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New Light on Cholera
167
intestinal cells. However, cyclic A.M.P. is formed by an enzyme, adenyl cyclase, and broken down by How esterase activity, and a further question arose: does cholera toxin raise the cyclic-A.M.P. concentration ? " The answer seems to be that it increases the activity of adenyl cyclase.6,7 About an hour after toxin is put into the lumen of the dog’s intestine 8 there is an increase in the activity of the enzyme and of the net flux of water and sodium into the lumen. These figures all rise steadily for 2-3 hours, are still raised after 24 hours, but are normal again at 48 hours. "
New
Light
on
Cholera
cholera illustrates the axiom that real of understanding a disease requires a spectrum or sequence of investigations ranging from the observational to the study of cell biology or, as HIMSWORTH would say, from the surface to the centre of the sphere of knowledge.! We start with clinical observations and epidemiology, such as those of JOHN SNOW and the Broad Street pump, which tell us that cholera is an infection, and we go on by finding that there are specific causative vibrios, that the disease is due almost entirely to fluid loss, and that this is due to a specific toxin produced by the vibrios 2-which do not enter the tissues at all. For some time the " question has been asked: How does this remarkable diarrhoea occur ? "-and we now have a clearer idea of the answer. There is normally a large flux of water and electrolytes from the plasma into the gut lumen and a very slightly larger flux from the lumen to the plasma, so that net absorption results. In cholera there is clearly a net loss, but this could be due to increased plasma-to-lumen flux, or to decreased lumen-to-plasma flux, or to a combination of the two. On p. 151 this week Dr. LovE and his co-workers shed light on the problem. Using 22Na and 24Na they have shown that both fluxes are reduced to about a half normal during the diarrhoeal phase of the disease, but that the gut-lumen-to-plasma flux is slightly more reduced than the plasma-to-gut flux. The net result is fluid loss. The situation is quite similar to the way in which a relatively small proportionate change in tubular reabsorption can increase urine flow manyfold. This is enough for most of us, but to the curious it prompts another question-namely, " Exactly what does the toxin do to the cells lining the lumen of the intestine ? " Lately, some answers to this have been given. It has been shown that not only cholera toxin but also prostaglandins and theophylline, which increase the concentration of adenosine 3’,5’-cyclic monophosphate, or cyclic A.M.P., in cells, stimulate an increase of flow of salts and fluid into the lumen of the intestine.3,4 Furthermore, there is an increase of cyclic A.M.P. in the mucosa treated with cholera toxin.5 Since cyclic A.M.P. is known to be the mediator of the action of many hormones in various tissues, it is plausible that it should also stimulate secretion in RESEARCH
1. 2. 3. 4. 5.
on
Himsworth, H. The Development and Organization of Scientific Knowledge. London, 1970. Pierce, N. F., Greenough, W. B., III, Carpenter, C. C. J. Bact. Rev. 1971, 35, 1. Field, M., Plotkin, G. R., Silen, W. Nature, 1968, 217, 469. Pierce, N. F., Carpenter, C. C. J., Jr., Elliott, H., Greenough, W. B., III. Gastroenterology, 1971, 60, 22. Schafer, D. E., Lust, W. D., Sircar, B., Goldberg, N. D. Proc. natn. Acad. Sci. U.S.A. 1970, 67, 851.
This raised adenyl-cyclase activity is found not only in animal cholera but also in cases of human cholera.9 There are still unanswered questions. What structure does the toxin interact with on the cell surface ? The fact that ganglioside inhibits its activity suggests that it may become attached to a similar substance in the cell membrane.10 How does this influence adenyl cyclase ? It seems that, once exposed to toxin, the cell undergoes typical changes even if it is washed, but we do not know if this means that toxin induces some slow reaction, or is transported elsewhere in the cell, or whether there is some other explanation. The exact cellular mechanism may be unravelled by investigation of the action of toxin on other more convenient cells-for example, adipose tissue, which shows the characteristic enzyme changes,10,11 or hela cells, which are unable to spread on a glass surface when treated with toxin.12 It is always satisfying if analysis of the disease process suggests useful lines of treatment, and in the case of cholera the study of fluid loss led to effective regimens of fluid replacement. Already the suggestion has been made that since aspirin prevents the synthesis of prostaglandins and their effect on cyclic A.M.P., it should be tried in cholera,13 but no results have yet been reported. Normal rat serum prevents the effect of toxin in intestinal and other preparations,14 and the effect of the toxin on hela cells can be reversed by human serum a-macroglobulins,l5 but these have not yet been proved to be effective in patients. We must continue to hope, therefore, that increasing understanding will give an improved treatment, one which really halts the physiological defect in the cells rather than correcting its secondary effects-namely, diarrhoea and dehydration. In fairness, however, it should be stated that we know how to prevent the disease very effectively-by applying basic sanitation Sharp, G. W. G., Hynie, S. Nature, 1971, 229, 266. Kimberg, D. V., Field, M., Johnson, J., Henderson, A., Gershon, E. J. clin. Invest. 1971, 50, 1218. 8. Guerrant, R. L., Chen, L. C., Sharp, G. W. G.J. infect. Dis. 1972, 125, 377. 9. Chen, L. C., Rohde, J. E., Sharp, G. W. G. Lancet, 1971, i, 939. 10. van Heyningen, W., Carpenter, C. J., Pierce, N. F., Greenough, W. B., III. J. infect. Dis. 1971, 124, 415. 11. Vaughan, M., Pierce, N. F., Greenough, W. B., III. Nature, 1970, 226, 658. 12. Inwood, J., Tyrrell, D. A. J. Br. J. exp. Path. 1970, 51, 597. 13. Bennett, A. Nature, 1971, 231, 536. 14. Aziz, K. M., Phillips, R. A. Proc. tech. Committee Rep. PakistanSeato Cholera Res. Lab. 1970, 1, 100. 15. Davies, J., Tyrrell, D. A. J., Ramsden, D. B., Louis, L. N., Milne, R. G. Exp. med. Path. (in the press). 6. 7.
168
It was the success achieved in the M.R.C. trial 15 years ago that provided W.H.O. with the factual foundation for their policy of stimulating B.c.G. vaccination throughout the world. There have been powerful opponents of vaccination, particularly in the U.S.A. But the evidence from this meticulously conducted trial was conclusive for the particular B.C.G. Vaccination population studied. It remained unknown whether 22 YEARS ago the Medical Research Council began a similar protection would be achieved in another popucontrolled trial of B.c.G. vaccination. Three reports lation in another environment. Unfortunately, it now have already been published.--3 Now the duration of seems likely that the degree of protection is, in fact, observation has been extended to 15 years.4 This, in related to environmental factors. So the protection to itself, is a remarkable achievement. The results of this be expected in a particular country cannot be accurately trial have the considerable effort important justified predicted from that demonstrated in Great Britain. involved. The variation in protective efficacy of B.c.G. vaccinaThere were 54,239 children aged 14-15 2 years in tion in the published reports of controlled trials has, the trial. Those with negative reactions to tuberculin indeed, been enormous. It ranges from 80% in a trial were vaccinated with B.c.G. or vole-bacillus vaccine or in North American Indians to nil in the first of the left unvaccinated, according to a method of random U.S. Public Health Service trials in Georgia. It has allocation. The annual incidence of tuberculosis among been suggested that at least part of the variation may those vaccinated with B.C.G. was 0-28 per 1000, combe due to different degrees of infection of the populapared with 1-28 per 1000 among the tuberculintion with non-tuberculous mycobacteria. Such negative children-a reduction of 78%. The annual infections confer some protection against infection by incidence among those vaccinated with vole-bacillus tubercle bacilli and limit the additional benefit which vaccine was 0-29 per 1000, compared with 1-50 per B.C.G. can confer. 1000 among the tuberculin-negative children admitted In the M.R.C. study, for instance, the incidence of to the trial concurrently with them-a reduction of tuberculosis among the children with low-grade 81%. The degree of protection was similar in the two tuberculin sensitivity-which in Britain is partly sexes. It decreased gradually during the 15-year period. tuberculous in origin and partly due to other mycoBut in the 10-15-year period after vaccination it was bacteria-was considerably lower than the incidence in still substantial; 59% for B.C.G. and 73% for volethe tuberculin-negative unvaccinated group. However, bacillus vaccine. HARTshowed in a detailed analysis that this factor A point of particular importance is that there were was inadequate completely to explain the great no cases among the vaccinated of the two most serious differences between the results of the British and the types of the disease-tuberculous meningitis and U.S. trials. He suggested that the U.S. vaccine may miliary pulmonary tuberculosis. Among the unnot have been as potent as that used in Great Britain. vaccinated there were 10 cases. Whatever the causes of the variations, their existence Vaccination with vole-bacillus vaccine has never got and magnitude have undoubtedly retarded the spread the in Great Britain, beyond of B.c.G. vaccination in the world. It has, for instance, experimental stage although it has been used extensively and routinely been estimated that in Latin America only 20% of the " elsewhere-for instance, in Czechoslovakia. Its use was susceptible " population has been vaccinated.7 The virtually discontinued after the report of very occasional situation is little better in the so-called developed development of lesions resembling lupus vulgaris at countries of Europe.88 Yet there is no doubt of the the vaccination site. This, however, happened only efficacy of B.c.G. vaccination in this region of the world. after vaccination with 3 substrains. The other substrain Apart from the M.R.C. controlled trial, evidence has been obtained, from the routine vaccination procedure produced low levels of post-vaccination skin sensitivity, but was subsequently shown to have produced a high in Birmingham, of its continuing efficacy in the degree of protection. This substrain is still available. prevention of tuberculosis.9 The same report has also In 1962, MiTCHELL 5 concluded that " the development shown that the Glaxo freeze-dried vaccine used in of lupus at the vaccination site may now be considered Britain has a high degree of efficiency, similar to that of to be of historical interest only and is no longer a the liquid vaccine from the Copenhagen strain formerly contra-indication to the use of vole bacillus vaccine ". used. The Glaxo vaccine is now used in many parts of The M.R.C. report states that it would " seem worth the world. considering the reintroduction of the vole bacillus as an Because of the uncertainty about the protective immunising agent against tuberculosis effect of B.c.G. vaccination in populations of tropical
and public health-but to apply these requires a modicum of organisation, good will, and money, which are probably harder to obtain than an understanding of the enzymes.
1. 2. 3. 4. 5.
Medical Medical Medical Medical
Research Council. Br. med. J. 1956, i, 413. Research Council. ibid. 1959, ii, 397. Research Council. ibid. 1963, ii, 973. Research Council. Bull. Wld Hlth Org. 1972, 46, 371. Mitchell, D. N. M.D. thesis, London, 1962.
Hart, P. D’A. Br. med. J. 1967, i, 587. Tubercle, 1971, 52, 303. Lotte, A., Perdrizet, S., Hatton, F. Bull. Wld Hlth Org. 1971, 44 (suppl.). 9. Springett, V., Sutherland, I. Br. med. J. 1970, iv, 148.
6. 7. 8.
intestinal cells. However, cyclic A.M.P. is formed by an enzyme, adenyl cyclase, and broken down by How esterase activity, and a further question arose: does cholera toxin raise the cyclic-A.M.P. concentration ? " The answer seems to be that it increases the activity of adenyl cyclase.6,7 About an hour after toxin is put into the lumen of the dog’s intestine 8 there is an increase in the activity of the enzyme and of the net flux of water and sodium into the lumen. These figures all rise steadily for 2-3 hours, are still raised after 24 hours, but are normal again at 48 hours. "
New
Light
on
Cholera
cholera illustrates the axiom that real of understanding a disease requires a spectrum or sequence of investigations ranging from the observational to the study of cell biology or, as HIMSWORTH would say, from the surface to the centre of the sphere of knowledge.! We start with clinical observations and epidemiology, such as those of JOHN SNOW and the Broad Street pump, which tell us that cholera is an infection, and we go on by finding that there are specific causative vibrios, that the disease is due almost entirely to fluid loss, and that this is due to a specific toxin produced by the vibrios 2-which do not enter the tissues at all. For some time the " question has been asked: How does this remarkable diarrhoea occur ? "-and we now have a clearer idea of the answer. There is normally a large flux of water and electrolytes from the plasma into the gut lumen and a very slightly larger flux from the lumen to the plasma, so that net absorption results. In cholera there is clearly a net loss, but this could be due to increased plasma-to-lumen flux, or to decreased lumen-to-plasma flux, or to a combination of the two. On p. 151 this week Dr. LovE and his co-workers shed light on the problem. Using 22Na and 24Na they have shown that both fluxes are reduced to about a half normal during the diarrhoeal phase of the disease, but that the gut-lumen-to-plasma flux is slightly more reduced than the plasma-to-gut flux. The net result is fluid loss. The situation is quite similar to the way in which a relatively small proportionate change in tubular reabsorption can increase urine flow manyfold. This is enough for most of us, but to the curious it prompts another question-namely, " Exactly what does the toxin do to the cells lining the lumen of the intestine ? " Lately, some answers to this have been given. It has been shown that not only cholera toxin but also prostaglandins and theophylline, which increase the concentration of adenosine 3’,5’-cyclic monophosphate, or cyclic A.M.P., in cells, stimulate an increase of flow of salts and fluid into the lumen of the intestine.3,4 Furthermore, there is an increase of cyclic A.M.P. in the mucosa treated with cholera toxin.5 Since cyclic A.M.P. is known to be the mediator of the action of many hormones in various tissues, it is plausible that it should also stimulate secretion in RESEARCH
1. 2. 3. 4. 5.
on
Himsworth, H. The Development and Organization of Scientific Knowledge. London, 1970. Pierce, N. F., Greenough, W. B., III, Carpenter, C. C. J. Bact. Rev. 1971, 35, 1. Field, M., Plotkin, G. R., Silen, W. Nature, 1968, 217, 469. Pierce, N. F., Carpenter, C. C. J., Jr., Elliott, H., Greenough, W. B., III. Gastroenterology, 1971, 60, 22. Schafer, D. E., Lust, W. D., Sircar, B., Goldberg, N. D. Proc. natn. Acad. Sci. U.S.A. 1970, 67, 851.
This raised adenyl-cyclase activity is found not only in animal cholera but also in cases of human cholera.9 There are still unanswered questions. What structure does the toxin interact with on the cell surface ? The fact that ganglioside inhibits its activity suggests that it may become attached to a similar substance in the cell membrane.10 How does this influence adenyl cyclase ? It seems that, once exposed to toxin, the cell undergoes typical changes even if it is washed, but we do not know if this means that toxin induces some slow reaction, or is transported elsewhere in the cell, or whether there is some other explanation. The exact cellular mechanism may be unravelled by investigation of the action of toxin on other more convenient cells-for example, adipose tissue, which shows the characteristic enzyme changes,10,11 or hela cells, which are unable to spread on a glass surface when treated with toxin.12 It is always satisfying if analysis of the disease process suggests useful lines of treatment, and in the case of cholera the study of fluid loss led to effective regimens of fluid replacement. Already the suggestion has been made that since aspirin prevents the synthesis of prostaglandins and their effect on cyclic A.M.P., it should be tried in cholera,13 but no results have yet been reported. Normal rat serum prevents the effect of toxin in intestinal and other preparations,14 and the effect of the toxin on hela cells can be reversed by human serum a-macroglobulins,l5 but these have not yet been proved to be effective in patients. We must continue to hope, therefore, that increasing understanding will give an improved treatment, one which really halts the physiological defect in the cells rather than correcting its secondary effects-namely, diarrhoea and dehydration. In fairness, however, it should be stated that we know how to prevent the disease very effectively-by applying basic sanitation Sharp, G. W. G., Hynie, S. Nature, 1971, 229, 266. Kimberg, D. V., Field, M., Johnson, J., Henderson, A., Gershon, E. J. clin. Invest. 1971, 50, 1218. 8. Guerrant, R. L., Chen, L. C., Sharp, G. W. G.J. infect. Dis. 1972, 125, 377. 9. Chen, L. C., Rohde, J. E., Sharp, G. W. G. Lancet, 1971, i, 939. 10. van Heyningen, W., Carpenter, C. J., Pierce, N. F., Greenough, W. B., III. J. infect. Dis. 1971, 124, 415. 11. Vaughan, M., Pierce, N. F., Greenough, W. B., III. Nature, 1970, 226, 658. 12. Inwood, J., Tyrrell, D. A. J. Br. J. exp. Path. 1970, 51, 597. 13. Bennett, A. Nature, 1971, 231, 536. 14. Aziz, K. M., Phillips, R. A. Proc. tech. Committee Rep. PakistanSeato Cholera Res. Lab. 1970, 1, 100. 15. Davies, J., Tyrrell, D. A. J., Ramsden, D. B., Louis, L. N., Milne, R. G. Exp. med. Path. (in the press). 6. 7.
168
It was the success achieved in the M.R.C. trial 15 years ago that provided W.H.O. with the factual foundation for their policy of stimulating B.c.G. vaccination throughout the world. There have been powerful opponents of vaccination, particularly in the U.S.A. But the evidence from this meticulously conducted trial was conclusive for the particular B.C.G. Vaccination population studied. It remained unknown whether 22 YEARS ago the Medical Research Council began a similar protection would be achieved in another popucontrolled trial of B.c.G. vaccination. Three reports lation in another environment. Unfortunately, it now have already been published.--3 Now the duration of seems likely that the degree of protection is, in fact, observation has been extended to 15 years.4 This, in related to environmental factors. So the protection to itself, is a remarkable achievement. The results of this be expected in a particular country cannot be accurately trial have the considerable effort important justified predicted from that demonstrated in Great Britain. involved. The variation in protective efficacy of B.c.G. vaccinaThere were 54,239 children aged 14-15 2 years in tion in the published reports of controlled trials has, the trial. Those with negative reactions to tuberculin indeed, been enormous. It ranges from 80% in a trial were vaccinated with B.c.G. or vole-bacillus vaccine or in North American Indians to nil in the first of the left unvaccinated, according to a method of random U.S. Public Health Service trials in Georgia. It has allocation. The annual incidence of tuberculosis among been suggested that at least part of the variation may those vaccinated with B.C.G. was 0-28 per 1000, combe due to different degrees of infection of the populapared with 1-28 per 1000 among the tuberculintion with non-tuberculous mycobacteria. Such negative children-a reduction of 78%. The annual infections confer some protection against infection by incidence among those vaccinated with vole-bacillus tubercle bacilli and limit the additional benefit which vaccine was 0-29 per 1000, compared with 1-50 per B.C.G. can confer. 1000 among the tuberculin-negative children admitted In the M.R.C. study, for instance, the incidence of to the trial concurrently with them-a reduction of tuberculosis among the children with low-grade 81%. The degree of protection was similar in the two tuberculin sensitivity-which in Britain is partly sexes. It decreased gradually during the 15-year period. tuberculous in origin and partly due to other mycoBut in the 10-15-year period after vaccination it was bacteria-was considerably lower than the incidence in still substantial; 59% for B.C.G. and 73% for volethe tuberculin-negative unvaccinated group. However, bacillus vaccine. HARTshowed in a detailed analysis that this factor A point of particular importance is that there were was inadequate completely to explain the great no cases among the vaccinated of the two most serious differences between the results of the British and the types of the disease-tuberculous meningitis and U.S. trials. He suggested that the U.S. vaccine may miliary pulmonary tuberculosis. Among the unnot have been as potent as that used in Great Britain. vaccinated there were 10 cases. Whatever the causes of the variations, their existence Vaccination with vole-bacillus vaccine has never got and magnitude have undoubtedly retarded the spread the in Great Britain, beyond of B.c.G. vaccination in the world. It has, for instance, experimental stage although it has been used extensively and routinely been estimated that in Latin America only 20% of the " elsewhere-for instance, in Czechoslovakia. Its use was susceptible " population has been vaccinated.7 The virtually discontinued after the report of very occasional situation is little better in the so-called developed development of lesions resembling lupus vulgaris at countries of Europe.88 Yet there is no doubt of the the vaccination site. This, however, happened only efficacy of B.c.G. vaccination in this region of the world. after vaccination with 3 substrains. The other substrain Apart from the M.R.C. controlled trial, evidence has been obtained, from the routine vaccination procedure produced low levels of post-vaccination skin sensitivity, but was subsequently shown to have produced a high in Birmingham, of its continuing efficacy in the degree of protection. This substrain is still available. prevention of tuberculosis.9 The same report has also In 1962, MiTCHELL 5 concluded that " the development shown that the Glaxo freeze-dried vaccine used in of lupus at the vaccination site may now be considered Britain has a high degree of efficiency, similar to that of to be of historical interest only and is no longer a the liquid vaccine from the Copenhagen strain formerly contra-indication to the use of vole bacillus vaccine ". used. The Glaxo vaccine is now used in many parts of The M.R.C. report states that it would " seem worth the world. considering the reintroduction of the vole bacillus as an Because of the uncertainty about the protective immunising agent against tuberculosis effect of B.c.G. vaccination in populations of tropical
and public health-but to apply these requires a modicum of organisation, good will, and money, which are probably harder to obtain than an understanding of the enzymes.
1. 2. 3. 4. 5.
Medical Medical Medical Medical
Research Council. Br. med. J. 1956, i, 413. Research Council. ibid. 1959, ii, 397. Research Council. ibid. 1963, ii, 973. Research Council. Bull. Wld Hlth Org. 1972, 46, 371. Mitchell, D. N. M.D. thesis, London, 1962.
Hart, P. D’A. Br. med. J. 1967, i, 587. Tubercle, 1971, 52, 303. Lotte, A., Perdrizet, S., Hatton, F. Bull. Wld Hlth Org. 1971, 44 (suppl.). 9. Springett, V., Sutherland, I. Br. med. J. 1970, iv, 148.
6. 7. 8.