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Summary of United States-Japan Cholera Symposium
Vol. 54, No.3 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1968 by The Williams & Wilkins Co.
COMMENTS Readers are invited to contribute Comments. If found suitable, they will be published promptly, subiect to the usual editing. They should be typewritten. double spaced, and sent to the Editor.
SUMMARY OF UNITED STATES-JAPAN CHOLERA SYMPOSIUM A symposium on cholera, sponsored by ippines, that had been free of it for decades. the Office of International Research, Na- Resultant dislocations of trade and tourism tional Institutes of Health, as a part of the add to the already great direct costs of United States-Japan Cooperative Medical cholera. Collection of world-wide statistics on the Sciences Program, was held in Palo Alto, California, July 26 to 28, 1967. Attendance incidence of cholera, as well as the definiwas limited to invited workers in the field; tion of quarantine regulations, is the rethe primary purpose of the meeting was to sponsibility of the World Health Organizafurther the exchange of information among tion, which was represented at the meeting active research workers representing the by Dr. D. Barua. Up to date information two countries. Those parts of the program on the geographic incidence of cholera and which may be of interest to gastroenterol- legal vaccination requirements is always ogists in general will be summarized here. available to American physicians through Complete proceedings of the meeting will W. H. O. or from the Communicable not be published. Disease Center, United States Public Cholera is an acute diarrheal disease, Health Service, Atlanta, Georgia. caused by intraluminal infection of the Cholera vaccines in current use consist gastrointestinal tract with a specific orga- merely of suspensions of killed whole cells nism, Vibrio cholerae, several serological of certain established laboratory strains. types of which can be distinguished. No Although it has recently been shown that natural host other than man has been iden- such vaccines are protective to a limited tified. Unlike shigellosis, salmonellosis, and degree and for a brief period of months, many other bacterial infections of the di- their use is associated with annoying 10gestive system, cholera causes little or no cal and systemic reactions, and they are pain, fever, or toxicity, and no ulceration clearly inadequate for complete control of of the gut or blood loss. The disease dis- cholera in the populations of de,-eloping abIes and kills only by way of the dehy- countries. The search for a better immunizdration and acidosis resulting from losses ing agent remains a prime object of cholera of water and electrolytes in the stool. It research under the United States-Japan may be prevented by adequate sanitation, Program. To that end, many studies have and individual cases may be treated very been directed toward the separation and successfully by parenteral fluid replace- characterization of antigenic components of ment and antibiotics. Neither measure, V. cholerae, and the determination of their however, is within the economic reach of antigenicity in man and experimental anilarge populations in densely inhabited, mals. Papers presented by Drs. Y. Watansemitropical parts of Asia, where major abe, W. Verwey, R. Pike, R. Finkelstein, epidemics of cholera with high mortality H. Ogonuki, J. Feeley, and J. Craig dealt still occur. Since 1958, the geographic with such research. spread of cholera has been progressive, and On the basis of serological studies carried the disease has once again become a prob- out in East Pakistan, Dr. W. H. Mosley lem in countries, such as Iran and the Phil- showed that resistance to naturally occur471
472
COMMENTS
ring infection is correlated with the circulating titer of vibriocidal antibodies measurable in vitro. In the endemic area, this antibody titer is higher in adults than in children, while the attack rate for clinical cholera decreases markedly with increasing age. Likewise, among the immediate family contacts of index cases of cholera, he could show an inverse relationship between the existing antibody titer and the probability of a secondary attack of clinical disease. Cholera vaccine of proven protective value elicited a rise in vibriocidal antibody titers, and, when different vaccinated populations were compared, there was a good correlation between vaccine effectiveness and the serological response. Thus it appears that a vaccine administered parenterally may protect against bacterial colonization of the lumen of the gut, and that circulating antibodies are concerned with the mediation of the effect. In studies carried out in experimental animals, Dr. R. Freter had also observed that antibacterial immunity was more important than antitoxic in protecting against infection. Further detailed study of the mechanisms of immunity in cholera should be of great gelleral interest. Dr. W. M. McCormack presented results of epidemiological surveillance of a rural population in East Pakistan during one cholera season, from November 1966 to March 1967. Although the villagers considered themselves to have been fortunate enough to have escaped cholera during that season, bacteriological study of 200 mild cases of diarrhea revealed that 5, at least, were infected with V. cholerae. An additional 30 persons out of a sample of 954 showed a sufficient rise in vibriocidal titers that they were felt to have had an inapparent infection. Thus it would appear that typical clinical cholera represents only one extreme of a spectrum of severity of infection, and that inapparent infections outnumber obvious ones by many fold. Much work remains to be done to determine quantitatively the significance of such inapparent infections and that of convalescent carriers in the propagation of cholera epidemics. The majority of the Japanese partici-
Vol. 54, No.3
pants in the meeting reported on in vitro bacteriological investigations, there being no clinical cholera in Japan. Studies of the effects of antibiotics on the bacterium, on antigenic classification of subtypes, on resistance of the organism to drying, and on cholera bacteriophage were presented by Drs. Takeya, Kuwabara, Sakazaki, Tamaoki, and Sato. Both Japanese and American investigators have recently obtained convincing evidence that the principal serotypes of V. cholerae, known as Ogawa and Inaba, are not genetically stable, but that mutations in either direction occur with sufficient frequency to be observable both in vivo and in vitro. These results were presented by Drs. Fukumi, Sasaki, Sack, and DeWitt. Of all the bacteriological studies, those of greatest potential significance to the field of gastroenterology were those dealing with the preparation of sterile toxic filtrates from cultures of V. cholerae. Such filtrates show a variety of interesting effects, and it remains to be learned whether one or more than one distinct toxin is involved. Dr. J. Craig described recent studies using rabbit skin as an assay system; cholera filtrates injected subcutaneously cause a delayed increase in capillary permeability with erythema, edema, and extravasation of plasma proteins. The responsible toxin appears to be a protein, is antigenic, and can be converted to a toxoid. Dr. R. Finkelstein has preferred to test toxicity in the intestines of infant rabbits; these animals respond to intraluminal toxin by accumulation of fluid, diarrhea, and death by dehydration. The responsible agent has been concentrated and partially characterized; it too is a protein and is antigenic. Whereas the infant rabbit is very sensitive to cholera culture filtrates introduced into the lumen of the upper small intestine, the adult rabbit is much more resistant unless the bowel is also obstructed. Such a loop of adult rabbit intestine, ligated at each end but left in situ, is used for the assay of cholera toxin by Dr. W. Burrows. He reported on the partial purification and characterization of the moiety responsible for this phe-
March 1968
COMMENTS
nomenon; he believes it to consist of 60% protein and 40% lipid, with no detectable carbohydrate. The isolation of the toxin or toxins responsible for these various effects, and their precise chemical characterization, is an immediate goal of current research. As part of the United States-Japan Cholera Program, supplies of crude toxic filtrates, free of viable organisms, are to be procured and made available to qualified investigators beginning in January 1968. The day devoted to pathogenesis and pathophysiology of cholera opened with a symposium on fluid transfer mechanisms in the gastrointestinal tract. Drs. P. Curran, H. Wheeler, and J. Fordtran reviewed their own recent studies of solute and water absorption in the intestine and gall bladder. Dr. M. Grossman summarized the scanty knowledge that exists in the literature of secretion by the mammalian small intestine. This part of the digestive system normally transports water and electrolytes only from the lumen to the circulation, although the use of radioisotopes has given firm evidence that such net transport is always the consequence of unidirectional fluxes in each direction, that from lumen to blood being greater than that from blood to lumen. Net transport of fluid into the lumen of the small bowel has been reported to occur after experimental denervation of the gut and in response to distension. Studies of the chemical composition of such fluid could not be found. Hypersecretion, presumably attributable to the small intestine, has also been reported in a few unusual patients with pancreatic islet cell adenomas and diarrhea, but without gastric hyperacidity or peptic ulceration. Whether or not the mechanism of diarrhea production in cholera has anything in common with these experimental and clinical situations remains to be learned. Work on the pathophysiology of cholera has been greatly facilitated by the recent observation that the dog is susceptible either to infection with large inocula of virulent V. cholerae, as reported to the meeting by Dr. B. Sack, or to the action of sterile toxic filtrates, as described by Dr. C.
473
Carpenter. Dogs with such experimental cholera appear satisfactorily to duplicate all the major clinical and physiological features of the naturally occurring disease in man. Studies in the dog model have so far revealed the following important points: (1) almost all of the diarrheal fluid originates in the jejunum and ileum, as may be shown by ligating the gut at appropriate levels and collecting the fluid between ligatures, and (2) lymph flow from the affected segment of canine intestine is markedly increased during cholera. Drs. H. Elliott and J. Yardley reported on microscopic and electron microscopic studies of tissues taken by biopsy from such dogs with experimental cholera. Whereas local hemorrhage and separation of the epithelial cell layer had been observed in some earlier specimens obtained by suction biopsy, no such changes were found in tissues excised surgically under direct vision. Histopathology in open biopsy tissues was limited to minimal degrees of acute inflammation, dilation of mucosal capillaries, edema of the lamina propria which may have been more related to fluid therapy than to cholera, dilation of the lumina of the crypts with associated flattening of the epithelium, and discharge of mucus from goblet cells. There was no alteration in the ultrastructure of the epithelial cells on electron micrography. Bacteria were observed in the gut lumen, but never appeared to invade tissue. These observers summarized their studies by stating that there was no significant structural change in the intestinal mucosa in experimental canine cholera, but that all the changes observed were consistent with alterations in the functional state of the tissues. Drs. A. Abraham and G. Dammin had had an opportunity to perform microscopic studies on the tissues of a relatively small number of human cholera victims who had succumbed to their disease in East Pakistan. Since over 99% of such patients coming to medical attention are cured, interpretation of the findings in the rare fatal cases will inevitably be complicated
474
COMMENTS
by the coexistence of other diseases, and perhaps by secondary effects of unsatisfactory therapy. The results of the study of human tissues were all consistent with the picture of dog cholera as presented above. With the greater availability of sterile toxic solutions produced in vitro by cholera cultures, it is natural that there should be increasing interest in the action of such preparations on isolated vertebrate tissues. To date, there has been no satisfactory and consistent demonstration of an effect of cholera toxin on epithelial transport functions in such systems. To this accumulation of negative information, Dr. G. Grady added a report of studies carried out on isolated human ileal mucosa obtained at surgery. Such tissues could be maintained in vitro for 1 hr or more, and showed rapid inhibition of active sodium transport when exposed to dinitrophenol. Cholera filtrates known to be effective in the production of fluid accumulation in experimental animals in vivo were without action on isolated human ileum. It should be noted, however, that Dr. Carpenter had reported that a latent period of several hours was required before cholera filtrate showed its characteristic effect on the ileum of the experimental dog, and that Dr. Grady was not able to maintain human ileum in vitro for so long a time. Reports on physiological studies in clinical cholera were presented by Drs. N. Pierce and J. Banwell of the Johns Hopkins-Calcutta School of Tropical Medicine International Center for Medical Research and Training, and by Drs. D. Sachar, N. Hirschhorn, and J. Kinzie of the Pakistan-SEATO Cholera Research Laboratory. In each of these laboratories, it has been found by intubation studies using nonabsorbable markers that net transport of fluid into the jejunal lumen occurs during cholera in man, and that this fluid is isotonic. When first formed, its ionic composition differs only little, if at all, from that of an ultra filtrate of plasma. More distally in the gut, concentrations of potassium and bicarbonate rise, and those of sodium and chloride fall.
Vol. 54, No.3
Introduction into the gut lumen of actively transported sugars (glucose and galactose, but not fructose) was shown to be followed by an increase in the electrical potential difference between the lumen and an indifferent electrode, a phenomenon that occurs normally and is attributed to coupling of active sugar absorption with that of sodium. It was further shown that net fluxes of sodium and water were favorably affected by the administration of glucose, with reduction of the efflux within study segments, or even a reversal of net flow. These reports were all consistent with the concept that the active absorption of glucose, and the transport of sodium associated with it, remain normal in spite of cholera, and that sodium absorption by this process can even match the cholera-induced transfer of sodium and water into the gut, whatever the nature of the latter process may be. This work, of course, also leads to hope that an effective oral therapy of cholera, suitable for use in rural areas of developing countries, may be a possibility. The session closed with a report presented by Dr. T. Bayless, showing that the effects of glucose on fluid and sodium absorption could also be demonstrated in the dog model, and with a discussion by Dr. L. Griffith of certain practical problems in the treatment of cholera in young children in the Philippines. In summary, research on cholera, motivated primarily by the practical problems of control and treatment of the disease in developing Asian countries, has led to findings of great potential significance in the understanding of intestinal physiology and of mechanisms that may be involved in other diarrheal conditions. A cell-free toxin can be prepared, and its chemical characterization is being undertaken. Further study of the action of this toxin on intestinal tissue preparations, both in vivo and in vitro, promises to shed new light on a hitherto mysterious subject, the secretory capacity of the small intestine. Under the United States-Japan Cooperative Medical Sciences Program, the National Institutes of Health are committed
March 1968
COMMENTS
to furthering research in this field. Pertinent research grant applications are being actively solicited.
Robert S. Gordon, Jr., M.D. National Institute of Arthritis and Metabolic Diseases Bethesda, Maryland 20014SUGGESTED BIBLIOGRAPHY FOR BACKGROUND READING
4.
5.
6.
1. Gordon, R. S., Jr., J. C. Feeley, W. B. Greenough, III, H. Sprinz, and R. Oseasohn. 1966.
Cholera. Combined Clinical Staff Conference at the National Institutes of Health. Ann. Intern. Med. 64: 1328-51. 2. Carpenter, C. C. J., P. P. Mitra, and R. B. Sack. 1966. Clinical studies in Asiatic cholera. I-VI. Bull. Hopkins Hosp. 118: 165245.
3. Sack, R. B., C. C. J. Carpenter, R. W. Steen-
7. 8.
47.5
berg, and N. F. Pierce. 1966. Experimental cholera-a canine model. Lancet 2: 206. Leitch, G. J., M. E. Iwert, and W. Burrows. 1966. Experimental cholera in the rabbit ligated ileal loop: toxin-induced water and ion movement. J. Infect. Dis. 116: 303-312. Oseasohn, R. 0., A. S. Benenson, and Md. Fahimuddin. 1965. Field trial of cholera vaccine in rural East Pakistan. First year of observation. Lancet 1: 450. Craig, J. P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature (London) 207: 614. Pollitzer, R. 1959. Cholera. World Health Organization, Series 43, 997 pp. World Health Organization, Geneva. Proceedings of the Cholera Research Symposium. 1965. Public Health Service Publication 1328. United States Government Printing Office, Washington, 397 p.
MILK INTOLERANCE AND LACTOSE TOLERANCE TESTS This comment is offered as an effort to correlate the results of lactose tolerance testing with the clinical problem of milk intolerance. The lactose tolerance test is a useful tool and arguments over interpretation, often with negative connotations, may have clouded this fact. There have been several articles in this and other journals that have attempted to establish "normal" and "abnormal" lactose tolerance test results and to correlate these with arbitrarily determined normal and abnormal jejunal lactase levels. 1-3 Delayed gastric emptying probably caused the fiat curves in normals in one paper.1 Some reports do not state whether normal people had a history of milk intolerance even though some had symptoms with the lactose loads. 2 • 4 One group stated that persons with less than 0.5 unit of lactase activity per g wet wt of mucosa were "lactase-deficient," 3 while another group chose 1 unit as the dividing line between normal and "abnormal." 2 Furthermore, variations in the technique of lactase assay in different laboAddress requests for reprints to: Dr. Theodore M. Bayless, Johns Hopkins Hospital, Baltimore, Maryland 21205.
ratories make an arbitrary normal level difficult to interpret. Our experience would indicate that this arbitrary approach does not correlate with the clinical histories of milk intolerance, is restrictive, and does not provide all of the useful information that can be obtained from the lactose tolerance tests. Because the main clinical problem is to establish the presence or absence of symptoms which might be attributable to milk and lactose ingestion, we have based our separation of patients on the presence or absence of a history of milk intolerance. 5 Forty healthy prisoner volunteers were studied by interview, lactose tolerance tests (50 g per m 2 of body surface with a maximum of 100 g in 400 cc of water), glucosegalactose tolerance tests, and disaccharidase assays of small intestinal biopsies (jejunum in 37 and distal duodenum in three). Twenty-one of the 40 subjects stated that milk acted as a laxative. This usually required only 1 or 2 glasses of milk but 6 subjects noted this effect only after 1 quart of milk. Nineteen individuals were able to consume milk without symptoms. Lactose tolerance tests produced symptoms of cramps and distension or diarrhea,
GASTROENTEROLOGY
Copyright © 1968 by The Williams & Wilkins Co.
COMMENTS Readers are invited to contribute Comments. If found suitable, they will be published promptly, subiect to the usual editing. They should be typewritten. double spaced, and sent to the Editor.
SUMMARY OF UNITED STATES-JAPAN CHOLERA SYMPOSIUM A symposium on cholera, sponsored by ippines, that had been free of it for decades. the Office of International Research, Na- Resultant dislocations of trade and tourism tional Institutes of Health, as a part of the add to the already great direct costs of United States-Japan Cooperative Medical cholera. Collection of world-wide statistics on the Sciences Program, was held in Palo Alto, California, July 26 to 28, 1967. Attendance incidence of cholera, as well as the definiwas limited to invited workers in the field; tion of quarantine regulations, is the rethe primary purpose of the meeting was to sponsibility of the World Health Organizafurther the exchange of information among tion, which was represented at the meeting active research workers representing the by Dr. D. Barua. Up to date information two countries. Those parts of the program on the geographic incidence of cholera and which may be of interest to gastroenterol- legal vaccination requirements is always ogists in general will be summarized here. available to American physicians through Complete proceedings of the meeting will W. H. O. or from the Communicable not be published. Disease Center, United States Public Cholera is an acute diarrheal disease, Health Service, Atlanta, Georgia. caused by intraluminal infection of the Cholera vaccines in current use consist gastrointestinal tract with a specific orga- merely of suspensions of killed whole cells nism, Vibrio cholerae, several serological of certain established laboratory strains. types of which can be distinguished. No Although it has recently been shown that natural host other than man has been iden- such vaccines are protective to a limited tified. Unlike shigellosis, salmonellosis, and degree and for a brief period of months, many other bacterial infections of the di- their use is associated with annoying 10gestive system, cholera causes little or no cal and systemic reactions, and they are pain, fever, or toxicity, and no ulceration clearly inadequate for complete control of of the gut or blood loss. The disease dis- cholera in the populations of de,-eloping abIes and kills only by way of the dehy- countries. The search for a better immunizdration and acidosis resulting from losses ing agent remains a prime object of cholera of water and electrolytes in the stool. It research under the United States-Japan may be prevented by adequate sanitation, Program. To that end, many studies have and individual cases may be treated very been directed toward the separation and successfully by parenteral fluid replace- characterization of antigenic components of ment and antibiotics. Neither measure, V. cholerae, and the determination of their however, is within the economic reach of antigenicity in man and experimental anilarge populations in densely inhabited, mals. Papers presented by Drs. Y. Watansemitropical parts of Asia, where major abe, W. Verwey, R. Pike, R. Finkelstein, epidemics of cholera with high mortality H. Ogonuki, J. Feeley, and J. Craig dealt still occur. Since 1958, the geographic with such research. spread of cholera has been progressive, and On the basis of serological studies carried the disease has once again become a prob- out in East Pakistan, Dr. W. H. Mosley lem in countries, such as Iran and the Phil- showed that resistance to naturally occur471
472
COMMENTS
ring infection is correlated with the circulating titer of vibriocidal antibodies measurable in vitro. In the endemic area, this antibody titer is higher in adults than in children, while the attack rate for clinical cholera decreases markedly with increasing age. Likewise, among the immediate family contacts of index cases of cholera, he could show an inverse relationship between the existing antibody titer and the probability of a secondary attack of clinical disease. Cholera vaccine of proven protective value elicited a rise in vibriocidal antibody titers, and, when different vaccinated populations were compared, there was a good correlation between vaccine effectiveness and the serological response. Thus it appears that a vaccine administered parenterally may protect against bacterial colonization of the lumen of the gut, and that circulating antibodies are concerned with the mediation of the effect. In studies carried out in experimental animals, Dr. R. Freter had also observed that antibacterial immunity was more important than antitoxic in protecting against infection. Further detailed study of the mechanisms of immunity in cholera should be of great gelleral interest. Dr. W. M. McCormack presented results of epidemiological surveillance of a rural population in East Pakistan during one cholera season, from November 1966 to March 1967. Although the villagers considered themselves to have been fortunate enough to have escaped cholera during that season, bacteriological study of 200 mild cases of diarrhea revealed that 5, at least, were infected with V. cholerae. An additional 30 persons out of a sample of 954 showed a sufficient rise in vibriocidal titers that they were felt to have had an inapparent infection. Thus it would appear that typical clinical cholera represents only one extreme of a spectrum of severity of infection, and that inapparent infections outnumber obvious ones by many fold. Much work remains to be done to determine quantitatively the significance of such inapparent infections and that of convalescent carriers in the propagation of cholera epidemics. The majority of the Japanese partici-
Vol. 54, No.3
pants in the meeting reported on in vitro bacteriological investigations, there being no clinical cholera in Japan. Studies of the effects of antibiotics on the bacterium, on antigenic classification of subtypes, on resistance of the organism to drying, and on cholera bacteriophage were presented by Drs. Takeya, Kuwabara, Sakazaki, Tamaoki, and Sato. Both Japanese and American investigators have recently obtained convincing evidence that the principal serotypes of V. cholerae, known as Ogawa and Inaba, are not genetically stable, but that mutations in either direction occur with sufficient frequency to be observable both in vivo and in vitro. These results were presented by Drs. Fukumi, Sasaki, Sack, and DeWitt. Of all the bacteriological studies, those of greatest potential significance to the field of gastroenterology were those dealing with the preparation of sterile toxic filtrates from cultures of V. cholerae. Such filtrates show a variety of interesting effects, and it remains to be learned whether one or more than one distinct toxin is involved. Dr. J. Craig described recent studies using rabbit skin as an assay system; cholera filtrates injected subcutaneously cause a delayed increase in capillary permeability with erythema, edema, and extravasation of plasma proteins. The responsible toxin appears to be a protein, is antigenic, and can be converted to a toxoid. Dr. R. Finkelstein has preferred to test toxicity in the intestines of infant rabbits; these animals respond to intraluminal toxin by accumulation of fluid, diarrhea, and death by dehydration. The responsible agent has been concentrated and partially characterized; it too is a protein and is antigenic. Whereas the infant rabbit is very sensitive to cholera culture filtrates introduced into the lumen of the upper small intestine, the adult rabbit is much more resistant unless the bowel is also obstructed. Such a loop of adult rabbit intestine, ligated at each end but left in situ, is used for the assay of cholera toxin by Dr. W. Burrows. He reported on the partial purification and characterization of the moiety responsible for this phe-
March 1968
COMMENTS
nomenon; he believes it to consist of 60% protein and 40% lipid, with no detectable carbohydrate. The isolation of the toxin or toxins responsible for these various effects, and their precise chemical characterization, is an immediate goal of current research. As part of the United States-Japan Cholera Program, supplies of crude toxic filtrates, free of viable organisms, are to be procured and made available to qualified investigators beginning in January 1968. The day devoted to pathogenesis and pathophysiology of cholera opened with a symposium on fluid transfer mechanisms in the gastrointestinal tract. Drs. P. Curran, H. Wheeler, and J. Fordtran reviewed their own recent studies of solute and water absorption in the intestine and gall bladder. Dr. M. Grossman summarized the scanty knowledge that exists in the literature of secretion by the mammalian small intestine. This part of the digestive system normally transports water and electrolytes only from the lumen to the circulation, although the use of radioisotopes has given firm evidence that such net transport is always the consequence of unidirectional fluxes in each direction, that from lumen to blood being greater than that from blood to lumen. Net transport of fluid into the lumen of the small bowel has been reported to occur after experimental denervation of the gut and in response to distension. Studies of the chemical composition of such fluid could not be found. Hypersecretion, presumably attributable to the small intestine, has also been reported in a few unusual patients with pancreatic islet cell adenomas and diarrhea, but without gastric hyperacidity or peptic ulceration. Whether or not the mechanism of diarrhea production in cholera has anything in common with these experimental and clinical situations remains to be learned. Work on the pathophysiology of cholera has been greatly facilitated by the recent observation that the dog is susceptible either to infection with large inocula of virulent V. cholerae, as reported to the meeting by Dr. B. Sack, or to the action of sterile toxic filtrates, as described by Dr. C.
473
Carpenter. Dogs with such experimental cholera appear satisfactorily to duplicate all the major clinical and physiological features of the naturally occurring disease in man. Studies in the dog model have so far revealed the following important points: (1) almost all of the diarrheal fluid originates in the jejunum and ileum, as may be shown by ligating the gut at appropriate levels and collecting the fluid between ligatures, and (2) lymph flow from the affected segment of canine intestine is markedly increased during cholera. Drs. H. Elliott and J. Yardley reported on microscopic and electron microscopic studies of tissues taken by biopsy from such dogs with experimental cholera. Whereas local hemorrhage and separation of the epithelial cell layer had been observed in some earlier specimens obtained by suction biopsy, no such changes were found in tissues excised surgically under direct vision. Histopathology in open biopsy tissues was limited to minimal degrees of acute inflammation, dilation of mucosal capillaries, edema of the lamina propria which may have been more related to fluid therapy than to cholera, dilation of the lumina of the crypts with associated flattening of the epithelium, and discharge of mucus from goblet cells. There was no alteration in the ultrastructure of the epithelial cells on electron micrography. Bacteria were observed in the gut lumen, but never appeared to invade tissue. These observers summarized their studies by stating that there was no significant structural change in the intestinal mucosa in experimental canine cholera, but that all the changes observed were consistent with alterations in the functional state of the tissues. Drs. A. Abraham and G. Dammin had had an opportunity to perform microscopic studies on the tissues of a relatively small number of human cholera victims who had succumbed to their disease in East Pakistan. Since over 99% of such patients coming to medical attention are cured, interpretation of the findings in the rare fatal cases will inevitably be complicated
474
COMMENTS
by the coexistence of other diseases, and perhaps by secondary effects of unsatisfactory therapy. The results of the study of human tissues were all consistent with the picture of dog cholera as presented above. With the greater availability of sterile toxic solutions produced in vitro by cholera cultures, it is natural that there should be increasing interest in the action of such preparations on isolated vertebrate tissues. To date, there has been no satisfactory and consistent demonstration of an effect of cholera toxin on epithelial transport functions in such systems. To this accumulation of negative information, Dr. G. Grady added a report of studies carried out on isolated human ileal mucosa obtained at surgery. Such tissues could be maintained in vitro for 1 hr or more, and showed rapid inhibition of active sodium transport when exposed to dinitrophenol. Cholera filtrates known to be effective in the production of fluid accumulation in experimental animals in vivo were without action on isolated human ileum. It should be noted, however, that Dr. Carpenter had reported that a latent period of several hours was required before cholera filtrate showed its characteristic effect on the ileum of the experimental dog, and that Dr. Grady was not able to maintain human ileum in vitro for so long a time. Reports on physiological studies in clinical cholera were presented by Drs. N. Pierce and J. Banwell of the Johns Hopkins-Calcutta School of Tropical Medicine International Center for Medical Research and Training, and by Drs. D. Sachar, N. Hirschhorn, and J. Kinzie of the Pakistan-SEATO Cholera Research Laboratory. In each of these laboratories, it has been found by intubation studies using nonabsorbable markers that net transport of fluid into the jejunal lumen occurs during cholera in man, and that this fluid is isotonic. When first formed, its ionic composition differs only little, if at all, from that of an ultra filtrate of plasma. More distally in the gut, concentrations of potassium and bicarbonate rise, and those of sodium and chloride fall.
Vol. 54, No.3
Introduction into the gut lumen of actively transported sugars (glucose and galactose, but not fructose) was shown to be followed by an increase in the electrical potential difference between the lumen and an indifferent electrode, a phenomenon that occurs normally and is attributed to coupling of active sugar absorption with that of sodium. It was further shown that net fluxes of sodium and water were favorably affected by the administration of glucose, with reduction of the efflux within study segments, or even a reversal of net flow. These reports were all consistent with the concept that the active absorption of glucose, and the transport of sodium associated with it, remain normal in spite of cholera, and that sodium absorption by this process can even match the cholera-induced transfer of sodium and water into the gut, whatever the nature of the latter process may be. This work, of course, also leads to hope that an effective oral therapy of cholera, suitable for use in rural areas of developing countries, may be a possibility. The session closed with a report presented by Dr. T. Bayless, showing that the effects of glucose on fluid and sodium absorption could also be demonstrated in the dog model, and with a discussion by Dr. L. Griffith of certain practical problems in the treatment of cholera in young children in the Philippines. In summary, research on cholera, motivated primarily by the practical problems of control and treatment of the disease in developing Asian countries, has led to findings of great potential significance in the understanding of intestinal physiology and of mechanisms that may be involved in other diarrheal conditions. A cell-free toxin can be prepared, and its chemical characterization is being undertaken. Further study of the action of this toxin on intestinal tissue preparations, both in vivo and in vitro, promises to shed new light on a hitherto mysterious subject, the secretory capacity of the small intestine. Under the United States-Japan Cooperative Medical Sciences Program, the National Institutes of Health are committed
March 1968
COMMENTS
to furthering research in this field. Pertinent research grant applications are being actively solicited.
Robert S. Gordon, Jr., M.D. National Institute of Arthritis and Metabolic Diseases Bethesda, Maryland 20014SUGGESTED BIBLIOGRAPHY FOR BACKGROUND READING
4.
5.
6.
1. Gordon, R. S., Jr., J. C. Feeley, W. B. Greenough, III, H. Sprinz, and R. Oseasohn. 1966.
Cholera. Combined Clinical Staff Conference at the National Institutes of Health. Ann. Intern. Med. 64: 1328-51. 2. Carpenter, C. C. J., P. P. Mitra, and R. B. Sack. 1966. Clinical studies in Asiatic cholera. I-VI. Bull. Hopkins Hosp. 118: 165245.
3. Sack, R. B., C. C. J. Carpenter, R. W. Steen-
7. 8.
47.5
berg, and N. F. Pierce. 1966. Experimental cholera-a canine model. Lancet 2: 206. Leitch, G. J., M. E. Iwert, and W. Burrows. 1966. Experimental cholera in the rabbit ligated ileal loop: toxin-induced water and ion movement. J. Infect. Dis. 116: 303-312. Oseasohn, R. 0., A. S. Benenson, and Md. Fahimuddin. 1965. Field trial of cholera vaccine in rural East Pakistan. First year of observation. Lancet 1: 450. Craig, J. P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature (London) 207: 614. Pollitzer, R. 1959. Cholera. World Health Organization, Series 43, 997 pp. World Health Organization, Geneva. Proceedings of the Cholera Research Symposium. 1965. Public Health Service Publication 1328. United States Government Printing Office, Washington, 397 p.
MILK INTOLERANCE AND LACTOSE TOLERANCE TESTS This comment is offered as an effort to correlate the results of lactose tolerance testing with the clinical problem of milk intolerance. The lactose tolerance test is a useful tool and arguments over interpretation, often with negative connotations, may have clouded this fact. There have been several articles in this and other journals that have attempted to establish "normal" and "abnormal" lactose tolerance test results and to correlate these with arbitrarily determined normal and abnormal jejunal lactase levels. 1-3 Delayed gastric emptying probably caused the fiat curves in normals in one paper.1 Some reports do not state whether normal people had a history of milk intolerance even though some had symptoms with the lactose loads. 2 • 4 One group stated that persons with less than 0.5 unit of lactase activity per g wet wt of mucosa were "lactase-deficient," 3 while another group chose 1 unit as the dividing line between normal and "abnormal." 2 Furthermore, variations in the technique of lactase assay in different laboAddress requests for reprints to: Dr. Theodore M. Bayless, Johns Hopkins Hospital, Baltimore, Maryland 21205.
ratories make an arbitrary normal level difficult to interpret. Our experience would indicate that this arbitrary approach does not correlate with the clinical histories of milk intolerance, is restrictive, and does not provide all of the useful information that can be obtained from the lactose tolerance tests. Because the main clinical problem is to establish the presence or absence of symptoms which might be attributable to milk and lactose ingestion, we have based our separation of patients on the presence or absence of a history of milk intolerance. 5 Forty healthy prisoner volunteers were studied by interview, lactose tolerance tests (50 g per m 2 of body surface with a maximum of 100 g in 400 cc of water), glucosegalactose tolerance tests, and disaccharidase assays of small intestinal biopsies (jejunum in 37 and distal duodenum in three). Twenty-one of the 40 subjects stated that milk acted as a laxative. This usually required only 1 or 2 glasses of milk but 6 subjects noted this effect only after 1 quart of milk. Nineteen individuals were able to consume milk without symptoms. Lactose tolerance tests produced symptoms of cramps and distension or diarrhea,