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THE PROPERDIN SYSTEM
1218
Under the first Medical Act the Council’s powers were indicated rather than defined; but, though these powers were scanty, great things had been achieved in the century. Referring to earlier leaders in the Council, including Brodie, Sir "Donald MacAlister, and Sir We are their debtors for their Herbert Eason, he said: ideal and their example." Lord Cohen of Birkenhead observed that, through the G.M.C., the Governments of Great Britain, Northern Ireland, and Eire met for a common purpose. Reciprocity in registration and temporary registration was one of the most abiding links of friendship with the Commonwealth. The history and present work of the Council are related in booklet by the Registrar, Mr. Walter Pyke-Lees (Centenary of the General Medical Council, 1858-1958). a
SEX AND AGE DIFFERENCES IN RADIATION-INDUCED MUTATION-RATES
A RATHER unexpected finding of the recent surveys of man’s exposure to artificial radiation was that the main Such radiation was source was clinical radiography. a much more important source of exposure of the gonads, in countries where X rays were much used medically, than, at any rate, the external radiation from atomic and thermonuclear devices so far exploded. Efforts, therefore, are now being made to reduce gonad exposure to medical X rays. For these efforts to be well directed it is important to know whether it is men or women who particularly need protection-by, for example, such devices as the ’Armadillo ’, recently described in our columns 1-and to know whether mutation is particularly likely after exposure in foetal, child, or adult life. Experiments in animals and plants show that radiationinduced mutation-rates may vary greatly, not only between male and female but also between different stages in the maturation of the germ cells in the same sex. In man there is no evidence yet on radiation-induced mutationrates, and no evidence even about spontaneous mutationrates for each sex separately. Haldane 2 has pointed out that the family patterns for conditions due to sex-linked mutations may provide evidence on the relative rates of spontaneous mutations; but no conclusive differences have yet been found by this method. Spontaneous mutationrates, too, are of only limited value in estimating the relative rates of radiation-induced mutation, since much, perhaps most, spontaneous mutation is not due to background mutation. Accordingly, Carter3 has designed and performed experiments on radiation-induced mutation-rates by sex and age in the mouse, the most suitable mammal for studies of this kind. In mammals the germ plasm in females is present in oogonia in foetal life, and mainly in oocytes in postnatal life. In males the germ plasm is present in spermatogonia in foetal life, and also in spermatogonia for most of postnatal life, the stages of spermatocyte and spermatozoon lasting, perhaps, only about a year. In mice the mutationrates for a number of gene-loci have already been established for adult males in America by Russell. Using Russell’s strains of mice, Carter looked for mutations in the offspring of irradiated foetal male mice and adult female mice. With the foetal male mice he was able to follow Russell’s method closely, using high-voltage X rays for a short period, though to avoid foetal damage the total 1. Tanner, J. 2. 3.
M., Whitehouse, R. H., Powell, J. H. Lancet, Oct. 11, 1958, p. 779. Haldane, J. B. S. Ann. Eugen. 1947, 13, 262. Carter, T. C. Brit. J. Radiol. 1958, 31, 407.
dose was 300 and not 600 rads. This technique was useless in adult females, since they were largely sterilised; so he used the same total dosage of 600 rads, but gave it by gamma-radiation over seven weeks. There is no reason to suppose that the lower dosage to the foetal male mice would affect a comparison with Russell’s findings. The comparability of the effects of X-radiation in a little over an hour with the same dose as gamma-radiation over some weeks is uncertain, but may well be valid. Carter’s findings were surprising. With a little over 10,000 offspring in each group he would have expected, if Russell’s findings on adult males applied, between 11 and 12 mutations in the offspring of the irradiated adult females, and 6 in those of the irradiated foetal males. In fact, only 1 mutation was recognised in each group. It is clear that these results need repeating with, if possible, different mammals and different techniques of irradiation. In particular, the question of the comparability of the same dose given at a high rate over a short period, and at a low rate over a long period, needs further investigation, But Carter’s findings already suggest that it is the postnatal male who particularly needs protection from irradiation of the gonads during radiography. It will be fortunate if this proves to be the case, since it is just this group that is most easily protected. THE PROPERDIN SYSTEM
SPECIFIC antigen-antibody reactions are not the only system of immunity in the body. A globulin fraction has been demonstrated in human serum which contributes to the destruction of bacteria of the shigella group, abnormal red blood-cells, and the inhibition of certain viruses.’ Properdin is a p-euglobulin with a high molecular weight; it represents no more than 0-03% of total serum-proteins, It is active only in a system which invokes complement and magnesium ions, and differs from the usual antigenantibody system in its lack of specificity and its ability to inactivate the C’3 component of complement-a component only partly affected by antigen-antibody reactions. This provides the basis for properdin assay: zymosan, consisting of purified cell walls obtained after tryptic digestion of yeast, forms an insoluble polysaccharide complex with properdin, which may thus be removed from serum; a unit of properdin is defined as the smallest amount then required to reduce the C’3 titre in properdindeficient serum from 120 to 0 units.2 The importance of the properdin system lies in its lack of specificity and therefore wide range of defence. Besides shigella, some strains of salmonella, pseudomonas, proteus,1 paracolon bacilli, and escherichia are properdin-sensitive,3! A relationship has been shown between survival and properdin levels in mice infected with Esch. coli and Friedlander’s bacillus; moreover intravenous administration of properdin altered the course of the infection.4 Rowley’5 has injected mice with cell walls prepared from Esch. coli and demonstrated that the animals then die after being infected by as few as 5 Esch. coli, whereas as many as 10,000 organisms may be needed to kill normal animals. This suggests that bacterial cell walls remove properdin just as zymosan does, the corollary being that the properdin system protects by combining with such substances. 1. Pillemer, L., Blum, L., Lepow, I. H., Ross, O. A., Todd, E. W., Wardlaw, A. C. Science, 1954, 120, 279. 2. Pillemer, L., Blum, L., Lepow, I. H., Wurz., L., Todd, E. W. J. Med. 1956, 103, 1. 3. Wardlaw, A. C., Pillemer, L. ibid. p. 553. 4. Pillemer, L. Trans. N. Y. Acad. Sci. 1955, 17, 526. 5. Rowley, D. Lancet, 1955, i, 233.
1219
In-vivo injections of zymosan cause a fall in serumproperdin, but the titres rise after two days to two to three times normal. This phenomenon has been used to protect laboratory animals against the septicaemia which follows irradiation; and it is significant that properdin levels fall after whole-body radiation6 and irreversible haemorrhagic shockand such falls are accompanied by loss of resistance to infection and to endotoxins. Serum-properdin titres
tend to be subnormal in man in association with infection and after gross tissue destruction. Pillemer et al.9 have shown that in mice polysaccharides of high molecular weight derived from normal and neoplastic tissue combine with properdin, thus altering serum levels and resistance to gram-negative infections; this observation, amplifying the findings in man, points to endogenous causes of We already have one example of lowered immunity. possible endogenous utilisation of the properdin system in paroxysmal nocturnal haemoglobinuria; for in this condition, which-brings into action no antibody reaction, properdin is found to be necessary for lysis in vitro. Hubay et al.1O have designed experiments to find out whether the properdin system accounts for the failure of homografts, since the development of antibodies to skin homografts has not been demonstrated. But skin-homograft survival was not prolonged by depressing the properdin system, nor were renal homografts affected. 11 It is interesting that Hubay et al. achieved depression of serum-properdin by injecting dextran B1355; this finding may point to a need for caution regarding the use of dextran therapeutically as a blood substitute. TREATMENT OF THE NEPHROTIC SYNDROME
IN the absence of an xtiological diagnosis, treatment of the nephrotic syndrome is necessarily empirical. The very multitude of remedies encourages the view that none is specific. It was early observed that spontaneous diuresis occasionally accompanied pyrexia; and artificial fever, produced either with T.A.B. or malaria, has been used successfully in some cases. Diuretics are commonly prescribed with no aim other than to reduce oedema, and administration of urea sometimes does this dramatically. Restriction of dietary sodium is widely practised, and is a logical way of preventing further accumulation of extracellular fluid. But none of these treatments aims at correcting the underlying cause of the oedema—namely, the reduced colloid-osmotic pressure of the blood-or at increasing the low plasma-volume, which is the probable stimulus to increased aldosterone secretion by the adrenal. Such the as have been made to expand plasma-volume attempts have usually been unsuccessful, 12but a recent case reported by Williams and Ryan 13’ is a noteworthy exception. These workers induced diuresis in a nephrotic by means of intravenous infusion of concentrated human serumalbumin after they had failed with steroids. The diuresis, which was accompanied by increased urinary excretion of protein, signifying that the renal lesion was unaffected, may well have been the result of reduced adrenal stimulation from increased plasma-volume. It is widely recognised that the nephrotic syndrome can be effectively treated only by correcting the cause of 6. Ross, O. A. Ann. N.Y. Acad. Sci. 1956, 66, 274. 7. Frank, E., Fine, J., Pillemer, L. Proc. Soc. exp. Biol., N.Y. 1955, 89, 223. 8. Benson, J. W., Holden, W. D. Surg. Form. 1958, 8, 121. 9. Pillemer, L., Landy, M., Shear, M. J. J. exp. Med. 1957, 106, 99. 10. Hubay, C. A., Wityk, J. J., Holden, W. D. Surg. Gynec. Obstet. 1958, 107, 311. 11. Hubay, C. A., Persky, L. Transplantation Bull. 1957, 4, 58. 12. Edwards, G. A. Lancet, 1957, i, 563. 13. Williams, R. S., Ryan, T. J. ibid. Nov. 8, 1958, p. 988.
the proteinuria; but in only occasional cases is the cause known with certainty, and even more rarely can a specific remedy be applied.14 In most cases of acquired nephrotic syndrome which are not due to poisoning by drugs or metals the lesions are glomerular. The only drugs which seem to affect the glomerulus directly are the steroid hormones, and their usefulness can hardly be exaggerated. These hormones may also act indirectly by reducing the secretion of aldosterone by the adrenals. They have been administered in two ways-either in short intensive courses when a withdrawal diuresis was planned, or else as continuous medication when it was hoped that some direct glomerular effect was being induced. Continuous therapy is complicated by side-effects and often has to be abandoned on this score. Clearly some cases-for instance, due to renal-vein thrombosis-will not be directly influenced by steroid hormones. In others-for example, systemic lupus erythematosus-the hormones may well act directly on the glomerulus. Short courses might suit the former type, and long-continued therapy the latter. The debate continues, and a report by Lange and Wasserman 15 is particularly welcome as it indicates a logical compromise. These workers report on 46 cases of nephrosis (35 children and 11 adults), having specifically excluded those caused by renal-vein thrombosis, amyloidosis, or diabetic nephropathy. The series includes all their cases of so-called pure or lipoid nephrosis, and of the nephrotic pure stage of glomerulonephritis. Whether there is a lipoid nephrosis " is undecided,14 but from the clinical and chemical data presented it is clear that all were examples of the nephrotic syndrome. Treatment was begun with aqueous corticotrophin for at least twelve days, and prolonged intermittent therapy with cortisone was then applied-the oedema disappearing in consequence. This treatment, which was started five days after the onset of the diuresis, consisted of cortisone orally for three days of each week for at least a year, regardless of clinical or chemical improvement. After a year the interval between maintenance courses was extended, and finally cortisone was withdrawn completely. Antibiotics and a potassium supplement were also given, and a diet containing less than 1 g. of salt daily was recommended. Fluid intake was not restricted. A control series was ingeniously selected from a group of nephrotics observed between 1946 and 1952, after the introduction of antibiotics but before steroids A remarkable difference was were widely available. demonstrated in the treated group. Instead of the 12-8 predicted deaths only 1 occurred in the first sixty months of observation. Moreover, most of the adults in the treated group were able to lead normal lives, and the children grew and behaved normally. No harmful side-effects were Other investigators 16 have reported similar good seen. results. In the present state of our knowledge this seems to be the treatment of choice for the nephrotic syndrome. "
MR. PERCY HODDER-WILLIAMS
WE regret to record the death of Mr. R. P. HodderWilliams, for twenty-one years chairman of The Lancet. In 1927 he succeeded his elder brother, Sir Ernest Hodder-Williams, as the senior member of our board of directors, and also as chairman of the publishing firm of Hodder & Stoughton. In both offices he had to cope with the added difficulties of the war, and in both he is remembered with affection and respect for his qualities of heart and mind. He died on Nov. 29 at the age of 78. 14. ibid. p. 1000. 15. Lange, K., Wasserman, E. J. Amer. med. Ass. 1958, 168, 377. 16. Piel, C. F., William, J. F. J. Amer. med. Wom. Ass. 1957, 12,
273
Under the first Medical Act the Council’s powers were indicated rather than defined; but, though these powers were scanty, great things had been achieved in the century. Referring to earlier leaders in the Council, including Brodie, Sir "Donald MacAlister, and Sir We are their debtors for their Herbert Eason, he said: ideal and their example." Lord Cohen of Birkenhead observed that, through the G.M.C., the Governments of Great Britain, Northern Ireland, and Eire met for a common purpose. Reciprocity in registration and temporary registration was one of the most abiding links of friendship with the Commonwealth. The history and present work of the Council are related in booklet by the Registrar, Mr. Walter Pyke-Lees (Centenary of the General Medical Council, 1858-1958). a
SEX AND AGE DIFFERENCES IN RADIATION-INDUCED MUTATION-RATES
A RATHER unexpected finding of the recent surveys of man’s exposure to artificial radiation was that the main Such radiation was source was clinical radiography. a much more important source of exposure of the gonads, in countries where X rays were much used medically, than, at any rate, the external radiation from atomic and thermonuclear devices so far exploded. Efforts, therefore, are now being made to reduce gonad exposure to medical X rays. For these efforts to be well directed it is important to know whether it is men or women who particularly need protection-by, for example, such devices as the ’Armadillo ’, recently described in our columns 1-and to know whether mutation is particularly likely after exposure in foetal, child, or adult life. Experiments in animals and plants show that radiationinduced mutation-rates may vary greatly, not only between male and female but also between different stages in the maturation of the germ cells in the same sex. In man there is no evidence yet on radiation-induced mutationrates, and no evidence even about spontaneous mutationrates for each sex separately. Haldane 2 has pointed out that the family patterns for conditions due to sex-linked mutations may provide evidence on the relative rates of spontaneous mutations; but no conclusive differences have yet been found by this method. Spontaneous mutationrates, too, are of only limited value in estimating the relative rates of radiation-induced mutation, since much, perhaps most, spontaneous mutation is not due to background mutation. Accordingly, Carter3 has designed and performed experiments on radiation-induced mutation-rates by sex and age in the mouse, the most suitable mammal for studies of this kind. In mammals the germ plasm in females is present in oogonia in foetal life, and mainly in oocytes in postnatal life. In males the germ plasm is present in spermatogonia in foetal life, and also in spermatogonia for most of postnatal life, the stages of spermatocyte and spermatozoon lasting, perhaps, only about a year. In mice the mutationrates for a number of gene-loci have already been established for adult males in America by Russell. Using Russell’s strains of mice, Carter looked for mutations in the offspring of irradiated foetal male mice and adult female mice. With the foetal male mice he was able to follow Russell’s method closely, using high-voltage X rays for a short period, though to avoid foetal damage the total 1. Tanner, J. 2. 3.
M., Whitehouse, R. H., Powell, J. H. Lancet, Oct. 11, 1958, p. 779. Haldane, J. B. S. Ann. Eugen. 1947, 13, 262. Carter, T. C. Brit. J. Radiol. 1958, 31, 407.
dose was 300 and not 600 rads. This technique was useless in adult females, since they were largely sterilised; so he used the same total dosage of 600 rads, but gave it by gamma-radiation over seven weeks. There is no reason to suppose that the lower dosage to the foetal male mice would affect a comparison with Russell’s findings. The comparability of the effects of X-radiation in a little over an hour with the same dose as gamma-radiation over some weeks is uncertain, but may well be valid. Carter’s findings were surprising. With a little over 10,000 offspring in each group he would have expected, if Russell’s findings on adult males applied, between 11 and 12 mutations in the offspring of the irradiated adult females, and 6 in those of the irradiated foetal males. In fact, only 1 mutation was recognised in each group. It is clear that these results need repeating with, if possible, different mammals and different techniques of irradiation. In particular, the question of the comparability of the same dose given at a high rate over a short period, and at a low rate over a long period, needs further investigation, But Carter’s findings already suggest that it is the postnatal male who particularly needs protection from irradiation of the gonads during radiography. It will be fortunate if this proves to be the case, since it is just this group that is most easily protected. THE PROPERDIN SYSTEM
SPECIFIC antigen-antibody reactions are not the only system of immunity in the body. A globulin fraction has been demonstrated in human serum which contributes to the destruction of bacteria of the shigella group, abnormal red blood-cells, and the inhibition of certain viruses.’ Properdin is a p-euglobulin with a high molecular weight; it represents no more than 0-03% of total serum-proteins, It is active only in a system which invokes complement and magnesium ions, and differs from the usual antigenantibody system in its lack of specificity and its ability to inactivate the C’3 component of complement-a component only partly affected by antigen-antibody reactions. This provides the basis for properdin assay: zymosan, consisting of purified cell walls obtained after tryptic digestion of yeast, forms an insoluble polysaccharide complex with properdin, which may thus be removed from serum; a unit of properdin is defined as the smallest amount then required to reduce the C’3 titre in properdindeficient serum from 120 to 0 units.2 The importance of the properdin system lies in its lack of specificity and therefore wide range of defence. Besides shigella, some strains of salmonella, pseudomonas, proteus,1 paracolon bacilli, and escherichia are properdin-sensitive,3! A relationship has been shown between survival and properdin levels in mice infected with Esch. coli and Friedlander’s bacillus; moreover intravenous administration of properdin altered the course of the infection.4 Rowley’5 has injected mice with cell walls prepared from Esch. coli and demonstrated that the animals then die after being infected by as few as 5 Esch. coli, whereas as many as 10,000 organisms may be needed to kill normal animals. This suggests that bacterial cell walls remove properdin just as zymosan does, the corollary being that the properdin system protects by combining with such substances. 1. Pillemer, L., Blum, L., Lepow, I. H., Ross, O. A., Todd, E. W., Wardlaw, A. C. Science, 1954, 120, 279. 2. Pillemer, L., Blum, L., Lepow, I. H., Wurz., L., Todd, E. W. J. Med. 1956, 103, 1. 3. Wardlaw, A. C., Pillemer, L. ibid. p. 553. 4. Pillemer, L. Trans. N. Y. Acad. Sci. 1955, 17, 526. 5. Rowley, D. Lancet, 1955, i, 233.
1219
In-vivo injections of zymosan cause a fall in serumproperdin, but the titres rise after two days to two to three times normal. This phenomenon has been used to protect laboratory animals against the septicaemia which follows irradiation; and it is significant that properdin levels fall after whole-body radiation6 and irreversible haemorrhagic shockand such falls are accompanied by loss of resistance to infection and to endotoxins. Serum-properdin titres
tend to be subnormal in man in association with infection and after gross tissue destruction. Pillemer et al.9 have shown that in mice polysaccharides of high molecular weight derived from normal and neoplastic tissue combine with properdin, thus altering serum levels and resistance to gram-negative infections; this observation, amplifying the findings in man, points to endogenous causes of We already have one example of lowered immunity. possible endogenous utilisation of the properdin system in paroxysmal nocturnal haemoglobinuria; for in this condition, which-brings into action no antibody reaction, properdin is found to be necessary for lysis in vitro. Hubay et al.1O have designed experiments to find out whether the properdin system accounts for the failure of homografts, since the development of antibodies to skin homografts has not been demonstrated. But skin-homograft survival was not prolonged by depressing the properdin system, nor were renal homografts affected. 11 It is interesting that Hubay et al. achieved depression of serum-properdin by injecting dextran B1355; this finding may point to a need for caution regarding the use of dextran therapeutically as a blood substitute. TREATMENT OF THE NEPHROTIC SYNDROME
IN the absence of an xtiological diagnosis, treatment of the nephrotic syndrome is necessarily empirical. The very multitude of remedies encourages the view that none is specific. It was early observed that spontaneous diuresis occasionally accompanied pyrexia; and artificial fever, produced either with T.A.B. or malaria, has been used successfully in some cases. Diuretics are commonly prescribed with no aim other than to reduce oedema, and administration of urea sometimes does this dramatically. Restriction of dietary sodium is widely practised, and is a logical way of preventing further accumulation of extracellular fluid. But none of these treatments aims at correcting the underlying cause of the oedema—namely, the reduced colloid-osmotic pressure of the blood-or at increasing the low plasma-volume, which is the probable stimulus to increased aldosterone secretion by the adrenal. Such the as have been made to expand plasma-volume attempts have usually been unsuccessful, 12but a recent case reported by Williams and Ryan 13’ is a noteworthy exception. These workers induced diuresis in a nephrotic by means of intravenous infusion of concentrated human serumalbumin after they had failed with steroids. The diuresis, which was accompanied by increased urinary excretion of protein, signifying that the renal lesion was unaffected, may well have been the result of reduced adrenal stimulation from increased plasma-volume. It is widely recognised that the nephrotic syndrome can be effectively treated only by correcting the cause of 6. Ross, O. A. Ann. N.Y. Acad. Sci. 1956, 66, 274. 7. Frank, E., Fine, J., Pillemer, L. Proc. Soc. exp. Biol., N.Y. 1955, 89, 223. 8. Benson, J. W., Holden, W. D. Surg. Form. 1958, 8, 121. 9. Pillemer, L., Landy, M., Shear, M. J. J. exp. Med. 1957, 106, 99. 10. Hubay, C. A., Wityk, J. J., Holden, W. D. Surg. Gynec. Obstet. 1958, 107, 311. 11. Hubay, C. A., Persky, L. Transplantation Bull. 1957, 4, 58. 12. Edwards, G. A. Lancet, 1957, i, 563. 13. Williams, R. S., Ryan, T. J. ibid. Nov. 8, 1958, p. 988.
the proteinuria; but in only occasional cases is the cause known with certainty, and even more rarely can a specific remedy be applied.14 In most cases of acquired nephrotic syndrome which are not due to poisoning by drugs or metals the lesions are glomerular. The only drugs which seem to affect the glomerulus directly are the steroid hormones, and their usefulness can hardly be exaggerated. These hormones may also act indirectly by reducing the secretion of aldosterone by the adrenals. They have been administered in two ways-either in short intensive courses when a withdrawal diuresis was planned, or else as continuous medication when it was hoped that some direct glomerular effect was being induced. Continuous therapy is complicated by side-effects and often has to be abandoned on this score. Clearly some cases-for instance, due to renal-vein thrombosis-will not be directly influenced by steroid hormones. In others-for example, systemic lupus erythematosus-the hormones may well act directly on the glomerulus. Short courses might suit the former type, and long-continued therapy the latter. The debate continues, and a report by Lange and Wasserman 15 is particularly welcome as it indicates a logical compromise. These workers report on 46 cases of nephrosis (35 children and 11 adults), having specifically excluded those caused by renal-vein thrombosis, amyloidosis, or diabetic nephropathy. The series includes all their cases of so-called pure or lipoid nephrosis, and of the nephrotic pure stage of glomerulonephritis. Whether there is a lipoid nephrosis " is undecided,14 but from the clinical and chemical data presented it is clear that all were examples of the nephrotic syndrome. Treatment was begun with aqueous corticotrophin for at least twelve days, and prolonged intermittent therapy with cortisone was then applied-the oedema disappearing in consequence. This treatment, which was started five days after the onset of the diuresis, consisted of cortisone orally for three days of each week for at least a year, regardless of clinical or chemical improvement. After a year the interval between maintenance courses was extended, and finally cortisone was withdrawn completely. Antibiotics and a potassium supplement were also given, and a diet containing less than 1 g. of salt daily was recommended. Fluid intake was not restricted. A control series was ingeniously selected from a group of nephrotics observed between 1946 and 1952, after the introduction of antibiotics but before steroids A remarkable difference was were widely available. demonstrated in the treated group. Instead of the 12-8 predicted deaths only 1 occurred in the first sixty months of observation. Moreover, most of the adults in the treated group were able to lead normal lives, and the children grew and behaved normally. No harmful side-effects were Other investigators 16 have reported similar good seen. results. In the present state of our knowledge this seems to be the treatment of choice for the nephrotic syndrome. "
MR. PERCY HODDER-WILLIAMS
WE regret to record the death of Mr. R. P. HodderWilliams, for twenty-one years chairman of The Lancet. In 1927 he succeeded his elder brother, Sir Ernest Hodder-Williams, as the senior member of our board of directors, and also as chairman of the publishing firm of Hodder & Stoughton. In both offices he had to cope with the added difficulties of the war, and in both he is remembered with affection and respect for his qualities of heart and mind. He died on Nov. 29 at the age of 78. 14. ibid. p. 1000. 15. Lange, K., Wasserman, E. J. Amer. med. Ass. 1958, 168, 377. 16. Piel, C. F., William, J. F. J. Amer. med. Wom. Ass. 1957, 12,
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