ibid. p. 978. 3. Chan, S., Simons, M. ibid. p. 1246. 4. De Cree, J., Verhaegen, H., De Cock, W., Vanheule, R., Brugmans, J., Schuermans, V. ibid. 1974, ii, 294. 5. Schuermans, Y. ibid. 1975, i, 111. 6. Dinai, Y., Pras, M. ibid. 1975, ii, 556. 1. 2.
Verhaegen, H.,
SIR Increased urinary excretion of 5-hydroxyindoleacetic acid (5-H.I.A.A.), a metabolite of 5-hydroxytryptamine (5-H.T.), has been reported in both adults and children with untreated cceliac disease.10-13Clinical recovery following the introduction of a gluten-free diet is accompanied by a fall in urinary 5-H.I.A.A.35 Raised blood-levels of 5-H.T. in coeliac disease also return to normal after gluten withdrawal from the diet." Since 5-H.T. is synthesised by enterochromaffin (E.c.) cells in the small intestine, hyperplasia and/or hyperactivity of these cells could explain the raised levels of both blood-5-H.T. and urinary 5-H.I.A.A. in coeliac disease. To examine this hypothesis we have used a morphometric method 16 to count E.c. cells in samples of duodenal mucosa obtained by peroral biopsy from a standard site between the third and fourth part of the duodenum in three groups of children. 17 E.c.-cell counts were com pared in 10 children with normal biopsy findings on light mic roscopy and 10 children with coeliac disease whose biopsy material showed flattened villi. A third group of 4 children with a clinical history suggestive of cceliac disease was also studied. The duodenal mucosa in these patients initially showed equivocal changes, but after 10 days on an oral gluten challenge (10 g gluten 3 times a day) a second duodenal-biopsy specimen showed more severe epithelial damage. These children subsequently responded well to gluten withdrawal. E.c. cells were stained with alkaline diazonium" and counted by inserting a grid into the eyepiece of a light microscope. Tlus gave a final image magification of x 250. The 1 cm square gnd contained 100 uniformly distributed points, and the section of duodenal mucosa was systematically covered by the grid unui the E.C. cells within tissue bounded by 1000 points had been 7. Tripodi, D., Parks, L., Brugmans, J. New. Engl. J. Med. 1973, 289, 354 8. Amery, W. Lancet, 1975, i, 389. 9. Webster, D., Hughes, L. ibid. 10. Haverback, B. J., Davidson, J. D. Gastroenterology, 1958, 35, 570. 11. Kowlessar, O. D., Williams, R. C., Law, D. H., Sleisenger, M. H. New Eng J. Med. 1958, 259, 340. 12. Challacombe, D. N., Brown, G. A., Black, S. C., Storris, M. H. Archs Dis. Childh. 1972, 47, 442. 13. Challacombe, D. N., Goodall, M., Gaze, H., Brown, G. A. ibid. 1935, 50, 779. 14. Sleisenger, M. H. New Engl. J. Med. 1961, 265, 49. 15. Pimparker, B. D., Senesky, D., Kaiser, M. H. Gastroenterology, 1961. 46 504. 16. Piris, J., Whitehead, R. J. clin. Path. 1975, 28, 636. 17. Challacombe, D. N., Robertson, K. Unpublished. 18. Pearse, A. G. E. Histochemistry, Theoretical and Applied. London. 1965
