Effect of Bacterial Enterotoxins on the Gastrointestinal Tract

GASTROENTEROLOG Y 65: 467- 497, 1973 Copyright© 1973 by The Willi ams & Wilkins Co.

Vol. 65 , No.3

Printed in U.S.A.

PROGRESS IN GASTROENTEROLOGY

EFFECT OF BACTERIAL ENTEROTOXINS ON THE GASTROINTESTINAL TRACT JOHN

G.

BANWELL ,

M.D .,

AND HOWARD SHERR,

M.D.

Division of Gastroenterology, Department of M edicine, Johns Hopkins Hospital, Baltimore, Maryland

The current level of interest in the effects of bacterial enterotoxins on the intestine can be traced back to early clinical experience with epidemic and endemic cholera diarrhea, which is carefully compiled and documented in the classic monograph of Pollitzer. 1 After identification and culture of the vibrio during the epidemics in Egypt and Calcutta in 1864, Koch 2 suggested that toxic materials, elaborated by this organism, were the cause of the disease, cholera. In later years Koch 3 and Metchnikoff, 4 working independently, were able to induce a similar diarrheal illness in the guinea pig and suckling rabbit, respectively. However, for unaccountable reasons, 5 75 years were to elapse before De and Chatterjee 6 and Dutta and Habbu 7 demonstrated that cell-free products (whole cell lysate or crude broth culture supernatant) from the vibrio would cause consistent fluid production in ligated ileal segments and severe diarrhea when administered directly into the rabbit stomach. The recognition that other organisms might also elaborate enterotoxins was derived from the work of Smith 8 during comRecei ved November 1, 1972. Address requests for reprints to : Dr. John G. Banwell, Gastroenterology Division, Department of Medi cine, University of Kentucky Medical Center, Lexington , Kentucky 40506. Supported in part by the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education and Welfare, and Grants AM 05095 and A 108187, and the Irwin Strasburger Memorial Fund.

parative studies of diarrhea in pigs and calves and the demonstration 9 that cellfree culture extracts of Escherichia coli, isolated from children with neonatal diarrhea, were able to induce fluid accumulation in the rabbit ileal loop preparation. Several recent review articles on cholera toxin 5 • 1 0 • 11 and its pathogenesis 1 2 - 15 provide excellent sources of information on the subject. Present concepts of intestinal E. coli infections are provided in a recent symposium. 16 The normal and abnormal intestinal flora, 17 • 18 characteristics of diarrhea in general 19 and bacterial diarrhea in particular, 20 as well as the concepts involved in secretion from the intestinal tract, 21 • 22 provide other sources of important information in this field. This article will be directed primarily to an analysis of the metabolic consequences arising from the contact of bacterial enterotoxins with the intestinal mucosa and the pathogenesis of enterotoxic diarrhea. The local and systemic immune response resulting from this exposure has been the subject of previous articles 23 " 28 and will not be discussed here. Moreover, since enterotoxins of Vibrio cholera and E. coli have received the most study, the mode of the action of these two agents will form the substance of this review. Characteristics of Bacterial Ente..otoxins (Table 1) Bacterial enterotoxins are a form of exotoxin elaborated by intact bacteria into broth cultures-the release of enterotoxin depending on outward diffusion or transport through the bacterial cell wall and not on cell lysis. In general, they possess

467

30

Cholera

Human intestinal disease

• Microliters per centimeter per hour. • Characteristics probably similar to cholera enterotoxin ."

+

+

28,494

Rabbit

Rabbit

+

+

35,000 6- 9

Rat

+

-

7- 8

Rabbit

155

< 1 '12 hr 72

Rabbit > 90 5-7 112 hr 2 112-5 hr 194 I min Rat < 10 < 20 min mm Ra bbit < 18 min

1l2 - 1 hr Dog < 15 min Rabbit < 15 < 1 V2-3 mm hr

Pig, dog, rabbit

600-700

Period Maximum of peak rate of response secretion a

7- 8

Duration of effect

Dog, guinea pig Dog15 min 24-36 hr 4-5 hr Rabbit, rat, man Rabbit < 15 > 12 hr 2- 4 hr min Rabbit

Time of onset

Secretory res ponse

8

Animal species

+

+

+

Protease

Optimum pH

+

+

+

+

Heat

Inactivation

55- 60,000

5 X 10 6

84,000

Mol wt

1. Characteristic features of bacterial enterotoxins modified from the table of Grady and Keusch "

Nonaggluttinating vibrio••• Food-poisoning gastroenteritis Escherichia coli 32 - " Neonatal diarrhea Traveler's diarrhea Acute undifferentiated diarrhea of adults Shigella dysenteriae" Bacillary dysentery Staphy loco ccus aureus'"· 37 Sta phylococcal food poisoning Clostridium perfringens 38 Food poisoning Enteritis necroticans Pseudomonas aeroginosa''

Vibrio cholerae"·

Bacterial organism

TABLE

Protein

225- 363

466

251-413

mg/ 100

cont ent

*'00"

"'

~

~

~

"<

<;)

a

t-<

a

::0

~ t;3

a

S2en ;5

~

~ en

::0

a<;)

;g

m

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PROGRESS IN GASTROENTEROLOGY

similar qualities of heat lability (heatstable fractions of staphylococcal and E. coli enterotoxins are recognized), sensitivity to Pronase and resistance to trypsin, nondialyzability, and inhibition below pH 6, as well as extreme potency in even small doses. The unique and most significant property of all enterotoxins is to cause fluid and electrolyte secretion on exposure to the small intestinal mucosa. This effect is primarily responsible for the severe diarrhea which accompanies infection with a particular organism and for secondary depletion of extracellular fluid. Cytotoxic properties of certain bacterial culture filtrates which cause damage to intestinal mucosal cells are best considered as a separate feature distinct from enterotoxic activity for the purpose of this review. Enterotoxins stimulate fluid and electrolyte secretion by a mechanism which is unrelated to damage of the mucosal epithelial cells caused by the enterotoxin molecule or associated bacterial products in the culture filtrates. Enterotoxic activity can be demonstrated in cell-free culture supernatant fluid from Vibrio cholerae, 29 • 30 nonagglutinable vibrios, 31 Escherichia coli, 3 2 - 3 4 Shigella dysenteriae, 3 5 Staphylococcus aureus, 36 • 37 Clostridium perfringens, 38 and Pseudomonas aeroginosa. 3 9 Salmonella typhi 40 induce intestinal secretion but the role of an enterotoxin in this process is uncertain. Only the characteristics of cholera and staphylococcal enterotoxins, and more recently, E. coli and Shigella enterotoxin, have received intensive study. Cholera enterotoxin has been isolated by Lospalluto and Finkelstein 29 in a highly purified crystalline form without loss of activity after successive attempts at purification. 30 During the recovery of the enterotoxin (choleragen) another antigenically identical, but essentially nontoxic, protein was also purified (choleragenoid) , which probably represented a spontaneously formed toxoid. Choleragen had a molecular weight of 84,000 on ultracentrifugation and consisted of 6 subunits, each with a mol wt of

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approximately 15,000. Choleragenoid consisted of a mixture of at least three proteins with a mol wt of 58,000 and 4 subunits of approximately equal size. Neither toxin nor choleragenoid protein contained significant lipid or hexose moieties. Amino acid analysis indicated differences between the toxin and choleragenoid in the content of arginine, lysine, aspartic acid, and tyrosine . Production of toxoid by formalinization did not alter the molecular weight on ultracentrifugation. 'T'he availability of the pure protein toxin and toxoid may now permit detailed studies to be made of the site of localization of the toxin on or within the mucosal epithelial cell, 41 exact definition of its chemical structure, determination of the relationship between chemical structure and activity of the toxin, and its utilization in analysis of the biochemical and physiological events accompanying fluid and electrolyte secretion. Pure choleragen (0.033 f.lg) causes secretion in the rabbit ileal loop, 0.0035 f.lg skin permeability changes, and 3 f.lg per ml, death in infant rabbits. In contrast to other enterotoxins, choleragen exhibits the unique feature of inducing capillary permeability changes on subcutaneous injection. 42 This permeability activity is closely related to enterotoxic activity during purification, 3 0 and the antibody response to both types of effects are parallel providing a useful epidemiological 43 and microbiological method 44 for evaluating features of the disease in man and toxin production in microbial cultures. Noncholera vibrios, which do not agglutinate with cholera 0 antiserum, also appear to be capable of causing a cholera-like illness. 4 5 A recent study has shown that they produce an enterotoxin which does possess the vascular permeability factor, is nondiffusible and heat-labile, and almost completely neutralized by antisera against purified cholera enterotoxin. 31 A variety of preparations of E. coli enterotoxin have been studied and both a heat-labile 32 • 3 3 · 46 • 47 and heat-stable 34 • 48 49 • form has been described. Recent attempts to establish the chemical nature of the E. coli enterotoxin resulted in a puri-

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fied material of high molecular weight (> 5 x 10 6 ) after ultrafiltration and gel filtration chromatography. It contained both endotoxic, as well as enterotoxin, activity which could not be separated by a variety of techniques . 50 This suggested that a single complex molecule was responsible for the different biological activities with a lipid carbohydrate moiety accounting for the classical endotoxin activity and a protein moiety responsible for the enterotoxin activity. These same authors (T. M. Jacks, B. J. Wu, A. C. Braemer, personal communication) and others 34 have shown that there is also a low molecular weight enterotoxin (mol wt 1,000 to 10,000) which is heat-stable. Recent work has demonstrated that cholera antitoxin and choleragenoid will diminish the fluid secretory response to E. coli enterotoxin 32 and conversely that V . cholerae enterotoxin was neutralized by antiserum against enterotoxigenic E. coli in swine. 33 This suggests that different enterotoxins may, in part, possess a similar molecular configuration and perhaps share similar active sites for binding to the same mucosal receptor (both cholera- and E. coli-induced secretion may also be mediated through the adenyl cyclase-cyclic adenosine monophosphate (AMP) system.) Although there is much evidence 5 • 20 to indicate that enterotoxins are exotoxins, there is no clear reason for their production. Cholera toxin is only formed at certain times when vibrio are grown in broth culture, appearing rapidly in the bacterial cells during the exponential and onset of the log phase of growth. 5 1 No known functions have been accorded to enterotoxins in the bacteria's own metabolism-a notable similarity to other exotoxins, such as botulinus and tetanus toxin. 52 Richardson 5 1 has proposed that cholera enterotoxin is the result of abnormal synthesis of a surface protein unable to fulfill its structural function , or, perhaps , that it develops by elaboration or release of a protein which is normally a component of the periplasmic complex. Further work along these lines may indicate which biochemical pathways are subverted into toxin synthesis in actively growing bacterial cultures.

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Staphylococcal enterotoxins are proteins produced by staphylococci in food and under certain conditions in culture media. 53 The ingestion of this material by man causes staphylococcal food poisoning. This type of food-borne illness is widespread and characterized by severe vomiting and diarrhea, occurring 2 to 6 hr after eating contaminated food. Four staphylococcal enterotoxins, A, B, C, and D, have been identified and the primary structure of enterotoxin B has recently been elucidated and shown to comprise 239 amino acid residues in the polypeptide chain with a mol wt of 28 ,494. 54 All four enterotoxins have been implicated in food poisoning and type B specifically in episodes of enteritis. 55 The enterotoxins are heat-stable and show resemblances in C- and N-terminal amino acid structure. 5 3 Shemano and co-workers 5 6 demonstrated that highly purified enterotoxin had a direct inhibitory effect on intestinal tone, contractility, and colonic transit in the dog. Another study 36 has recorded alterations in intestinal fluid transport. Pure staphylococcal enterotoxin B has been shown to reverse net absorption of Na, K, Cl, and H 20 in a control period to net secretion after toxin addition in the rat upper small intestine. 3 7 A return to net water absorption occurred rapidly after toxin was removed from the perfusion solution. No changes were observed in potential difference (PD) in vivo, or in short circuit- current or resistance in vitro 3 7 after exposure to staphylococcal enterotoxin. It was concluded that enterotoxin triggered a nonelectrogenic secretory mechanism in the small intestine leaving the absorption mechanism intact. However, a feature of severe food poisoning is enteritis. Enterotoxin B administered orally causes an acute enteritis in monkeys and dogs. 5 7 • 5 8 The toxin also caused pronounced mucosal damage to the villus crest cells of the monkey intestine. 5 9 In addition to its effect on fluid transport, therefore, staphylococcal enterotoxin causes significant cytotoxic damage to the intestinal mucosa. An enterotoxin has been isolated from Shigella dysenteriae and partially purified by ultrafiltration and Sephadex gel filtra-

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tion. It has an estimated mol wt of 50,000 to 60,000. 35 A similar material has not been isolated to date from other Shigella strains, Shigella enterotoxin may be the same material previously identified as a neurotoxin in mouse lethality testing. 3 5 The enterotoxin is heat-labile and elicited a fluid response in the rabbit ileal loop, but less than to an equal weight of cholera enterotoxin. Morphological damage to the villi occurred in the ligated rabbit loop exposed to this toxin, 6 0 although in experiments lasting 6 to 9 hr in which ·the same toxin was perfused, minimal morphological damage was detected. 61 Fluid secretion developed only after 1- to 2 1/z -hr latent period and the rate diminished after 5 to 6 hr. Formal and co-workers 6 2 compared the disease-producing capacity in animal models of a wild type Shigella dysenteriae strain capable of penetrating the mucosa and of producing toxin, with mutant strains altered in either of these cell properties. The disease which resulted from the nontoxigenic penetrating mutant was not easily distinguished from that of the original toxin-producing parent strain. These findings suggested that the more important property for causing disease was the ability to penetrate and multiply in colonic mucosa. Although the enterotoxin may have a role in causing the watery diarrhea which may precede dysentery for 1 to 2 days, fever, colicky abdominal pain, hemorrhage, and ulceration result from epithelial cell penetration which also causes the characteristic dysenteric stool containing blood, pus, and mucus. Clostridia perfringens is a common cause of food-borne disease manifested as abdominal colic and diarrhea. 2 0 • 63 Infection with type A strains usually results in a self-limited diarrheal disease. 6 3 An enterotoxin has been isolated from some type A strains. 3 8 Type C strains may cause a more severe hemorrhagic necrotizing enteritis related more to tissue invasion and localized intestinal gas gangrene. 64 · 65 Pseudomonas aeruginosa also produces an enterotoxin, 39 the characteristics of which are shown in table 1. Powell and co-workers 6 6 ' 69 utilized the

471

rat as a model for studying the effects of Salmonella typhimurium infection. In the infected animals with diarrhea, jejunal and large intestinal transport was similar to infected animal without diarrhea. However, in all animals with diarrhea ileal secretion of H 2 0, Na, K, and Cl was observed. The net fluid accumulation in the bowel was accompanied by a 2- to 3-fold increase in protein content compared with control animals. Both protein accumulation and histological evidence of blunted fused villi and elongated hyperplastic crypts were evidence of tissue damage. Nevertheless , the protein content was lower than expected for an exudative secretion. Active electrolyte secretion may, therefore, be a factor in fluid accumulation in addition to tissue damage. At this time, no enterotoxin has been isolated from this organism, although positive rabbit loop responses occur similar to other pathogens which do produce enterotoxins 4 0 When Gangarosa and colleagues 7 0 in 1960 demonstrated that the small intestinal mucosal epithelium was intact in human cholera, the established but not totally accepted 71 • 72 dogmas of Virchow and of Koch 3 that cholera diarrheal fluid was the result of epithelial desquamation and denudation, were largely rejected. Many additional studies since 7 3 • 7 5 have confirmed that only minimal morphological change in goblet cells and dilation of the crypt lumen occur in the intestinal mucosa exposed to purified V. cholerae and E. coli enterotoxins. Thus, V. cholerae represents in the purest form an organism which is noninvasive where the effects of the disease after intestinal colonization result from the metabolic effects of enterotoxin on the intestinal mucosa. In contrast, indubitable cytotoxic damage occurs in the small and large intestinal mucosa associated with shigella, staphylococcal, clostridia, and salmonella infections in man and on exposure to culture filtrates in animals. The cytotoxic factors elaborated by these invasive bacteria which allow them to cause focal disruption of mucosal cell microvilli and terminal web, 7 6 adherence to the cell, 77 mucosal penetration, 7 8 and intraepithelial multiplication prior to dissemination into

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the tissues , 79 lie beyond the scope of this review. However, there is definite evidence40· 62 that some or all of these invasive organisms may elaborate enterotoxin(s) in addition which evoke fluid and electrolyte secretion. The relative contribution made by each type of enterotoxin to the diarrheal fluid loss in the particular disease caused by these organisms is unclear at this time, but appears to be important in staphylococcal food poisoning and the prodromal phase of infection due to shigella. Genetic determinants of enterotoxin formation. A number of factors which contribute to the pathogenesis and dissemination of enteric bacterial species are now known to be determined by plasmids. 80 Plasmids are extrachromasomal hereditary determinants of cytoplasmic inheritance which reproduce in an autonomous fashion. 81 Their importance was initially recognized in relation to multiple drug resistance of Enterobacteriaceae and the transfer of the resistance factor by cell to cell contact. · They are now known to control a wide variety of bacterial products, including production of hemolysins, 82 colicins, 83 and the bacterial fimbria! antigen, 84 as well as the formation of enterotoxin. 85 Enterotoxin, hemolysin production, and the fimbria! antigen are associated with plasmids in enteropathogenic E. coli isolated from swine .86 Such plasm ids can be selectively introduced into other bacterial cells by conjugation , 87 and selectively removed from cells by treat ment with antibiotics . 88 Smith and Lingood 86 have shown that the K88 plasmid which codes for fimbria! antigen is involved in the ability of enteropathogenic organisms to colonize the upper intestine, perhaps, by favoring adherence to the epithelium, whereas the enterotoxin plasmid is responsible for fluid production. Furthermore, enterotoxin formation and mucosal cell penetrance 85 are genetically controlled transmissible factors in several forms of E. coli which indicate that a variety of similar synthetic processes may be interrelated in order to facilitate invasion of the host epithelium by the organism. E . coli, for instance, may express

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both enterotoxic and cytotoxic properties in different strains 76 and perhaps even in the same strain, 89 providing a close resem blance to known pathogenetic characteristics of Shigella . The ease with which gramnegative bacteria transfer genetically de termined characteristics is dramatically provided by the recent outbreak of epidemic typhoid in Mexico in which the pattern of antibiotic resistance was identical to that of the pandemic strain of Shigella dysenteriae I which had caused dysentery in neighboring Guatemala. 90

Pathogenesis of Human Enterotoxic Diarrhea Current concepts of human cholera and E. coli diarrhea are largely derived from simultaneous bacteriological and metabolic measurements made on patients 91-96 and human volunteers 97· 98 with diarrhea. In the pathogenesis of cholera diarrhea (fig. 1) the live organism is ingested in food or water and usually only reaches the upper small intestine in sufficient concentration if gastric juice is buffered to an alkaline pH or if gastric secretory function is impaired. 98 · 99 Ingestion of preformed toxin may play a role in Staphylococcal food poisoning, but does not appear to be important in cholera or E. coli disease. 100 Bacteria colonize the entire small intestine, 92 · 95 overcoming the normal antibacterial and mechanical mechanisms 101 for
S eptertiber 1973

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473

V. Cholerae Diarrhea

Oral inge s tion o f ba c t e rial ~ (97, 98) ( o r 1 pr efo r med ( 104 ) e n t e r o t ox in

Proli fe rati on and co l o ni z at ~al l i nte s tine (92)

Enterot ox in f or mation by org~44)

Fluid and Ele c troly te se c re ti on by small int e stinal (9 3 )I mucosa

I DIA RRH EA I

A Es c he richia Col i Diarrhea

EXOGENOUS I NFECTION

ENDOGENOUS I NI'ECTION

Toxigeni c s t rains of E. Col i by or al r oute ( 89)

Tox i genic s trains of E. . Col i

i n d i sta l i leum a nd co l on ( Retro{;rode Spreod) ( 17)

A u

t

Delaye d Onset

0 n

e t

I mplantatio n i n Upper G-I Tract (95 ) ' Paralysis of Antibacteria l. Mechanisms Toxi n Proct r t ion ( 47 )

Flu i d Accumula t i on ( 96 )

B

I DIJRH!£]

FIG. 1. Represents, in a sc hem atic m anner, the pathogen etic steps in developm ent of (A) Vibrio cholerae diarrhea, (B) E scherichia coli diarrhea. B is derived from Gorbach and Levitan. 17

duration of effect resulting from exposure of the intestine to toxin alone. 103 It is not known whether continued toxin production by enteric vibrios play a role in the persistent secretion which occurs in the disease, or whether duration of secretion is limited by duration of cell turnover in the mucosa or metabolic factors within the epithelium. The response to tetracycline in rapidly reducing small bowel bacterial concentrations, as well as curtailing the duration

of diarrheal fluid loss, suggests that continued toxin production (and mucosal fixation of the toxin) do play a role in the continuing fluid secretion. 2 A similar pathogenetic schema can be derived for E. coli diarrhea in which exogenous enteropathogens enter by the oral route. However, Gorbach 1 8 has speculated that a retrograde spread may also occur from established resident strains in the colon if conditions are favorable to their spread

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proximally into the small intestine (fig. 1). Evidence from animal models of cholera and E. coli diarrhea clearly incriminates the small intestine as the source of fluid and electrolyte loss in the disease. In both man 104 and animals, 105 the diarrheal process can be reproduced entirely by ingestion of preformed toxin. In man, transmucosal fluid movement measured by the marker perfusion technique was greater in jejunum than ileum, and, indeed, in some patients net ileal absorption was demonstrated in the course of cholera diarrhea when the jejunum exhibited net fluid secretion. 93 The average net fluid movements are shown in table 1. In the jejunum, net transmucosal fluid movement correlated with the rate of fluid flow in the bowel lumen and the diarrheal stool loss measured at the time. A gradual return to net fluid absorption occurred in the jejunum over the succeeding 5 to 7 days which paralleled the clinical recovery and clearance of vibrio from the intestine . Similar findings were made in adult acute undifferentiated diarrhea attributed to enterotoxic strains of E. coli. However, net fluid secretion was generally less and of shorter duration than in cholera. 95 Although the colon does not contribute to fluid loss in the dog exposed to these enterotoxins, its role in man has not been studied. Normal colonic reabsorptive capacity is limited to 2500 ml per day , 106 • 107 but this may be augmented by effects of secondary hyperaldosteronism associated with dehydration 108 (colonic Na and Cl reabsorption is increased by acute and chronic 109 aldosterone administration). During severe cholera diarrhea, ileal and fecal bicarbonate concentrations are similar. With recovery from the disease and gradual reduction of diarrheal fluid production, stool bicarbonate concentrations progressively increased 11 0 • 111 which suggested that normal colonic Cl-HC0 3 exchange mechanisms become effective as colonic fluid flow rates and diarrheal fluid production diminish. However, the exact extent to which the colon modifies the composition and reduces the total loss of ileal effluent entering it during cholera,

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remains to be established. Therapeutic enhancement of normal colonic reabsorptive capacity might be a feasible experimental approach to further reduce fluid loss in diarrheal states. Thus, the role of the colon in enteropathogenic diarrhea requires further investigation.

Additional Effects of Cholera and E. coli Enterotoxins Systemic effects of enterotoxin. Single intravenous doses of 100 J.Lg of purified choleragen or more, usually caused death of dogs within 2 to 10 days but none developed diarrhea. 112 This observation, and others, 3 1 make it unlikely that cholera enterotoxin is absorbed from the gastrointestinal tract and transported in blood to effect secretion in distant segments of the bowel as proposed by one recent study. 11 3 Smaller doses cause hyperglycemia, hyponatremia, and elevated hepatic alkaline phosphatase and glutamic oxaloacetic transaminase levels which persisted for more than 5 days. The rise in alkaline phosphatase after intravenous challenge is similar to the effects of bile duct ligation which is attributed to enzyme induction. 11 4 Intravenous injection of formalinized cholera toxoid did not cause these effects. The development of fluid secretion from intestinal mucosa remote from the site of direct mucosal content by the cholera toxin has been observed. 11 5 It was suggested that absorbed toxin accounts for this phenomenon , but since even large doses of choleragen injected intravenously 112 fail to cause diarrhea, this seems unlikely. Panse and Dutta 11 6 suggested that histamine might be produced, and others 115 have postulated that a hormonal agent is released into the blood stream, and, indeed, an uncharacterized choleragenic agent can be detected during cross perfusion experiments. 117 Further evidence for the nature of this agent will be awaited with interest. Intestinal motility and muscle wall. Small intestinal jejunal flow, transit time, and volume relationships in human cholera were studied using an indicator dilution technique during segmental perfusion of the bowel. 11 8 The flow of intestinal con-

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tents in acute cholera showed considerable variability. Segment volume was closely correlated to flow rates in both acute disease and convalescence, suggesting that bowel wall compliance was similar in both phases of the disease. Although the "normal" convalescent jejunum was able to maintain a fairly constant mean transit time over a wide range of flow rates, the acutely affected jejunum was less capable of performing this function. It has been a frequent clinical observation that cholera diarrhea is painless once acid-base deficiencies and dehydration have been corrected, 119 whereas acute undifferentiated diarrhea in the tropics (attributable to E . coli enterotoxin) is often accompanied by cramping abdominal pain. In the laboratory, jejunal loops exposed to cholera toxin appear flaccid while E. coli exposed loops retain their motility, which may help explain the differences in symptoms. The effect of enterotoxins on small intestinal muscle and sphincter function remains to be studied. Enterotoxins may have direct effects on myoneural elements in the bowel wall distinct from secondary changes induced by fluid secretion and electrolyte depletion. Mucus secretion. A reduced content of mucus in goblet cells after exposure of the small intestine to cholera enterotoxin has been observed in dogs , 74 rabbits, 1 20 and pigs. 75 Depletion of mucus in response to live cholera vibrio was accompanied by ultrastructural evidence of heightened synthetic activity in goblet cells. 1 2 0 It was observed in both jejunum and ileum, and was independent of the mode of exposure and not induced by hypertonicity of intraluminal fluid. Neither heat-inactivated preparations of toxin nor plain culture broth caused release of mucus which made it unlikely that constituents of the culture media other than cholera toxin caused the discharge of mucus. The final mucus content in goblet cells assessed histologically is the combined result of mucus release and synthesis and further work will be neces sary to clarify which of these two processes were affected by cholera toxin . Cholera toxin did not cause mucus or fluid secre-

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tion in the colon; a further indication that the toxin did not act in the manner of a nonspecific irritant, such as mustard oil or pilocarpine. 12 1 Osmoregulation in the jejunum. Hypertonic electrolyte solutions containing mannitol instilled into in vivo canine jejunal loops approach the osmolality of plasma slower than do dilute solutions. After exposure to cholera enterotoxin the readjustment towards isotonicity occurred more slowly and Na + concentration of the fluid remained higher in all cases . 12 2 In a comparison of the jejunal response of the rabbit to cholera toxin and hypertonic mannitol singly and in combination, 1 23 net fluid production in response to cholera enterotoxin differed in electrolyte composition from that in response to a hypertonic stimulus. However, when the two stimuli were combined, the observed response, both in amount of fluid produced and electrolyte composition, was equivalent to the sum of the responses to the individual stimuli. Moreover, calculated sodium freewater clearance in response to hypertonic mannitol was not increased in loops preexposed to cholera enterotoxin. The data from both studies can best be interpreted to mean that fluid production in response to cholera enterotoxin is independent of the response to an osmotic gradient, does not involve an increase in transmucosal permeability, and the response to either stimulus of fluid production (enterotoxin or osmotic) is unaltered by the other. Gastric secretion. Gastric acid production in response to submaximal doses of Histalog is reduced during human cholera diarrhea and achlorhydria was more frequently detected during convalescence than in control subjects. 99 In another study, 12 4 using maximum histamine infusion (40 ,ug per kg per hr) only 1 of 16 patients had acid secretion below the normal range. While the role of impaired gastric acid production in predisposing to natural infection with acid-labile Vibrio cholerae is uncertain, human volunteer studies indicate that consistent and significant infection will reproducibly occur only when 108 to 10 10 vibrio are administered

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with antacids. 8 9 The role of cholera enterotoxin inhibition of gastric secretion requires further evaluation in the experimental animal. It has been suggested that cholera enterotoxin may release hormones 103 • 105 such as secretin, cholecystokinin, or enterogastrone 125 on direct contact with small intestinal epithelium which secondarily inhibit gastric secretion through as yet uncertain pathways, possibly involving cyclic adenosine monophosphate release or competitive inhibition of gastrin. A direct effect of the minute quantities of absorbed enterotoxin on gastric function or adenyl cyclase activity in gastric mucosa, although unlikely, might also play a role in inhibition of gastric secretion. Gallbladder. Schafer and co-workers 126 reported that crude cholera enterotoxin caused a secretory response in the dog gallbladder after 1- to 1 1/z -hr delay which was sustained for up to 24 hr at a rate of 0.5 to 1.0 ml per hr. Secreted fluid was viscid with a bicarbonate concentration of 40 to 50 mEq per liter. Viscosity increased with time and total protein concentration fell from 2.5 to 3.5 mg per ml initially to less than 0.5 mg per ml. Although fluid secretion rates were too low to make significant contributions to diarrheal fluid loss, the study raised the question, in the light of stimulation of adenyl cyclase by cholera enterotoxin in many other types of tissue homogenate, 10 whether relative lack of responsiveness of other regions of the gastrointestinal tract might be attributed to the presence or absence of suitable receptors to facilitate the response. Intestinal lymphatics. Dilation of the central lacteals of villi was observed after cholera toxin exposure in the dog. 74 Lymph flow rates increased in the thoracic duct in experimental cholera and were further augmented by saline repletion. 103 Total protein concentrations of lymph were inversely related to the flow rates in the thoracic duct but never exceeded normal values; electrolytes and reducing substances were in similar concentration to plasma. Lee and Silverberg 1 27 measured

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villus lymph pressure in vitro in the rabbit intestine after in vivo exposure to cholera enterotoxin, by a micro-injection technique. They recorded that villus lymph pressure was reduced approximately 50% by a wide range of crude cholera toxin dosages even though similar dosages enhanced fluid secretion in vivo. Formation of lymph is a primary result of water absorption, and, in the rat, lymph flow may account for 85% of absorbed water. 128 The authors assumed that reduced lymph pressure in cholera was evidence for reduced lymph flow, and, therefore, reduced transmucosal fluid absorption. The low lymph pressure in the presence of 2, 4-dinitrophenol and absence of glucose from the mucosal bathing solution supported this point of view; but, it is also possible that cholera enterotoxin may have had additional effects on secretion by the epithelium which could alter hydraulic permeability of the lymphatic wall and the normal relationship between lymph pressure and water flux. Intestinal capillaries. Cholera toxin causes rapid increase in skin capillary permeability on intracutaneous injection. 5 Intestinal exposure to toxin increased permeability to ferritin, 1 29 although no increase was detected for Evans blue and saccharrated iron oxide. 130 Histological study of intestinal mucosa after toxin challenge showed minimal morphological change in the capillaries which were not probably attributable to altered blood flow. 74 Another study in experimental cholera with parenteral horseradish peroxidase indicated an accelerated transfer out of mucosal capillaries , particularly evident in the crypts. 131 Intravascular materials are thought to cross the capillary wall by passing through pores in the fenestra estimated to be 70 to 90 A in the intestine. 1 32 It is unlikely, therefore, that the capillary wall represents a significant impedance to the passage of low molecular weight substances, such as water and electrolytes, entering the bowel lumen in response to enterotoxins. No other enterotoxins have the skin permeability factor of cholera

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enterotoxin. 20 The response of intestinal microvasculature to other enterotoxins has not been studied.

477

studies were unable to confirm this and the permeability changes he observed may have occurred after long incubation (10 to 12 hr) and distention of the closed loops Pathogenesis of Enterotoxin-induced employed in the study. The other studies in Small Intestinal Secretion human and experimental cholera all indiThe role of increased intestinal capillary cate that relative permeability is unhydrostatic pressure and trans mucosal changed. Sherer and co-workers 1 3 7 demonpermeability in enterotoxin-induced secre- strated that the relative movements of tion. Increased intestinal capillary hydro- uncharged water soluble molecules ( 14 Cstatic pressure and alteration in trans- urea, 14 C-creatinine, and 14 C-lactose) mucosal permeability do not appear to be through the rabbit jejunum in vivo were the mechanisms for fluid secretion in re- unchanged by cholera toxin exposure. sponse to enterotoxin. Although no accu- Moore and co -workers 1 38 noted that cholera rate measurements of villus capillary blood toxin caused no change from normal in the flow or pressure are available, several com- ratio of 14 C-urea to 3 H -arabinose across pelling arguments are evidence against the rabbit ileum. Gordon and co-workers 13 4 "filtration" hypothesis. Intestinal fluid demonstrated in human cholera that the secretion and diarrhea continue in the rate of clearance of 14 C-mannitol after cholera patient who is pulseless and with- intravenous injection into stool water was out recordable systemic blood pressure. 11 9 inconsistent with an ultrafiltration process. Intestinal secretion in response to cholera In addition, relative absorption rates of toxin in the dog continues unabated even 1 4 C-urea and 3 H-arabinose 1 39 during experwhen superior mesenteric blood flow is imental canine and human cholera were reduced to less than 30% of normal. 13 3 unchanged, and in another recent study, Gordon and co-workers 1 3 4 calculated that if Lifson and co-workers 14 0 utilized an in vivo the filtration hypothesis were correct and canine loop to study passive permeability hydraulic permeability of the entire human of urea, arabinose, and 14 C-glucose after intestine were 0.3 ml per min per millios- exposure to crude cholera enterotoxin. mole, an osmotic gradient of 20 millios- Both diffusive and convective permeability moles which is thermodynamically equiva- were measured for net fluid absorption and lent to a hydrostatic pressure gradient of secretion and subjected to analysis with a one-half atmosphere or 380 mm Hg would previously verified mathematical model for be required to achieve a flow rate of 6 ml passive permeability. Values for diffusive per min. Such pressures are extremely clearance were similar during net absorpunlikely to be achieved in vivo. Moreover, tion and net cholera enterotoxin-induced Hakim and Lifson 13 5 have shown that the secretion. dog intestine in vitro will only support Animal Models of Enterotoxigenic serosal pressure up to 20 mm Hg before Diarrhea damage and permeability alterations ocDog. Klemperer 14 1 observed that dogs cur. If transmucosal permeability were developed diarrhea in response to live cholgreatly reduced, however, theoretical esti- era vibrio. The subsequent development of mates of the filtration pressure required a suitable animal model resulted from the could also be lowered towards normal. It intensive study by Sack and Carpenter 10 5 • was suggested by Love 1 36 in early experi- 14 2 • 14 3 and Carpenter and Greenough. 1 44 ments in which bulk water flow in response Such an animal is responsive to live vibrios to osmotic gradients in closed rabbit jeju- or cholera enterotoxin administered by nal loops exposed to cholera vibrios were mouth or intraluminally by intestinal tube. measured, that permeability was in- Exposed animals develop features of diarcreased. However, a variety of subsequent rhea, dehydration, metabolic acidosis, and

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death , comparable to the human disease, as well as agglutinating, vibriocidal, and antitoxic antibody responses. The model has been useful for studying therapeutic and immunization regimes, as well as pathogenetic mechanisms involved in cholera infections. 14 5 The dog also develops diarrhea on oral challenge with enterotoxin producing strains of E. coli. Net fluid secretion occurs in response to E. co li enterotoxin in isolated intestinal loops. 33 The response was more transient than with cholera enterotoxin, occurring within 90 min of exposure and ending rapidly after perfusion of toxin ceased. The canine colon was relatively unresponsive to both cholera 146 and E. coli enterotoxin. Rabbit . The isolated in vivo rabbit ileal loop of infant or adult rabbits has been used extensively to detect and assay enterotoxin activity. 147 • 148 Accurate criteria have been evolved for evaluating the biological response which also allow for quantitative measurement of the secretory response to cholera enterotoxin. 24 Leitch et al. 149 - 151 employed an in vivo rabbit loop to measure fluid secretion in response to crude cholera toxin and live vibrios. Rabbit intestinal loops have proved useful for many other studies of intestinal secretory activity involving simultaneous measurement of electrical potential difference and fluid secretion, 152 the effects of metabolic inhibitors on net fluid secretion 153 and the response to E. coli enterotoxin. 154 · 155 The magnitude of the response to E. coli was less than crude cholera toxin and the over-all response more evanescent. The secreted fluid was isotonic and low in protein content, similar to the canine response to this toxin. 33 Secretion developed within 15 min of exposure, appeared to reach a maximal rate rapidly although secretion was poorly maintained and diminished after 2 hr. Net absorption of d-glucose and glycine were inhibited in response to this toxin preparation. Rat. Several recent reports 156-158 indicate that the rat intestine will respond to crude cholera enterotoxin although responsiveness is not present in the fetal state. 158 An early report indicated the need for a 72-hr

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glucose-water diet prior to experiments to achieve secretion. 156 Another recent study 157 showed that a consistent secretory response to cholera toxin can be obtained which is dose-dependent, greater in jejunum than ileum, but which did not occur in the isolated gut sac preparation. Other investigators 11 have suggested that the rat intestine has a natural inhibitor which must be overcome by increased concentration of enterotoxin. Cat and guinea pig. Both species were shown to respond to cholera infection but have been employed little in recent work. 1 The cat intestine has been studied in a recent experiment to investigate the inhibitory effects of aspirin on cholera-induced secretion. 159 The guinea pig as a model for fluid secretion 160 has been carefully studied and detailed physiological measurements are available which would prove valuable for a comparison of enterotoxins. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin has been demonstrated in guinea pig mucosal homogenates . 161 Pig. Coliform diarrheal disease in the pig is a major problem in veterinary practice. 162 Recent interest in enterotoxin-producing strain of E. coli has demonstrated that similar secretory responses to cholera and E. coli enterotoxin can be obtained in this species without significant change in intestinal morphology.16, 32, 1s. 163-165 Mouse. The infant mouse has been used to study oral administration of vibrio 166 and the protective effects of antibody and detection of enteropathogenic E. co li in field studies. 167 Calves and lambs. These species are also susceptible to E. coli infection and enterotoxin. 163 In vivo studies of animal models of enterotoxin-induced small intestinal secretion. The perfused jejunal and ileal intestinal loop preparation has been used to define how enterotoxins cause the intestinal mucosa to secrete. Moore and coworkers138 measured ion and water transport rates in the normal and purified choleragen-treated dog ileum. After exposure to toxin, fluid was absorbed for the first 4

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hr and secretion developed progressively afterwards. Bicarbonate and potassium concentrations rose (HCOa - : 29 _, 80, K +: 5 _, 13 mEq per liter) and Cl - concentration fell (110 _, 65 mEq per liter) in the perfusion fluid. PD was + 4 mv (lumen negative) in the resting state and fell steadily to reach - 16 mv by the 6th hr. Addition of glucose to the perfusion solution to achieve a 50 mM concentration in the solution reduced PD further to -20 mv. Isotonic mannitol perfusion only reverted the PD to that accompanying electrolyte perfusion ( - 19 to -15 mv), whereas in the normal untreated ileum perfused with mannitol solution, the PD changed considerably more, from - 4 to + 35 mv. These findings appear to indicate that HC0 3 was secreted actively against both an electrical and chemical concentration gradient and that N a + movement could be attributed entirely to passive diffusion in response to the negative luminal PD. Cl secretion also occurred against an electrochemical gradient, and, although solvent drag forces may have contributed to the Cl - movement, the normal ileum usually has low passive permeability for Cl ions. 168 Evidence from the PD response to mannitol perfusion might indicate that selective ionic permeability of the ileum for anions, such as chloride, was altered by choleragen. However, the current hypothesis to account for ileal transport favor a coupled anion-cation exchange without an active bicarbonate or hydrogen ion transport system, which could explain the electrolyte movements observed in response to choleragen in the dog ileum. 168 • 169 Moritz and co-workers 152 studied cholera toxin-induced secretion in the in vivo perfused rabbit jejunum using a crude cholera culture broth supernatant . Control and cholera-treated loops were perfused with a chloride solution (Na +, 140, K +, 10, HCO a- , 30, Cl - , 130 mEq per liter, respectively) or bicarbonate solution (N a +, 140, K +, 10, HC0 3 - , 150 mEq per liter, respectively). Net water secretion followed toxin exposure with both perfusion solutions and became constant after 2 to 3 hr, although the onset was delayed in those loops ex-

479

posed to the bicarbonate solution. Transmural PD again became progressively negative with time in cholera-treated chloride loops ( -9 -+ -3.0 mv after 5 hr) and more profoundly in HC0 3 loops ( - 6.1 -+ -0.95 mv after 5 hr) indicating that the transmural PD was influenced by cholera toxin as well as the ionic constitution of the perfusion solution. No consistent correlation was observed between mean PD and net H 20 flux. In other studies 170 • 171 utilizing the inhibitory effects of cyclohexamide on cholera-induced secretion, unidirectional fluxes changed but the transmural PD did not reflect either the magnitude or the direction of net H 2 0 or ion flux across the mucosa. This indicated that some electrical effects were attributable to the influence of cyclohexamide and raised the question of how closely PD and fluid secretion were related in cholera-induced secretion, although other in vivo studies 172 do demonstrate a relationship between PD change and fluid movement. In vitro studies. In vitro experiments provided the earliest direct evidence that intestinal mucosa secretes actively when stimulated by cholera enterotoxin. Field and co-workers 17 3 • 1 H in a series of studies have employed an aerated lucite chamber with a cross sectional area of 1.13 em 2 across which a strip of rabbit ileum is suspended. Physical, electrical , and chemical gradients across the mucosa can be neutralized by appropriate control over the composition of the bathing solutions and by applying a short circuit current sufficient to nullify the electrical potential difference across the mucosa. Both surfaces were bathed in a bicarbonate-Ringer solution. Ion transport processes were examined by measuring fluxes of 22 Na, 2 4 Na, and 36 Cl. In most studies, the mucosa stripped of muscularis and serosa by rapid blunt dissection has been used . This preparation maintained a higher and more stable short circuit current (SCC) and a greater sec response to d-glucose than the tillstripped mucosa. This tissue preparation remains viable for 5 to 6 hr. When normal ileal tissue is suspended in this way, active

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net Na + and Cl - absorptive fluxes are detected which can be augmented by the addition of an actively transported sugar or amino acid to the mucosal solution (substrate-independent and -dependent transport, respectively.) Indirect evidence for another active transport process into the mucosal solution is also provided by the finding of a residual ion flux. The SCC must equal the sum of all net ion fluxes across the short circuited mucosal membrane but is not fully accounted for by the net sodium and chloride fluxes . Since there is evidence that the mammalian ileum may "secrete" bicarbonate actively in vivo, 168 • 169 this residual flux has been attributed to active HCO 3 - transport, serosa _, mucosa (S _, M) , but could be accounted for by any active anion movement from S _, M or cation from M _, S. It is known that adenosine 3' - , 5' -cyclic phosphate (cyclic AMP) enhanced fluid absorption in the toad bladder, 175 frog skin, 176 and renal collecting tubule, 177 primarily by increasing luminal membrane permeability to water and Na; whereas, net secretion was increased in stomach, 178 salivary gland, 179 and pancreas. 180 In the in vitro rabbit ileum preparation, 174 cycle AMP, and many other nucleotides, as well as pyrophosphate, caused a net S _, M chloride flux and the disappearance of a net M _, S Na flux which was glucose-independent. Theophylline which inhibited phosphodiesterase conversion of cyclic AMP to 5' -AMP, thus elevating intracellular cyclic AMP, had the same effect . Similarly, adenosine triphosphate, cyclic guanosine phosphate, and dibutyryl cyclic AMP caused these effects. Due to the complexities of ion transport processes and the recognition that ion fluxes in the system are determined by three major factors: (a) passive or exchange diffusion , (b) solvent drag, and (c) active transport of electrolytes, the demonstrated changes in flux do not necessarily indicate a change in active transport. For instance, in results obtained with cyclic AMP, decreased net Na flux may have occurred by inhibition of active Na absorption or by a stimulation of active Na secretion (inhibition of Na ab-

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sorption being the preferred mechanism to the authors because of the reduction which occurred in M _, S flux.) Stimulation of a Na pump, another possibility, would imply that sec changes in response to cyclic AMP were due to stimulation of active Na absorption , and, this was considered less likely than anion secretion because substitution of sulfate for chloride in the Ringer's solution abolished the sec response. A crude dialyzed filtrate of cholera enterotoxin (2 mg per ml) , as well as purified 172 • 181 choleragen (1 J,Lg per J,Lliter) , caused a stimulatory effect when applied to the luminal surface. Changes in PD and sec were smaller with purified choleragen and when toxin was applied to the mucosa in vivo prior to mounting the mucosa in the chamber. The SCC gradually increased to a peak at 2.5 hr, the increase really beginning 40 min after addition of toxin and reaching a plateau at 1 1/z to 2 hr. Ion fluxes measured 3 to 4 hr after addition of toxin indicated that there was a net Cl flux S _, M and that net Na flux M _, S was reduced to zero. Residual ion flux did not change although tissue electrical resistance was increased, especially after in vivo exposure to toxin. Cholera enterotoxin also reduced the sec response to theophylline an d dibutyryl cyclic AMP in vitro. A major discrepancy between these in vitro results and the in vivo studies described relate to HC0 3 - transport , and inhibition of the active Na absorption pump. If residual ion flux in vitro can be equated with HC0 3 - flux , cholera toxin does not influence HC0 3 - transport, although, in man and animal models, net HC0 3 - secretion into the bowel lumen occurs in both jejunum and ileum in response to cholera toxin. Quantitive measurements of HC0 3 - fluxes are needed to assess the separate contributions of active and passive forces to resolve this difference. Also in conflict is the apparent inhibition of active sodium pump absorption by choleragen in the presence of normal substrate-dependent Na transport. In vivo studies 103 • 182 and, indeed, the major recent development in the therapeutic management of cholera diarrhea 111 • 18 3 - 18 8 re-

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cord that net sodium absorption, as well as both glucose- and glycine-stimulated Na absorption, are normal. An appai'ent reconciliation of these conflicts lie in the work of Powell and co-workers, 189 who restudied the effects of purified choleragen on rabbit ileal mucosa in vitro and measured bidirectional fluxes only 70 min after addition of choleragen. Control values for net Na, Cl transport, and SCC were similar to the data of Field and co-workers. 172 However, after choleragen addition (L7 1-tg per ml), net Na and Cl transport were both stimulated to an equal degree; the sec effect was small and comparable to the effect observed by Field et al. with purified choleragen. Flux changes were inhibited if Ringer's solution was changed to sodium isethionate (Cl - + HCO 3 - free solution) and only minimal increase in SCC occurred in a Na-free solution. Choleragen may have altered residual flux (HC0 3 secretion?) as well. These results would suggest that choleragen might activate a neutral sodium chloride pump instead of an anionic pump. This would be easier to reconcile with the in vivo findings and would not require a direct inhibitory effect on · the active sodium absorption pump. E. coli toxin 190 as the crude filtrate has been shown to cause a rapid increase in sec and ion flux changes in vitro after 1 hr exposure which resembled the effects of cholera toxin. A crude E. coli toxin has been shown to inhibit glucose and glycine absorption 1 54 in vivo, although glucose facilitated sodium and water absorption was unaffected. 191 ' 192 Shigella enterotoxin failed to enhance sec in this system and caused no inhibition of the response to theophylline (M. Field, personal communication.) Metabolic changes in intestinal mucosa exposed to enterotoxin. Direct evidence that enterotoxins utilize energy dependent processes while exerting their secretory effect is not yet available. Keusch and co-workers 1 93 have investigated the effect of a purified cholera enterotoxin on oxidative metabolism of everted segments of infant rabbit jejunum.

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Oxygen consumption remained unchanged, whether the enterotoxin was added in vitro or in vivo. Mitochondria from infant rabbit liver showed normal oxidative phosphorylation and swelling phenomena on exposure to cholera toxin. Intestine mucosal adenosine triphosphatase and Na +-K + stimulated adenosine triphosphatase activity was the same in cholera-treated tissue as in control tissue, although another study 161 demonstrated marked inhibition of adenosine triphosphatase by cholera toxin. The failure to demonstrate effects of cholera toxin in these systems serves to further the argument that changes in fluid transport are not due to inhibition of active transport processes involving oxidative metabolism. In other studies, 194 it was observed that the production of lactic acid by jejunal segments from infant rabbit with experimental cholera was higher than that of segments from control animals. Cholera enterotoxin added in vitro with measurements made 1 hr later did not affect glycolytic activity. The importance of glycolysis as an energy source is known to vary with the animal specie, 195 degree of maturity, 196 and region of the gastrointestinal tract involved. 197 These metabolic effects of enterotoxin require further investigation. The ability to prepare mucosal cells isolated from the intestine of the chicken 198 • 199 and guinea pig 200 and the demonstration of their metabolic viability for at least 4 hr should enable metabolic substrates for enterotoxin secretion to be defined. It is of interest that provision of acetate to the mucosa in the form of ethyl acetate (which being lipid-soluble facilitated entry and subsequent breakdown by epithelial cell esterases to free intracellular acetate) , was shown to stimulate net water transport in the rat jejunum. 201 A study of the effects of inhibitors of anaerobic glycolysis, such as oligomycin 202 and the utilization of specific metabolic substrates during exposure of mucosal cells to cholera toxin might be rewarding in the further study of this problem. Net fluid secretion cannot be demonstrated in the everted gut sac exposed to cholera enterotoxin. 156

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Mode of Action of Enterotoxins on the Small Intestine

Enterotoxin-mucosal cell interaction. Direct contact of cholera toxin with the mucosal cell surface is required to cause secretion. The duration of exposure can be short. Goodgame et al. 203 and McGonagle and co-workers 204 showed that 5-min exposure was sufficient to cause a near-maximal secretory response in the perfused rabbit jejunal loop. In other studies it has been observed that intestinal secretion can be stimulated by less than one minute contact of cholera enterotoxin with the mucosal surface. 205 Once secretion has begun, removal of toxin solution from the loop and repetitive washing of the mucosa with saline does not cause secretion to cease. Cholera enterotoxin must be firmly bound to mucosa to sustain continued secretion or alternatively may stimulate a self-perpetuating secretory response in the mucosal cell. The rapidity of response , large molecular weight of cholera enterotoxin, and in vivo localization of the toxin to mucosal surface 41 suggest that its effect must be mediated on contact with glycocalyx or surface membrane of the microvilli. Large macromolecular proteins, such as horseradish peroxidase (mol wt 40,000), when placed in the intestinal lumen, 206 will pass into the cytoplasmic canalicular and vesicular network of the mucosal cell, as well as intercellular spaces and the submucosa within 1 hr of exposure. However, the major distribution of this enzyme, as well as impure toxin, 207 was found along the surface of absorptive cells in the area of the brush border membrane, the greatest concentration being on mature cells towards the tips of villi, less along the sides of villi and none was seen on crypt cells. Granules were also distributed all along the luminal surface of the intermicrovillus pits. The distribution of choleragen in intestinal mucosa after intraluminal injection has been demonstrated elegantly by immunohistochemical techniques (fluorescein- and horseradish peroxidase-labeled antibody) and autoradiography with tritium-labeled toxin. 41 Both choleragen and choleragenoid were specifically and se-

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lectively absorbed uniformly to the entire mucosal surface of the crypts and villi. Ultrastructured studies demonstrated this to be on the membranes of the microvilli. Specific absorption of toxin to the brush border membrane appears to be the initial step in stimulating fluid secretion, although another step of activation of secretion (effected by choleragen but not choleragenoid) is also essential to the process. 208 Leitch 209 • 210 studied the effect of crude and purified cholera toxin on the brush border enzymes of rabbit intestinal mucosal homogenates and compared this response to the effects of exposure to a commercial neuraminidase preparation. Results indicated that enterotoxin caused a reduction in alkaline phosphatase, as well as in brush border sialic acids and phospholipid, protein ratios differing quantitatively and qualitatively from the effects due to neuraminidase. Toxin affinity for binding sites is high. 211 Role of Cyclic AMP as a Mediator of Enterotoxin-Induced Secretion The biological role of cyclic AMP as the "second messenger system" in the transmission of the effects of a variety of hormones (the first messenger) are now well known and characterized. 212 The effect of hormones on the specific target cell involves interaction with specific receptors and activation of a membrane enzyme system, adenyl cyclase, which catalyze the transformation of adenosine triphosphate to cyclic AMP. The increased levels of cyclic AMP acting intracellularly modulates the hormonal effect by affecting enzymes, 213 permeability processes, 214 or the synthesis and release of other hormones. 215 Degradation of cyclic AMP to 5' AMP is caused by phosphodiesterases 216 located in the cytoplasm and to a lesser extent on the membrane. Thus, the level of cyclic AMP is controlled by release from an intracellular pool or new synthesis of adenyl cyclase, as well as by the rate of degradation by phosphodiesterases. Phosphodiesterase activity, like adenyl cyclase, is hormone-sensitive, and, therefore, may decrease or increase in response to agents such as xan-

September 1973

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thine, methylxanthine, and imidazole. However, in the subsequent discussion of the effects of enterotoxin on the adenyl cyclase system , it would appear that levels of phosphodiesterase are unaffected by cholera or E. coli enterotoxin so that this aspect of cyclic AMP turnover will not be discussed further. Hormones attach to receptor sites on the target cell membranes in close spatial proximity to adenyl cyclase. The nature of the relation between hormone receptors and adenyl cyclase is obscure. However, hormonal sensitivity or binding and the catalytic activity which evolves do not always develop in parallel. 217 This has permitted the development of a two-component model comprising a regulatory receptor component on the outside of the cell and a catalytic cyclic AMP generating system in an intracellular location. 218 In many tissues, liver, fat cells, and muscle, in particular, cyclic AMP has its effect by serving as a kinase activator. 219 The effect of cyclic nucleotides on the enzymes involved in glycogenolysis, now appear to involve a cascade of enzymatic events leading to the activation of a protein kinase. 217 Kinases are enzymes which serve to catalyze the transfer of phosphate from adenosine triphosphate to many proteins. They have two components, a catalytic portion and an associated inhibitory component. Cyclic AMP appears to attach to the inhibitory component, causing its disassociation from the catalytic portion, thereby, allowing it to catalyze protein phosphorylation. The phosphorylation of proteins results in their conversion to more active forms. A wide spectrum of biological activities are enhanced by cyclic AMP as a co-factor in enzymatic transfer of phosphate from adenosine triphosphate to proteins. Specificity of action is probably determined both by the clinical properties of the receptor-cyclase system in any tissue, as well as the intracellular compartment or spatial orientation of enzyme on which the effect is exerted (fig. 2). Many observations now suggest that cholera enterotoxin and perhaps E. coli enterotoxin exert their effect on fluid and

[E_~ ~~~O}_~x_r_Nj

~~~~~~~/,j/,,j;..:..::..::...IRECE~TOR~'l/I//II////~;I;/~:~~::!/!1//////////J/;

1////rAoENYL CYCLASE l'l!!//111111/1111!.iid/11////ii///' {7

ATP

c 'AMP

/

PROTEIN

+

+

CYCLIC AMP

- -

--

5' AMP

t

PHOSPHOOlESTE RASE

PHOSPHO PRO TEIN I
[ili] ATP

_______.. PROTEIN-P + AOP

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AL TERED CELL FUNCTION (Fitn d Secre11onJ

FIG. 2. Schematic diagram of the postulated in· teraction of cholera enterotoxin with t he mucosal cell. The activation of protein kinase has not been demonstrated to date and is presented in slanting type. Adenosine triphosphate, A TP; adenosine monophosphate, AMP; adenosine diphosphate, ADP.

electrolyte transport through activation of adenyl cyclase. Most criteria demanded for mediation through the adenyl cyclase system have been fulfilled. 217 (a) Cholera toxin induces a measurable change in the concentration of cyclic AMP in intact mucosal cells in the experimental animal. 161 • 220 (b) Cyclic AMP and analogues mimic the effect of cholera toxin when added to intact cells. 161 • 174 • 221 (c) The action of cholera enterotoxin is potentiated by agents which inhibit phosphodiesterase action (i.e., theophylline). 161 • 172 (d) When added to broken cell preparations of mucosa, cholera toxin stimulates conversion of adenosine triphosphate to cyclic AMP 161 (e) Cholera toxin stimulates adenyl cyclase activity in man 222 • 223 and animals. 224 - 227 (f) In dog intestine 161 • 226 exposed to cholera toxin, increase in adenyl cyclase activity parallels the time course of fluid secretion observed for cholera toxin-induced secretion in vitro. Whether the changes in cyclic AMP concentration precede the metabolic response, which is the sequence of events in response to natural hormones, 212 remains uncertain. No clear demonstration of an orderly response of adenyl cyclase or cyclic AMP to graduated doses of enterotoxin has been described so far, nor has it been shown that cholera enterotoxin has its effect through activation of protein kinase.

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The prolonged duration of elevation of adenyl cyclase by cholera toxin also differs considerably from the usual mode of stimulation by hormones in which return of activity to normal occurs early after removal of the stimulating agent. There are, therefore, differences in the mechanisms of adenyl cyclase activation by cholera enterotoxin. However, there is little doubt that this is a specific response to enterotoxin and sufficient explanation for the ion transport defects. Kimberg and coworkers161 were unable to demonstrate intestinal mucosal adenyl cyclase stimulation by a variety of other agents, including insulin, glucagon, epinephrine, and parathormone. Moreover, cholera enterotoxin causes stimulation of the adenyl cyclase system in other tissues, such as fat cells, 228 platelet, 229 and leukocyte. 230 The possibility remains that adenyl cyclase activation by enterotoxin may be a by-product of the secretory phenomenon. Kinzie and Alpers, 231 reported recently that cyclic AMP may act in regulating amino acid absorption by intestinal villus cells. Intestinal absorption of short chain alcohols may also result in stimulation of mucosal adenyl cyclase elevation. 232 It is conceivable that changes in membrane protein anabolism or the process of secondary reabsorption of secreted fluid in response to enterotoxin may also cause adenyl cyclase elevations. There are precedents for alteration in cyclic AMP metabolism in other disease states. Urinary cyclic AMP is elevated in hyperparathyroidism and , low in hypoparathyroidism. 233 A congenital defect in the receptor or catalytic subunit of adenyl cyclase may be the explanation for the inability of patients with pseudohyparathyroidism to mobilize calcium and excrete phosphorus and raise urinary cyclic AMP in response to parathyroid hormone. Thyroid adenomatous nodules have been shown to have an intact thyroid-stimulating hormone (TSH)-responsive adenyl cyclase-cyclic AMP system although such nodules do not concentrate 1' 31 after TSH administration, indicating that in this tissue the action of cyclic AMP on iodine metabolism may be impaired. 234 Schorr

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and Ney 235 have also shown that adrenal cancer cells of the rat, unlike normal adrenals, lose their specificity for a single tropic hormone, such as adrenocorticotropin, and become responsive to epinephrine and TSH. They suggested that the adenyl cyclase receptors of the tumor cells might have undergone structural alteration with loss of specificity or alternatively that tumor cells possessed several cyclase receptors. There is evidence, therefore, that in various diseases, the normal receptor-adenyl cyclase effect or response can be disordered and as Liddle and Hardman 209 point out, an autonomous adenyl cyclase system can become perverted in its responses. In cholera the converse situation may apply whereby the intestinal adenyl cyclase is responding to an abnormal stimulus not usually encountered under physiological conditions. Does the cholera stimulus cause a normal mechanism for intestinal fluid secretion to function excessively, or does it change a normal intracellular process in the manner of a toxin or virus, to another end, namely fluid secretion? Florey et al. 121 in 1941 reviewed evidence for a secretory function of the normal small intestine and there is now increasing evidence that fluid and electrolyte secretion may be a normal physiological process in man. (1) Intestinal fluid secretion is well documented in herbivorous animals, such as the guinea pig, 160 where a significant portion of total body water is in the intraluminal compartment. 236 (2) Intestinal secretion is a prominent feature in many different pathological states associated with diarrhea. 19 In association with medullary tumors of the thyroid, excessive prostaglandin release may be the causative factor 237 ; in association with gastrin 238 and nongastrin 239 · 240 secreting adenomas of the pancreas, other humoral factors may cause secretion. Other tumors may exert a trophic influence on the intestinal mucosa. 241 (3) Experimentally, diarrhea associated with net fluid transport into the small bowel lumen may be produced by gastrin, glucagon, and gastric inhibitory peptide in experimental animals and man. H2, 243 The effects of steroid hormones, such as

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estrogens and TSH is directed through the regulation of protein synthesis. They regulate synthesis of all types of ribonucleic acid under different experimental conditions and , may have a direct action at the level of transcription of deoxyribonucleic acid. 244 It is of some interest, therefore, that a protein produced by Clostridium perfringens has the ability to stimulate incorporation of 32P into phospholipid and the formation of intracellular colloid drops in thyroid slices, a combination of metabolic responses specific for THS. 245 The relationship of this to the enterotoxic protein of Clostridia 246 will require further elucidation, but raises the interesting speculation that a bacterial metabolic product, such as enterotoxin, might interact with cellular deoxyribonucleic acid with transcription of m-ribonucleic acid and synthesis of new polypeptide by the intestinal mucosal cells in such a way as to stimulate secretion of fluid and electrolytes. Interrelation of prostaglandins and the cyclic AMP system. Infusion of prostaglandins A, E , and F 2" into the canine superior mesenteric artery induced net secretion of water and electrolytes from the jejunum. 247 The effect of infusion into the jejunal lumen had a lesser effect upon net fluid movement than did prostaglandin infusion into the superior mesenteric artery. The protein content of prostaglandininduced fluid was higher than in cholera fluid, and epithelial cell necrosis of the villus tips occurred with higher rates (8 p,g per min) of infusion. Since prostaglandins have effects on gastrointestinal smooth muscle contractions, 248 splanchnic vascular tone, and blood flow, 249 some of the in vivo effects inay be mediated in this manner. There is, however, evidence that the effect of prostaglandins on fluid and electrolyte movement may occur by direct interaction with the adenyl cyclase system . This interaction can be observed in many tissues such as toad bladder, 250 stomach, 251 and fat cell. 252 Prostaglandin applied to the ileal serosal surface in the in vitro chamber caused changes in sec and anion flux similar to those after application of cyclic AMP. 25 3 Prior addition of theophylline reduced the subsequent sec response to

485

prostaglandin (PG). Kimberg and coworkers1 61 also showed that PG added to intestinal homogenates increased adenyl cyclase activity without affecting phosphodiesterase activity. The increase in activity caused by PG and NaF were similar in normal and cholera-treated mucosa which implied that cholera enterotoxin and prostaglandin probably stimulated the existing or preformed pool of adenyl cyclase rather than causing synthesis of new enzyme. Since the same adenyl cyclase-cyclic AMP mechanism may mediate the effect of cholera enterotoxin and prostaglandins, it has been proposed that cholera toxin may activate the adenyl cyclase system by first stimulating the release or synthesis of prostaglandins. 254 There is considerable evidence in other tissues to support the role of prostaglandins as intracellular mediators. 255 Bennett 254 speculated that aspirin, an inhibitor of prostaglandin synthesis might inhibit cholera-induced fluid secretion if PG had such a role in cholera toxin-mediated secretion. Utilizing the cat, and dosage levels similar to those employed in patients with rheumatoid arthritis, Fink and Katz 159 have recently confirmed this, showing a marked inhibition of cholera toxin-induced secretion after exposure to aspirin. Although aspirin could have exerted a direct effect on adenyl cyclase and phosphodiesterase enzyme activity, their observation may have direct therapeutic importance, as well as implications for defining the role of prostaglandins in the sequence of enzymatic events which develop after cholera toxin exposure to the mucosa.

Some Inhibitors of Enterotoxin-induced Secretion Various agents have been studied for their ability to inhibit enterotoxin-induced fluid secretion . Cholera toxin can be neutralized by immune serum or specific antitoxin antibody but not by control sera. 147 Furthermore, Van Heyningen and co-workers 256 have demonstrated deactivation of cholera toxin by water-soluble lipid components of intestinal mucosa and brain. The capacity

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to deactivate toxin resided primarily in the upper phase of the lipid fraction where gangliosides are known to partition. The toxin was fixed as well as deactivated, precipitating from a mixed solution of ganglioside and choleragen. Minute amounts (30 to 40 ng) of ganglioside were able to deactivate 2 mg of crude cholera toxin when incubated prior to exposure and even when added to the rabbit jejunal loop within 1 min after exposure to the cholera toxin. Ganglioside added to the loop 5 min after toxin exposure either failed to inhibit or was effective only in greatly increased quantities. These studies suggest that cholera toxin interacts with a lipid component of the cell wall which may be ganglioside, in the early phase of activating the mechanism which produces fluid secretion. 208 Another factor in determining the interaction between cholera enterotoxin and intestinal mucosal epithelium has been studied recently. Kunin, 257 Newton, 258 and Koike et a!. 259 have shown that the polypeptide antibiotic polymixin binds tightly to cell membranes and the antibacterial effect of this substance has been shown to be partly dependent on the binding between its free amino groups and phosphalipids of the bacterial cell membrane. Maimon and co-workers 260 have shown that pre-exposure of rabbit jejunal loops with polymixin (1 mM per liter) inhibited cholera enterotoxin-induced secretion by 96%. 'I_'he inhibition was dependent on a dire~t interaction between polymixin and the epithelial brush border without significant damage occurring to the mucosal cell. Polymixin also blocked the effect of E . coli enterotoxin in in vivo experiments. They suggested that polymixin might have a therapeutic role in the management of acute enterotoxigenic diarrhea which would merit further investigation in a suitable animal model. Diamox (20 to 50 mg per kg) was shown to inhibit fluid, sodium , and bicarbonate in in vivo rabbit ileal loop preparations and in augmenting the effects of glucose in restoring net fluid absorption. 150 • 192 • 261 The effect may have been due to inhibition of carbonic anhydrase. No inhibition was ob-

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served in the rat. 15 7 Further investigation of the mode of action of this agent in in vitro systems might be interesting. In chronic canine jejunal loops preexposed to cholera enterotoxin, intraluminal (750 mg) or intravenous (250 or 750 mg) ethacrynic acid administration caused a 50 to 60% reduction in fluid secretion. 262 Furosemide in a concentration which caused similar urinary output had no effect on intestinal secretion. In vitro, ethacrynic acid inhibits active anion secretion in rabbit ileum 263 and it is possible that this may be mediated by direct inhibition of adenyl cyclase . Another recent study 264 has shown that a variety of anti-inflammatory agents are effective in inhibiting fluid secretion in response to choleragen. In addition to confirming Fink and Katz 's 15 9 observation with aspirin, indomethacin, phenylbutazone, salicylate, dexamethasone, prednisone, and ethacrynic acid showed a 30 to 80% inhibition related to dosage. The mechanism of their effects was not defined. Colchicine and serotonin also inhibit cholera toxin-induced fluid production. 265 Role of membrane transport proteins. Inhibition of protein synthesis by cycloheximide pretreatment prevented cholera toxin-induced secretion by in vivo jejunal loops. 1 5 3 • 171 • 265 - 267 It was suggested that synthesis of a protein mediator might be required during the initiation of fluid secretion . Membrane transport proteins have been shown to be an essential feature for transport of organic and inorganic molecules across the bacterial wall.and have, in many instances, been characterized in detail. 268 Specific intestinal electrolyte transport defects in man, such as congenital chloridorrhea, 269 probably represent a congenital defect in such a membrane protein or transport process. For these reasons it is likely that transport proteins may be involved in fluid and electrolyte secretion response to enterotoxin. This will only be clearly defined where individual steps in the secretory process are known. An interesting technique was used to demonstrate that the action of aldosterone on sodium transport in the toad bladder was

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dependent on protein synthesis. 270 Two criteria were used to demonstrate that de novo protein synthesis occurred; (a) the ability of an ribonucleic acid inhibitor to block aldosterone at low concentrations which had no significant effect on the rate of sodium transport in control (steroiddepleted) tissue, and (b) inhibition of the action of aldosterone on sodium transport by a variety of inhibitors each acting on successive steps in the pathway of protein synthesis. A similar approach might be applicable to studies in the intestine.

A Unified Concept of Enterotoxin-induced Fluid Secretion Present knowledge of how enterotoxin causes fluid secretion may be related to two general concepts of fluid secretion. The first proposes that there is a general mucosal secretory process in the majority of cells lining the crypts and villus. In this model, enterotoxin, by stimulating the release of cyclic AMP from adenosine triphosphate 271 after initial access to receptors on the mucosal surface, causes the subsequent event of fluid secretion which may occur either across the mucosal cell membrane or via interepithelial or shunt pathways. Study of transmural unidirectional or bidirectional fluxes in experimental 272 • 273 and human cholera, 273 have demonstrated that during net fluid secretion serosal to mucosal flux (Js > m) for Na and H 20 increased. Similar findings have been made in E. coli diarrhea 96 and experimentally in response to the toxin. 154 Most studies have shown no alteration in mucosal to serosal flux (Jm > s), although in the in vitro chamber 172 • 181 and one human study, 274 studies with crude and purified cholera enterotoxins have demonstrated a reduction in (Jm > s) flux. The limitation of these transmucosal flux measurements for defining motive forces for ion movement is well recognized: active Na transport represents only a small contribution to the total measured unidirectional flux because the bidirectional diffusional component in most tissues is large. 275 In order to gain further understanding, the separate

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flux of ions through extracellular, interepithelial, or shunt pathways, and across mucosal or serosal membranes, respectively, have to be known. 276 Active transcellular sodium absorption from the lumen involves a two-step process: (a) entry across the brush border into the cell down an electrochemical gradient, possibly involving a protein synthetic carrier mechanism in the brush border 275 ; (b) exit of sodium from the cell into the lateral intercellular space through an active transport process which was ouabain-sensitive and dependent on Na +-K + adenosine triphosphatase located in the lateral and serosal borders. 277 Other experiments 265 have indicated that sodium diffusion through shunt pathways normally accounts almost entirely for the unidirectional flux from serosa to mucosa (Js > m). Thus, in the normal intestine, the entire Na flux (Js -. m) appeared to traverse extracellular shunt pathways with little or rio movement directly through epithelial cells, i.e., the active Na pump was completely rectified 278 • 279 and the serosal membrane was impermeable to Na. It was argued that net movement of Na and H 2 0 into the lumen in response to an enterotoxin, such as cholera, must involve either: (1) a change in serosal membrane permeability, (2) an increase in shunt conductance, (3) an increase in transepithelial PD, or (4) a new population of secretory cells. All evidence from studies of permeability of cholera exposed intestine are against a change in membrane permeability 134 • 13 7 • 139 or hydraulic or shunt conductance. 140 An increase in transepi thelia! resistance 17 3 with cholera toxin and cyclic AMP might even indicate a decrease in shunt conductance. An increase in potential differences also occurs after enterotoxin exposure and because of the normal high permeability of this shunt pathway, only a small driving force might be required to cause large ionic shifts through it. Indeed, extracellular volume expansion in dogs resulted in intestinal fluid secretion which may have occurred through such interepithelial shunt pathways. 280 According to the standing gradient hypothesis, 28 1 fluid absorption de-

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pends on active sodium pumping in a "forward" direction from the lateral membrane of the cells into the intercellular spaces. However, in some epithelia, such as the blow-fly rectum, 282 there is a "backward" functioning standing gradient mechanism and it is conceivable that heterogeneity of lateral membrane transport systems in the same mucosa allows for mucosa to serosa transport, as well as active serosa to mucosa movement, to occur via shunt pathways. In favor of this possibility are recent observations 22 7 which demonstrated that adenyl cyclase was not present in the brush border (although others 231 have found it to be present in these regions of the cell and stimulated during protein synthesis), but was mainly located in the basal and lateral margin of the cells. It remains to be proved, however, that there is significant active transport in addition to diffusion via shunt pathways from serosa to mucosa. The demonstration in the short circuited preparation 172 of net S _, M transport of Cl - (and Na +) is further presumptive evidence that there is active S _, M transport in addition to movement by shunt pathways. The second model proposes that secretion of fluid is a specific crypt cell process-a function of a specific area of the small intestine, the villus crypts, and a feature of a separate population of intestinal cells. Evidence for this point of view derives from a variety of observations. ( 1) Dilation of the crypts is a consistent feature of experimental cholera. (2) Undifferentiated crypt cells have morphological characteristics which are compatible with a secretory function. 283 (Undifferentiated crypt cells are rich in enzymes for cell division; microvilli are blunt and sparse and the cytoplasm conta ins abundant ribosomes) . (3) At a dose which only caused loss of mitotic figures from the crypts, cycloheximide inhibited cholera-induced fluid production without impairing glucose absorption in the villus crests. 15 3 ( 4) In the converse experiment, 284 damage to the villus crest by hypertonic sodium sulfate solution impaired glucose absorption without altering the secretory response to chol-

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era toxin. (5) The crypt region has been shown to have distinct metabolic, 285 vascular, 286 and circulatory 287 characteristics different hom the villus crest region. (6) Intraepithelial electrical potential difference was lower ( - 16.1 mv) in cells towards the base of the villus than at the tips ( - 18.7 mv). 288 (7) The crypts fulfill the criteria for a standing gradient system and might be expected to have a secretory role provided the dimension of the channels conform to the predictions of Diamond 281 and the active secretory pump can be located in the basal cells: cholera enterotoxin caused fluid secretion even when intestinal villi have been damaged by prior exposure to hypertonic sodium sulfate. 284 Technical difficulties, however, have so far precluded effective measurement of ionic flux or potential difference in the crypts, and , there is a paucity of information on the special metabolic characteristics of this region. Recent development of techniques 289 to obtain separation of crypt cells from the villus crest may permit experiments to be performed which will better assess which of the two regions, crypt or villus crest, primarily accounts for fluid secretion in response to enterotoxin. Indeed, in view of recent work it is quite possible that cholera toxin inhibits active absorption of N a+ and Cl - by villus cells and stimulates active secretion of anions and, perhaps, even Na+ by crypt cells. 290 REFERE NCES 1. Pollitzer R: Cholera . World Health Organi zat ion. Palais de Nations. Geneva Monogra ph Series. No. 43, 1959, p l - 1019

2. Chambers JS : The Conquest of Cholera -America's Greatest Scourge. New York, Macmillan Co, 1938 3. Koch R: Zwe ite seri e der Conferenzen zur erorterung der cholerafrage . Dtsch Med Wochenschr 11: 329, No. 37 A, 1885 4. Metchnikoff E: Recherches sur le cholera et les vibrions sur l'immuni te et la recept ivite vis-avis du cholera intestin al. Ann Inst P asteur 8:529- 542, 1894 5. Craig JP: Cholera toxins, chap 5, Bac teri a l Protein Toxins, vol 2a. Microbial Toxins. Edited

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by S Kadis, TC Montie, SJ Ajl. New York, Academic Press, 1971, p 189-254 6. De SN, Chatterjee DN: An experimental study of the mechanism of action of vibrio cholerae on the intestinal mucous membrane. J Pathol Bacterial 66:559-562, 1953 7. Dutta NK, Habbu MK: Experimental cholera in infant rabbits: a method for chemotherapeutic investigation. Br J Pharmacal 10:153-159, 1955 8. Smith T: Parasitism and disease. Princeton, Princeton University Press 1934, p 1- 196 9. Taylor J, Maltby MP, Payne JM: Factors influencing the response of ligated rabbit gut segments to injected Escherichia Coli. J Pathol Bacterial 76:491- 499, 1958 10. Pierce NF, Greenough WB III, Carpenter CCJ Jr: Vibrio Cholerae enterotoxin and its mode of action. Bacterial Rev 35:1-13, 1971 11. Carpenter CCJ: Cholera enterotoxin-recent investigations yield insights into transport processes. Am J Med 50:1-7, 1971 12. Greenough WB, Carpenter CCJ, Bayless TM, et al: The role of cholera exotoxin in the study of intestinal water and electrolyte transport, chap 14, Progress in Gastroenterology, vol 2. Edited by GBJ Glass. New York, Grune and Stratton, 1970, p 236-251 13. Hirschorn N, Greenough WB III: Cholera. Sci Am 225:15- 21, 1971 14. Hendrix TR, Ban well JG: Pathogenesis of cholera. Gastroenterology 57:751-755, 1969 15. Hendrix TR: The pathophysiology of cholera. Bull NY Acad Sci 47:1169-1180, 1972 16. Tennant B: Neonatal enteric infections caused by Escherichia Coli. Ann NY Acad Sci 176:1401, 1971 17. Gorbach SL, Levitan R: Intestinal flora in health and in gastrointestinal diseases, chap 15, Progress in Gastroenterology, vol 2. Edited by GBJ Glass. New York, Grune and Stratton, 1970, p 252-275 18. Gorbach SL: Intestinal microflora. Gastroenterology 60:1110- 1129, 1971 19. Phillips SF: Diarrhea: a current view of the pathophysiology. Gastroenterology 63:495-518, 1972 20. Grady GF, Keusch GT: Pathogenesis of bacterial diarrheas . N Eng! J Med 285 :831- 841; 891-900, 1971 21. Fordtran JS: Speculations on the pathogenesis of diarrhea. Gastroenterology 26:1405-1414, 1967 22. Hendrix TR, Bayless TM: Digestion: intestinal

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secretion . Ann Rev Physiol 32:139- 164, 1970 23. Benenson AS , Saad A, Mosley WH , et al: Serological studies in cholera. 3. Serum toxin neutralization -rise in titer in response to infection with Vibrio cholerae and the level in the normal population of East Pakistan. Bull WHO 38:287-295, 1968 24 . Burrows W: Cholera toxins. Ann Rev Microbial 22:245- 268, 1968 25. Pierce NF, Banwell JG, Sack RB, et al: Magnitude and duration of antitoxic response to human infection with Vibrio cholerae. J Infect Dis (in press) 26. Curlin GT, Craig JP, Subong A, et al: Antitoxic immunity m experimental canine cholera. J Infect Dis 121:463- 470, 1970 27 . Northrup RS , Bienenstock J, Tomasi TB: Immunoglobulins and antibody activity m the intestine and serum in cholera. I. Analysis of immunoglobulins in cholera stool. J Infect Dis 121(suppl) :S137- S141 , 1970 28. Fubara ES, Freter R: Source and protective function of coproantibodies in intestinal disease. Am J Clin Nutr 25 :1357- 1363, 1972 29. Lospalluto JJ , Finkelstein RA: Chemical and physical properties of cholera exo-enterotoxin (choleragen and its spontaneous formed toxoid choleragenoid.) Biochim Biophys Acta 257: 158- 166, 1972 30. Finkelstein RA, Lospalluto JJ: Pathogenesis of experimental cholera: preparation and isolation of choleragen and choleragenoid. J Exp Med 130:185- 202, 1969 31. Zinnaka Y, Carpenter CCJ Jr: An enterotoxin produced by non-cholera vibrios. Bull Johns Hopkins Hosp 131:403- 411, 1972 32. Gyles CL, Barnum DA: A heat labile enterotoxin from strains of Escherichia Coli enteropathogenic for pigs. J Infect Dis 120:419- 426, 1969 33. Pierce NF, Wallace CK : Stimulation of jejunal secretion by a crude Escherichia Coli enterotoxin. Gastroenterology 63:439- 448, 1972 34. Bywater RJ: Dialysis and ultrafiltration of a heat stable enterotoxin from Escherichia Coli. J Med Microbial 5:337- 343, 1972 35. Keusch GT, Grady GF, Mata LJ, et al: The pathogenesis of Shigella Diarrhea. I. Enterotoxin production by Shigella dysenteriae 1. J Clin Invest 51:1212-1218, 1972 36. Sullivan R: Effects of Enterotoxin B on intestinal transport in vitro Proc Soc Exp Bioi Med 131:1159-1162, 1969

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37. Sullivan R, Asano T: Effects of staphylococcal enterotoxin B on intestinal transport in the rat. Am J Physiol 220:1793-1797, 1971 38. Duncan CL, Strong DH: Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfrin!fens. J Bacteriol 100:86-94, 1969 39. Kubota Y, Liu PV: An enterotoxin of Pseudomonas aeruginosa . J Infect Dis 123:97- 98, 1971 40. Giannella RA, Formal SB, Dammin GJ, et al: Pathogenesis of Salmonellosis. Studies of fluid secretion, mucosal invasion and morphologic reaction in the rabbit ileum. J Clin Invest 52:441- 462, 1973 41. Peterson JW, Lospalluto JJ, Finkelstein RA: Locali zation of cholera toxin in vivo . J Infect Dis 126:617- 628, 1972 42. Craig JP: A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature (Lond) 207:614- 616, 1965 43. Mosley WH, Aziz KMS, Ahmed A: Serological evidence for the identity of the vascular permeability factor and ileal loop toxin of Vibrio cholerae. J Infect Dis 121:243- 250, 1970 44. Aziz KMS, Mosley WH: Quantitative studies of toxin in the stools and jejunal aspirates of patients with cholera. J Infect Dis 125:36- 44, 1972 45. Mcintyre OR, Feeley JC, Greenough WB, et al: Diarrhea caused by non-cholera vibrios. Am J Trop Med Hyg 14:412-418, 1965 46. Moon HW, Whipp SC , Engstrom GW, et al: Response of the rabbit ileal loop to cell-free products from Eschericia Coli enteropathogenic for swine. J Infect Dis 121:182-187, 1970 47. Sack RB, Gorbach SL, Banwell JG, et al: Enterotoxigenic Escherichia coli isolated from patients with severe cholera-like disease. J Infect Dis 123:378-385, 1971 48. Kohler EM: Observations on enterotoxins produced by enteropathog-enic Escherichia Coli. Ann NY Acad Sci 176:212- 219, 1971 49. Smith HW, Gyles CL: The relationship between two apparently different enterotoxins produced by enteropathogenic strains of Escherichia coli of porcine origin. J Med Microbiol 3:387- 401 , 1970 50. Jacks TM, Wu JB, Braemer AC, eta!: Properties of the enterotoxic component in Escherichia coli enteropathogenic for swine. Infect Immun 7: 178-211, 1973

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51. Richardson SH: Factors influencing in vitro skin permeability factor production by Vibrio cholerae. J Bacterial 100:27-34, 1969 52. Borsoff DA, DasGupta BR: Botulinus toxin , chapt 1, Bacterial Protein Toxins, vol 2a. Microbial Toxins. Edited by S Kadis, TC Montie, SJ Ajl. New York, Academic Press, 1971, p 1- 68 53. Bergdoll SM: Staphylococcal Enterotoxins, Biochemistry of Some Food Borne Microbial Toxins. Edited by RI Mateles, GN Wogan. Cambridge, Mass, MIT Press, 1967, p 1-25 54. Huang IY, Bergdoll MS: The primary structure of staphylococcal enterotoxin B. J Bioi Chern 245: 3493-3510, 3511-3517, 3518- 3525, 1970 55. Surgalla MJ , Dack GM: Enterotoxin produced by micrococci from cases of enteritis after antibiotic therapy. JAMA 158:649- 650, 1955 56 . Shemano I, Hutchens JT, Beiler JM: Paradoxical intestinal inhibitory effects of staphylococcal enterotoxin . Gastroenterology 51:71- 77, 1967 57. Kent TH: Staphylococcal enterotoxin gastroenteritis in Rhesus monkeys. Am J Pathol 48:387- 407, 1966 58. Kocandrle V, Houttuin E, Prohaska JV: Acute Hemodynamic and gastrointestinal changes produced by staphylococcal exotoxin and enterotoxin in dogs. J Surg Res 6:50- 57, 1966 59. Merrill TG, Sprinz H: The effect of staphylococcal enterotoxin on the fine structure of the monkey jejunum. Lab Invest 18:114- 123, 1968 60. Keusch GT, Grady GP, Takeuchi A, et al: The pathogenesis of Shigella diarrhea. II. Enterotoxin induced acute ileitis in the rabbit ileum. J Infect Dis 126:92- 95, 1972 61. Steinberg S, Banwell JG, Keusch GT, et al: The response of the rabbit jejunum to Shigella enterotoxin (abstr). Gastroenterology 62:816, 1972 62. Formal SB, Gemski J, Giannella RA, et al: Mechanisms of Shigella pathogenesis. Am J Clin Nut.r 25:1427- 1432, 1972 63. Hobbs BC, Smith ME, Oakley CL et a!: Clostridium Welcheii food poisoning. J Hyg (Camb) 51:75-101, 1953 64 . Murrell TGC, Roth L, Egerton J, et al: Pig-Bel: Enteritis necroticans. Lancet 1:217-222, 1966 65. Murrell TGC: Pig-bel: epidemic and sporadic necrotizing enteritis in the Highlands of New Guinea. Australas Ann Med 16:4- 10, 1967 66. Maenza RM, Powell OW, Plotkin GR, et al: Experimental diarrhea: salmonella enterocolitis in the rat. J Infect Dis 121:475-485, 1970 67. Powell OW, Plotkin GR, Maenza RM , et al:

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Experimental diarrhea. I. Intestinal water and electrolyte transport in rat salmonella enterocolitis. Gastroenterology 60:1053- 1064, 1971 68 . Powell DW, Plotkin GR, Solberg LI, et al: Experimental diarrhea. II. Glucose-stimulated sodium and water transport in rat salmonella enterocolitis. Gastroenterology 60:1065- 1075, 1971 69. Powell DW, Solberg LI, Plotkin GR, et al: Experimental diarrhea. III. Bicarbonate transport in rat salmonella enterocolitis. Gastroenterology 60:1076-1086, 1971 70. Gangarosa EJ, Beisel WR, Benyajati C, et a!: The nature of the gastrointestinal lesion in Asiatic cholera and its relation to pathogenesis: a biopsy study. Am J Trop Med Hyg 9:125-135, 1960 71. Goodpasture EW: Histopathology of intestine in cholera. Phillipine J Sci 22:413-434, 1923 72. Cohnheim JF: Lectures in general pathology. A handbook for practitioners and students, sect 3: The Pathology of Digestion, vol133.London, New Syndenham Society, 1890, p 949-960 73. Fresh JW, Versage PM, Reyes F: Intestinal morphology in human and experimental cholera. Arch Pathol 77 :529- 537, 1964 74. Elliott HL, Carpenter CCJ Jr, Sack RB , et al : Small bowel morphology in experimental canine cholera. A light and electron microscopic study. Lab Invest 22:112-120, 1970 75. Moon HW, Whipp SC, Baetz AI: Comparative effects of enterotoxins from Escherichia Coli and Vibrio Cholerae on rabbit and swine small intestine. Lab Invest 25:133-140, 1971 76. Staley TE, Wynn Jones E, Corley LD: Attachment and penetration of Escherichia Coli into intestinal epithelium of the ileum in newborn pigs. Am J Pathol 56:371 - 392, 1969 77. Hampton JC, Rosario B: The attachment of micro-organisms to epithelial cells in the distal ileum of the mouse. Lab Invest 14:1464- 1481, 1965 78. Takeuchi A: Electron microscope studies of experimental salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium. Am J Pathol 50:109-136, 1967 79. Takeuchi A, Formal SB, Sprinz H: Experimental acute colitis in the Rhesus monkey following peroral infection with Shigella flexneri. An electron microscope study. Am J Pathol52:503- 529, 1968 80. Watanabe T : Infectious drug resistance in enteric bacteria. N Eng! J Med 275:888-894, 1966 81. Hayes W: Bacterial episomes and plasmids .

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115. Serebro HA, McGonagle T, Iber FL, et a! : An effect of cholera toxin on small intestine without direct mucosal contact. Johns Hopkins Med J 123:229- 232, 1968 116. Panse MV, Dutta NK: Release of histamine by cholera toxin. Arch Int Pharmacodyn Ther 145:479-688, 1963 117. Vaughan WEM, Dohadwalla AN : The appearance of a choleragenic agent in the blood of infant rabbits infected intestinally with Vibrio cholerae demonstrated by cross circulation. J Infect Dis 120:658- 663, 1969 118. Brigham KL, Banwell JG, Pierce NF, et al : Indicator dilution studies in the small bowel of patients with cholera diarrhea. Johns Hopkins Med J 127:97-106, 1970 119. Carpenter CCJ, Monda! A, Sach RB, et a!: Clinical studies in Asiatic cholera. Bull Johns Hopkins Hosp 118:165-245, 1966 120. Yardley JH, Bayless TM, Luebbers EH, et a!: Goblet cell mucus in the small intestine. Findings after net fluid production due to cholera toxin and hypertonic solutions. Johns Hopkins MedJ 131:1-10,1971 121. Florey HW, Wright RD , Jennings MA: The secretions of the intestine. Physiol Rev 21:36- 69, 1941 122. Nalin DR, Aly K, HareR, et al: Effect of cholera toxin on jejunal osmoregulation of mannitol solutions in dogs. J Infect Dis 125:528- 532, 1972 123. Halsted CH, Bright LS, Luebbers EH, et al : A comparison of jejunal response to cholera exotoxin and to hypertonic mannitol. Johns Hopkins Med J 129:179-189, 1971 124. Cash RA, Alam J, Toaha KM: Gastric acid secretion in cholera patients. Lancet 2:1192, 1970 125. Johnson LR, Grossman MI: Intestinal hormones as inhibitors of gastric secretion . Gastroenterology 60:120-144, 1971 126. Schafer DE, Nicoloff DM , Gleason DP, et a!: Gallbladder secretion induced by an enterotoxin-like fraction of crude V. cholerae supernatant. Gastroenterology 56:1195, 1969 127. Lee JS, Silverberg JW: Effect of cholera toxin on fluid absorption and villus lymph pressure in dog jejunal mucosa. Gastroenterology 63:993- 1000, 1972 128. Lee JS, Duncan KM: Lymphatic and venous transport of water frr.m rat jejunum: a vascular perfusion study. Gastroenterology 54:559-567, 1968 129. Dalldorf FG, Keusch GT, Livingston HL: Transcellular permeability of capillaries in experimental cholera . Am J Pathol 57:153-160, 1969 130. Norris HT, Majno G: On the role of the ileal epithelium in the pathogenesis of experimental cholera. Am J Pathol 53:263- 279, 1968 131. Yardley JH: Intestinal microcirculatory changes

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