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EFFECT OF BORDETELLA PERTUSSIS VACCINE ON GROWTH OF HEPATOMA
402 that
one
of the
deciding factors
is the side
on
which the
gonad is situated. Fetal testes in rats 10 and mice 11 are markedly larger than ovaries, and this leads to the question whether the larger size is the cause or the result of testicular differentiation. The present data favour a causal relation. Indeed, if the development of testicular rather than ovarian tissue can be affected by so feeble a force as lateral asymmetry, it would seem unlikely that the information normally provided by the Y chromosome is very specific. Fetal testes are distinguished by their precocious growth and differentiation,12 in contrast to the slow development characteristic of the ovary. The early growth of the testis is normally ensured by the presence of a Y chromosome, 10 while in hermaphrodites the likelihood of the fetal gonad developing into a testis is increased by being situated on the right side of the body. I thank Dr Sylvia Lawler and the staff of the Fetal Tissue Bank, Royal Marsden Hospital, for supplying the fetal gonads, and Mr George Barrett for serial sectioning. This work was supported by a grant from the Science Research Council. Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE.
URSULA MITTWOCH.
BETA-BLOCKADE AND HEART WEIGHTS SIR,-The conclusions of Dr Vaughan Williams and Dr Raine (Nov. 2, p. 1048) are very interesting. It is, however, regrettable that no blood-pressures and bloodpressure changes were reported, since chronic administration of P-blockers may have had a hypotensive effect. This in turn could have been the cause for the lower ventricular weights of the treated rabbits, compared with the controls. 13 It is still unclear, therefore, which is the egg and which is the chicken. Beilinson Hospital, Petah Tiqva, Israel.
YEHUDA M. TRAUB.
BLOOD AND URINARY NICOTINE IN NON-SMOKERS was SIR,—I pleased to see the article on nicotine levels in non-smokers by Dr Russell and Mr Feyerabend (Jan. 25, p. 179). They find that the urinary nicotine in nonsmokers has a mean level of 12°4 16°9 ng. per ml. (standard deviation) (table 11). That means that the 15% or so of the population who lie one standard deviation below the mean must have negative quantities of nicotine in the urine. What do negative quantities of nicotine look like ? Sadly, on looking at the individual values and the figure I see that it is nothing new. Only the usual mistake has been made. The authors clearly recognise in their figure that urinary nicotine levels are logarithmically distributed, not normally distributed on an arithmetic scale. Yet they use The same mistake is constantly arithmetic statistics. being made with serum-enzyme activity and many other measurements. Biological measurements are more
often
Statistical treatments that ignore this are metic form. erroneous and misleading. Unfortunately the conclusions drawn in the particular case of nicotine transfer to nonsmokers do not depend on the statistical analysis and remain disturbingly valid. University College Hospital Medical School, University Street, London WC1E 6JJ.
A. E. M. MCLEAN.
EFFECT OF BORDETELLA PERTUSSIS VACCINE ON GROWTH OF HEPATOMA SIR,-Several adjuvants, such as B.C.G. and Corynebacterium parvum, inhibit tumour growth.1-5 Depending upon the route of injection, Bordetella pertussis also inhibits tumour growth. 6,7 Although the mechanism remains undefined, it functions as an immune potentiator. The growth of well-established hepatoma 5123 tumours was inhibited by either intravenous or subcutaneous injections of hepatoma cells and pertussis vaccine.’7 Conversely, growth was enhanced if the combined treatment was administered before tumour transplantation. The effects seemed to be tumour specific.’7 Moloney lymphoma isografts were facilitated in mice (strain C57L/KL) after intraperitoneal injection of pertussis vaccine. 8 B. pertussis vaccine (P.v.) has been used for active immunotherapy in patients with acute leukaemia.9 The results of these treatments suggest that this vaccine inhibits tumour growth. In an effort to determine the role of P.v. in tumour inhibition, female Buffalo rats, weighing 230-260- g., were inoculated with cells from Morris hepatoma 7777. Isografts of Morris hepatoma 7777 were obtained from 1. 2.
3. 4. 5. 6. 7. 8. 9.
Zbar, B., Bernstein, I., Tanaka, T., Rapp, H. J. Science, 1970, 170, 1217. Zbar, B., Bernstein, I. W., Rapp, H. J. J. natn. Cancer Inst. 1971, 46, 831. Zbar, B. Natn. Cancer Inst. Monogr. 1972, 35, 341. Woodruff, M. F. A., Boak, J. L. Br. J. Cancer, 1966, 20, 245. Halpern, B. N., Biozzi, G., Stiffel, C., Mouton, D. Nature, 1966, 212, 853. Likhite, V. V. Cancer Res. 1974, 34, 1027. Wissler, R. W., Craft, K., Kesden, D., Polisky, B., Dzoga, K. Proc. First Congr. Transplant Soc. 1968, p. 539. Floersheim, G. L. Nature, 1967, 216, 1235. Guyer, R. J., Crowther, D. Br. med. J. 1969, iv, 406.
logarithmically
than arithmetically distributed. Statistics based on normal distribution can be used if the log of the values is taken and treated in the usual way. The anti-log of the mean is then the geometric mean and one standard deviation either side can be given in the log form or in its unsymmetrical arithMittwoch, U. Genetics of Sex Differentiation. New York, 1973. Mittwoch, U., Buehr, M. L. Differentiation, 1973, 1, 219. Jost, A. in Schering Workship on Contraception: The Masculine Gender (edited by G. Raspé), p. 3. Oxford, 1973. 13. Hall, O., Hall, C. E., Ogden, E. Am. J. Physiol. 1953, 174, 175. 10. 11. 12.
Fig. 1-Effect of P.V. All rats received
cutaneously.
on
growth of hepatomas.
transplantable
Morris
hepatoma
7777 sub-
403
Although extensive proliferation of some cells may follow P.v. injections, the newly circulating lymphocytes are not the product of recent cell divisions.16 Morse and Barron suggested that P.v. induced the alteration of lymphocyte cell membranes which are partly responsible for the failure of the cells to recirculate properly and may be involved in the initial redistribution process.15 Evidence suggests that there may be a compartmentalisation of cell-mediated immune responses and transplantation immunity in monkeys. 17 ,188 These studies suggest the importance of the availability of the participating T lymphocytes at the local site. An attractive hypothesis of one aspect of the mechanism of action of this immunopotentiator (P.v.) would be the redistribution of a lymphocyte subpopulation (due to the initial triggering mechanism) resulting in the attraction of sensitised killer T lymphocytes at the P.v. inoculation site. By such a mechanism, the tumour would be directly attacked. The killer T cells are less available at the tumour site when the adjuvant is given intraperitoneally, thus facilitating the growth of the tumour. We are currently examining these hypotheses. We thank M. Parmely for measuring a-fetoprotein. This work was supported by a clinical investigatorship given to T. J. Yoo.
Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, U.S.A.
MOTOR-NEURONE DISEASE IN ISRAEL SIR,-Comparing the variations in the incidence and the natural history of an illness as they occur in different countries and populations, helps to clarify the nature of certain diseases. The gain in our knowledge concerning
Fig. 2-Level of oc-fetoprotein in tumour-bearing rats. All the rats received transplantable Morris hepatoma 7777
subcutaneously. Dr John S.
Thompson, University of Iowa. The solid hepatomas were excised and cut into small pieces of equal size (0-5 cm. in diameter) in Eisen’s culture medium. The implants were inserted subcutaneously on the flank of each rat approximately medial to the thigh. Five rats used in each group. The controls received no P.v. Of the other two groups, one was injected with Pv. at the tumour site and the other was injected with vaccine intraperitoneally within an hour of the inoculation of the tumour. In each group the rats received 1 ml. of vaccine containing 2 x 109 cells per ml. B. pertussis vaccine, strain 18334, was obtained from Connaught Laboratories, Toronto. The tumour was measured three times a week with a calliper in three axes to determine its rate of growth. Tail bleedings were also performed. The levels of m-fetoprotein in the sera were assayed by radioimmunoassay 10 and served as another indicator of tumour growth. The results of tumour growth and ex-fetoprotein levels are presented in figs. 1 and 2, respectively. This study demonstrated that P.v. inhibited tumour growth if it was administered at the tumour site at implantation, thus confirming observations by Guyer and Wissler.’9 If the vaccine was given intraperitoneally, the growth of the tumour was facilitated as reported by Floersheiin.8 Injection of P.v. mobilises lymphocytes and induces leucocytosis and lymphocytosis in ariimals.11-14 The mechanism of lymphocyte mobilisation by P.v. is not known. Its mobilising activity may be due to the abnormal redistribution of lymphocytes from various lymphoid organs and bone-marrow into the peripheral circulation.15
multiple sclerosis is 69 patients with
10. 11. 12. 13.
Sell, S. Cancer Res. 1973, 31, 1010. Morse, S. I., Bray, K. K. J. exp. Med. 1969, 129, 523. Morse, S. I. ibid. 1965, 121, 49. Rai, K., Chanana, A. D., Cronkite, E. P., Joel, D. D., Stevens, J. B. Blood, 1971, 38, 49. 14. Phanuphak, P., Moorhead, J. W., Claman, H. N. Int. Archs Allergy appl. Immun. 1972, 43, 305. 15. Morse, S. I., Barron, B. A. J. exp. Med. 1970, 132, 663.
good example of such results. disease (M.N.D.) were seen over the years departments of neurology of the Tel-Aviv University (58 in the Beilinson Hospital and 11 in the Ichilov Municipal Hospital). The notes of these patients were reviewed, and, where possible, their families were contacted and questioned; the evolving clinical data were analysed. The male/female ratio was 2/1, the average age of onset being somewhat lower in females. This finding, as well as data concerning age of onset, presenting symptoms, laboratory findings, and final outcome, did not differ significantly in our material from a
motor-neurone 1955-67 in two
were
.
T. J. Yoo C. Y. KUO J. I. STAGNER.
°
the data in the literature. The distribution of the disease within the different ethnic groups (immigrants from Europe, immigrants from the Afro-Asian countries, and the Israeli-born), was as expected, according to the ethnic ratio of the population of Israel as well as of the hospital admissions in that age-group. On the other hand, confirming our initial impression, the average duration of the different forms of M.N.D. was found to be considerably longer (7-4 years) than is described in the literature (Bonduelle 19 2-7 years, Elizan et a1.20 4-0 years, Friedman and Freedman 213 years). The longest duration of illness in our series was in patients with spinal muscular atrophy: average 10-7 years. In patients with bulbar features as presenting signs, the average duration was 5-5 years; while in patients in whom the disease was 16. 17.
18. 19. 20.
21.
Morse, S. I., Riester, S. K. ibid. 1967, 125, 401. Barclay, W. R., Dallgard, D. W., Good, R. C., Janicki, B. W., Kasik, J. E., Ribi, E., Ulrich, C. E., Wolinsky, M., Busey, W. M. Am. Rev. resp. Dis. 1973, 107, 351. Simmons, E. Personal communication. Bonduelle, M., Bouygues, P., Lormeau, G., Keller, J. Presse méd. 1970, 78, 827. Elizan, T. S., Hirano, A., Abrams, B. M., Need, R. L., Van Nuis, C., Kurland, L. T. Archs Neurol., Chicago, 1966, 14, 356. Friedman, A. P., Freedman, D. J. nerv. ment. Dis. 1950, 111, 1.
one
of the
deciding factors
is the side
on
which the
gonad is situated. Fetal testes in rats 10 and mice 11 are markedly larger than ovaries, and this leads to the question whether the larger size is the cause or the result of testicular differentiation. The present data favour a causal relation. Indeed, if the development of testicular rather than ovarian tissue can be affected by so feeble a force as lateral asymmetry, it would seem unlikely that the information normally provided by the Y chromosome is very specific. Fetal testes are distinguished by their precocious growth and differentiation,12 in contrast to the slow development characteristic of the ovary. The early growth of the testis is normally ensured by the presence of a Y chromosome, 10 while in hermaphrodites the likelihood of the fetal gonad developing into a testis is increased by being situated on the right side of the body. I thank Dr Sylvia Lawler and the staff of the Fetal Tissue Bank, Royal Marsden Hospital, for supplying the fetal gonads, and Mr George Barrett for serial sectioning. This work was supported by a grant from the Science Research Council. Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE.
URSULA MITTWOCH.
BETA-BLOCKADE AND HEART WEIGHTS SIR,-The conclusions of Dr Vaughan Williams and Dr Raine (Nov. 2, p. 1048) are very interesting. It is, however, regrettable that no blood-pressures and bloodpressure changes were reported, since chronic administration of P-blockers may have had a hypotensive effect. This in turn could have been the cause for the lower ventricular weights of the treated rabbits, compared with the controls. 13 It is still unclear, therefore, which is the egg and which is the chicken. Beilinson Hospital, Petah Tiqva, Israel.
YEHUDA M. TRAUB.
BLOOD AND URINARY NICOTINE IN NON-SMOKERS was SIR,—I pleased to see the article on nicotine levels in non-smokers by Dr Russell and Mr Feyerabend (Jan. 25, p. 179). They find that the urinary nicotine in nonsmokers has a mean level of 12°4 16°9 ng. per ml. (standard deviation) (table 11). That means that the 15% or so of the population who lie one standard deviation below the mean must have negative quantities of nicotine in the urine. What do negative quantities of nicotine look like ? Sadly, on looking at the individual values and the figure I see that it is nothing new. Only the usual mistake has been made. The authors clearly recognise in their figure that urinary nicotine levels are logarithmically distributed, not normally distributed on an arithmetic scale. Yet they use The same mistake is constantly arithmetic statistics. being made with serum-enzyme activity and many other measurements. Biological measurements are more
often
Statistical treatments that ignore this are metic form. erroneous and misleading. Unfortunately the conclusions drawn in the particular case of nicotine transfer to nonsmokers do not depend on the statistical analysis and remain disturbingly valid. University College Hospital Medical School, University Street, London WC1E 6JJ.
A. E. M. MCLEAN.
EFFECT OF BORDETELLA PERTUSSIS VACCINE ON GROWTH OF HEPATOMA SIR,-Several adjuvants, such as B.C.G. and Corynebacterium parvum, inhibit tumour growth.1-5 Depending upon the route of injection, Bordetella pertussis also inhibits tumour growth. 6,7 Although the mechanism remains undefined, it functions as an immune potentiator. The growth of well-established hepatoma 5123 tumours was inhibited by either intravenous or subcutaneous injections of hepatoma cells and pertussis vaccine.’7 Conversely, growth was enhanced if the combined treatment was administered before tumour transplantation. The effects seemed to be tumour specific.’7 Moloney lymphoma isografts were facilitated in mice (strain C57L/KL) after intraperitoneal injection of pertussis vaccine. 8 B. pertussis vaccine (P.v.) has been used for active immunotherapy in patients with acute leukaemia.9 The results of these treatments suggest that this vaccine inhibits tumour growth. In an effort to determine the role of P.v. in tumour inhibition, female Buffalo rats, weighing 230-260- g., were inoculated with cells from Morris hepatoma 7777. Isografts of Morris hepatoma 7777 were obtained from 1. 2.
3. 4. 5. 6. 7. 8. 9.
Zbar, B., Bernstein, I., Tanaka, T., Rapp, H. J. Science, 1970, 170, 1217. Zbar, B., Bernstein, I. W., Rapp, H. J. J. natn. Cancer Inst. 1971, 46, 831. Zbar, B. Natn. Cancer Inst. Monogr. 1972, 35, 341. Woodruff, M. F. A., Boak, J. L. Br. J. Cancer, 1966, 20, 245. Halpern, B. N., Biozzi, G., Stiffel, C., Mouton, D. Nature, 1966, 212, 853. Likhite, V. V. Cancer Res. 1974, 34, 1027. Wissler, R. W., Craft, K., Kesden, D., Polisky, B., Dzoga, K. Proc. First Congr. Transplant Soc. 1968, p. 539. Floersheim, G. L. Nature, 1967, 216, 1235. Guyer, R. J., Crowther, D. Br. med. J. 1969, iv, 406.
logarithmically
than arithmetically distributed. Statistics based on normal distribution can be used if the log of the values is taken and treated in the usual way. The anti-log of the mean is then the geometric mean and one standard deviation either side can be given in the log form or in its unsymmetrical arithMittwoch, U. Genetics of Sex Differentiation. New York, 1973. Mittwoch, U., Buehr, M. L. Differentiation, 1973, 1, 219. Jost, A. in Schering Workship on Contraception: The Masculine Gender (edited by G. Raspé), p. 3. Oxford, 1973. 13. Hall, O., Hall, C. E., Ogden, E. Am. J. Physiol. 1953, 174, 175. 10. 11. 12.
Fig. 1-Effect of P.V. All rats received
cutaneously.
on
growth of hepatomas.
transplantable
Morris
hepatoma
7777 sub-
403
Although extensive proliferation of some cells may follow P.v. injections, the newly circulating lymphocytes are not the product of recent cell divisions.16 Morse and Barron suggested that P.v. induced the alteration of lymphocyte cell membranes which are partly responsible for the failure of the cells to recirculate properly and may be involved in the initial redistribution process.15 Evidence suggests that there may be a compartmentalisation of cell-mediated immune responses and transplantation immunity in monkeys. 17 ,188 These studies suggest the importance of the availability of the participating T lymphocytes at the local site. An attractive hypothesis of one aspect of the mechanism of action of this immunopotentiator (P.v.) would be the redistribution of a lymphocyte subpopulation (due to the initial triggering mechanism) resulting in the attraction of sensitised killer T lymphocytes at the P.v. inoculation site. By such a mechanism, the tumour would be directly attacked. The killer T cells are less available at the tumour site when the adjuvant is given intraperitoneally, thus facilitating the growth of the tumour. We are currently examining these hypotheses. We thank M. Parmely for measuring a-fetoprotein. This work was supported by a clinical investigatorship given to T. J. Yoo.
Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, U.S.A.
MOTOR-NEURONE DISEASE IN ISRAEL SIR,-Comparing the variations in the incidence and the natural history of an illness as they occur in different countries and populations, helps to clarify the nature of certain diseases. The gain in our knowledge concerning
Fig. 2-Level of oc-fetoprotein in tumour-bearing rats. All the rats received transplantable Morris hepatoma 7777
subcutaneously. Dr John S.
Thompson, University of Iowa. The solid hepatomas were excised and cut into small pieces of equal size (0-5 cm. in diameter) in Eisen’s culture medium. The implants were inserted subcutaneously on the flank of each rat approximately medial to the thigh. Five rats used in each group. The controls received no P.v. Of the other two groups, one was injected with Pv. at the tumour site and the other was injected with vaccine intraperitoneally within an hour of the inoculation of the tumour. In each group the rats received 1 ml. of vaccine containing 2 x 109 cells per ml. B. pertussis vaccine, strain 18334, was obtained from Connaught Laboratories, Toronto. The tumour was measured three times a week with a calliper in three axes to determine its rate of growth. Tail bleedings were also performed. The levels of m-fetoprotein in the sera were assayed by radioimmunoassay 10 and served as another indicator of tumour growth. The results of tumour growth and ex-fetoprotein levels are presented in figs. 1 and 2, respectively. This study demonstrated that P.v. inhibited tumour growth if it was administered at the tumour site at implantation, thus confirming observations by Guyer and Wissler.’9 If the vaccine was given intraperitoneally, the growth of the tumour was facilitated as reported by Floersheiin.8 Injection of P.v. mobilises lymphocytes and induces leucocytosis and lymphocytosis in ariimals.11-14 The mechanism of lymphocyte mobilisation by P.v. is not known. Its mobilising activity may be due to the abnormal redistribution of lymphocytes from various lymphoid organs and bone-marrow into the peripheral circulation.15
multiple sclerosis is 69 patients with
10. 11. 12. 13.
Sell, S. Cancer Res. 1973, 31, 1010. Morse, S. I., Bray, K. K. J. exp. Med. 1969, 129, 523. Morse, S. I. ibid. 1965, 121, 49. Rai, K., Chanana, A. D., Cronkite, E. P., Joel, D. D., Stevens, J. B. Blood, 1971, 38, 49. 14. Phanuphak, P., Moorhead, J. W., Claman, H. N. Int. Archs Allergy appl. Immun. 1972, 43, 305. 15. Morse, S. I., Barron, B. A. J. exp. Med. 1970, 132, 663.
good example of such results. disease (M.N.D.) were seen over the years departments of neurology of the Tel-Aviv University (58 in the Beilinson Hospital and 11 in the Ichilov Municipal Hospital). The notes of these patients were reviewed, and, where possible, their families were contacted and questioned; the evolving clinical data were analysed. The male/female ratio was 2/1, the average age of onset being somewhat lower in females. This finding, as well as data concerning age of onset, presenting symptoms, laboratory findings, and final outcome, did not differ significantly in our material from a
motor-neurone 1955-67 in two
were
.
T. J. Yoo C. Y. KUO J. I. STAGNER.
°
the data in the literature. The distribution of the disease within the different ethnic groups (immigrants from Europe, immigrants from the Afro-Asian countries, and the Israeli-born), was as expected, according to the ethnic ratio of the population of Israel as well as of the hospital admissions in that age-group. On the other hand, confirming our initial impression, the average duration of the different forms of M.N.D. was found to be considerably longer (7-4 years) than is described in the literature (Bonduelle 19 2-7 years, Elizan et a1.20 4-0 years, Friedman and Freedman 213 years). The longest duration of illness in our series was in patients with spinal muscular atrophy: average 10-7 years. In patients with bulbar features as presenting signs, the average duration was 5-5 years; while in patients in whom the disease was 16. 17.
18. 19. 20.
21.
Morse, S. I., Riester, S. K. ibid. 1967, 125, 401. Barclay, W. R., Dallgard, D. W., Good, R. C., Janicki, B. W., Kasik, J. E., Ribi, E., Ulrich, C. E., Wolinsky, M., Busey, W. M. Am. Rev. resp. Dis. 1973, 107, 351. Simmons, E. Personal communication. Bonduelle, M., Bouygues, P., Lormeau, G., Keller, J. Presse méd. 1970, 78, 827. Elizan, T. S., Hirano, A., Abrams, B. M., Need, R. L., Van Nuis, C., Kurland, L. T. Archs Neurol., Chicago, 1966, 14, 356. Friedman, A. P., Freedman, D. J. nerv. ment. Dis. 1950, 111, 1.