M. M. Drucker, 1. Goldhar, P. L. Ogra, E. Neter
The Effect of Attapulgite and Charcoal on Enterotoxicity of Vibrio cholerae and Escherichia coli Enterotoxins in Rabbits Summary: Vibrio cholerae and certain strains of Escherichia coli produce heat-labile enterotoxins which play a significant role in the pathogenesis of the intestinal disease. Activated attapulgite, a heated magnesium aluminum silicate, was previously shown to prevent the toxic effects of endotoxin. The present study has revealed that this drug inhibits the toxic effects of cholera and E. coli enterotoxins in the intestinal loop of rabbits, when toxin and attapulgite are pre-incubated prior to injection. Up to 50 to 100 minimal effective doses are inhibited. Attapulgite is effective also when injected separately, albeit simultaneously, into the intestinal loops, but not when administered after the toxin. Since supernates of toxinattapulgite mixtures are non-toxic, it is postulated that attapulgite acts by adsorption and that the attached enterotoxin is no longer toxic to the rabbit intestine. The previously reported effect of charcoal on V. cholerae enterotoxin, paralleling that of attapulgite, was confirmed. In contrast to the effects of these adsorbents on isolated toxin, both failed to prevent enterotoxicity in the rabbit model of an enterotoxin-producing strain of E. coll.
Zusammenfassung: Die Wirkung yon AttapuIgit und KohIe auJ die Enterotoxittit yon Vibrio choleme- und Escherichia eoIi-Enterotoxine bei Kaninchen. Vibrio cholerae und bestimmte Stiimme yon Escherichia coli produzieren ein hitzeempfindliches Enterotoxin, das eine bedeutende Rolle in der Pathogenese der Darmerkrankungen spielt. Aktiviertes Attapulgit, ein erhitztes Magnesium-Aluminium-Silikat, inhibiert die toxische Wirkung des Endotoxins. Mindestens 100 Dosen yon Cholera-Enterotoxin werden neutralisiert. Attapulgit ist ebenso wirksam, wenn es getrennt, jedoch simultan verabreicht wird, jedoch nicht, wenn es 10 Minuten nach dem Toxin injiziert wird. Da die Oberfliiche der Toxin-AttapulgitMischung unschiidlich ist, kann man annehmen, da6 Attapulgit durch Adsorption wirkt und das adsorbierte Enterotoxin nicht mehr toxisch ist. Die bereits friiher bekanntgegebene Wirkung yon aktivierter Kohle auf das V. choleraeEnterotoxin, welche mit der yon Attapulgit parallel geht, wurde bestiitigt. Im Gegensatz zu der Wirkung dieser adsorbierenden Drogen auf isoliertes Toxin waren beide unwirksam gegen einen Enterotoxin-produzierenden Stamm yon E. coti in diesem Modell.
Introduction
fasted for 48 hr prior to surgery; water was given ad lib. The animals were operated under pemothal anaesthesia and infused intravenously with 100-200 ml of physiologic saline solution; 6 to 9 segments of small intestine, measuring approximately 6-12 cm each, were ligated; separated from each other by approximately 3 cm of intestine. Unless stated otherwise, toxin was mixed with equal amounts of saline solution or 10% attapulgite and incubated for 1 hr at room temperature; the mixtures in 2 ml amounts were injected into alternate segments. In additional experiments enterotoxin and drugs were injected simultaneously, albeit separately, into the loops or the adsorbents were injected either 10 min prior to or following the enterotoxin. Further studies were carried out with an enterotoxin-producing strain of E. coli. For each experiment two rabbits were used and the samples injected in reverse order in the second rabbit. Following closure of the abdomen, the animals were kept in a fasting state and were sacrified approximately 18 hr following surgery. In addition to gross inspection, length and volume of the segments were determined. The ratio of volume to length was defined as the index of fluid accumulation (V/L). Figure 1 shows the striking fluid accumulation caused by cholera enterotoxin (A, C, E) and partial (B) as well as complete (D, F) inhibition by attapulgite. E. coli strain H339 (O I5 : K?), isolated from an adult human subject was kindly obtained from Dr. C. Gyles, University of
Intensive research carried out during the past few years has resulted in the identification of heat-labile enterotoxins of Vibrio cholerae and Escherichia coli as the most important factors of pathogenicffy. These enterotoxins are heat-labile, antigenically similar and have a similar or identical mode of action (1, 2, 3, 12). The physiologic alterations caused by these toxins are due to their effect on cyclic A M P (4-6). Some years ago, it was reported from this laboratory that activated attapulgite, a heated magnesium a l u m i n u m silicate, inhibits the lethal effects of endotoxin in mice, presumably by adsorption (7). I n the present investigation it is shown that this material Mso prevents the characteristic intestinal lesions produced by V. choIerae and E. coli ente.rotoxins in the rabbit. This effect parallels that of charcoal as reported several years ago by Finkelstein (8). As shown by De (9), injection of V. cholerae and its filtrates results in fluid accumulation in the ligated intestinal loop of rabbits. This method has been widely used for the study of enterotoxins of this microorganism and of E. coli as well (12). The technique was employed to study the effect of attapulgite on enterotoxins produced by both V. cholerae and E. coll.
Materials and Methods Two V. cholerae preparations and one E. coli enterotoxin preparation were used in the present study. Adult albino New Zealand rabbits, weighing approximately 2-2.5 kg, were
Received: 1 June 1977 Dr. M. M. Drucker, Dr. ar. Goldhar, Dr. P. L. Ogra, Dr. E. Neter, Departments of Pediatrics and Microbiology, State University of New York at Buffalo and Children's Hospital, 219 Bryant Street, Buffalo, New York 14222, USA. Reprints: Dr. E. Neter
Infection 5 (t977) Nr. 4
211
M. M, Drucker, ]. GoIdhar, P. L. Ogra, E. Neter: Enterotoxicity of Vibrio cholerae and Escherichia coli in Rabbits
Figure 1: Fluid accumulation caused by cholera enterotoxin (A, C, E), and partial (B) as well as complete (D, F) inhibition by attapulgite. Guelph, Guelph, Ontario, Canada. The strain produces adequate amounts of heat-labile enterotoxin to yield positive reactions in rabbit loops. The strain was maintained on Kauffmann's egg medium. For the experiment it was grown in brain heart infusion broth for 6 hr at 37 °C and then transferred to trypticase soy broth; the latter was incubated for 18 hr on a rotator at 37 °C; it was used in a dilution of 1:100 in physiologic saline solution for the injection of the intestinal loops.
Results The ileal loop model was used to determine whether the adsorbents attapulgite and charcoal affect the toxicity of V. cholerae and E. coli enterotoxins. The results are summarized in Table 1. It is evident that significant protection was afforded when the drugs were pre-incubated with all three enterotoxins used, two obtained from V. cholerae and one from E. coll. Protection was afforded even when up to 50 to 100 effective doses of V. cholerae toxin were
used. Although attapulgite did not prevent the toxic effects in all animals, it is evident from the data shown on Table 1 that it materially reduced fluid accumulation (V/L) when comparison is made with the appropriate controls. Additional experiments were carried out to determine whether the adsorbents are effective also when injected separately from the toxin. The results are summarized in Table 2. It is evident that both adsorbents are effective when given simultaneously with, albeit separately from, the enterotoxin. They are essentially ineffective when given after toxin injection and of moderate effectiveness when given prior to enterotoxin. Whether the failure of attapulgite and charcoal to protect more effectively when given prior to enterotoxin is due to blockage of the inhibitors by materials in the lumen remains to be determined. A limited number of experiments were carried out with viable E. coil producing heat-labile enterotoxin. When used in a dilution of 1 : 100 in physiologic saline solution it produced positive loops in 7 out of 10 instances; when mixed with attapulgite in 4 out of 6 instances and when mixed with charcoal in 3 out of 6. There were no substantial differences in the indices of fluid accumulation, the figures being 1.3, 1.4, and 1.0, respectively. It is evident that neither attapulgite nor charcoal prevented the effects of enterotoxin under these conditions. To provide evidence that, as postulated, attapulgite and charcoal inhibit the effects of enterotoxin by adsorption, supernates of enterotoxin-adsorbent mixtures were injected into intestinal loops. It was shown that these superhates did not produce fluid accumulation in contrast to the toxin given alone. Thus, it is also evident that the enterotoxin attached to the adsorbent is no longer toxic to the rabbit intestine. It will be of interest to learn from
Table 1 : Effect of attapulgite and charcoal on toxicity of V. cholerae and E. coli enterotoxins Toxin and saline
Enterotoxins (rag per loop)
Mean V/L* Cholera (NIH)
Toxin and attapulgite (1070)
No. of pos. loops/total
1.5-3.0 0,6 0.12-0.3 0.06 0.01-0.03 0.007
2.40 2/2 } 2.51 II/I1 2.58 6/6 2.13 13/13 1.52 17/18 0.280/3
Cholera (Formal)
1.0 0.5 0.1
1.8 1.5 1.6
5/5 7[7 4/4
E. coli
8.0 4.0 2.0 1.0 0.5
1.9 1.7 0.95 0.97 0.84
414 } 6/7 5/9 4/8 4/9
(Gylies)
* Volume (ml)/length (em) of loops
212
Infection 5 (1977) Nr. 4
49/50
} 16/16
15/20
- ~ not done
Mean V/L*
Toxin and charcoal (10 ~)
No. of pos. loops/total
0.93 3/6] 0.84 5/10 0.53 1/8 0.56 1/7 0.33 2/14 0.240/2 }
0.45 0.55 0.32 . .
}
. .
No. of pos. loops/total
0.87 0.67 0.48 0.29 -
3/6} 2/t0 1/6 0/7 -
4/16
-
-
2/20
-
-
12/45
0.8 2/5 0.5 2/7 0.250/4 0/4 2/7 0/9
Mean V/L*
. .
. .
6/29
M. M. Drucker, I. Goldhar, P. L. Ogra, E. Neter: Enterotoxicity of Vibrio cholerae and Escherichia coli in Rabbits Table 2: Effect on toxicity of attapulgite m~d chargoal given prior to, simultaneous with, or after V. Choterae enterotoxin. Drugs Attapulgite
Charcoal
Saline
Injection order
Enterotoxin** simultaneous with adsorbent Enterotoxin followed by adsorbent Absorbent followed by enterotoxin
Mean V/L*
No. of pos. loops/total
Mean V/L*
No. of pos. loops/total
Mean V/L*
No. of pos. loops/total
0.51
2/13 6/8 2/5
0.50 1.60 0.77
0/6 4/5 2/5
1.47
7/7
-
-
-
-
1.56
1.15
* Volume (ml/length (cm) of loops future experiments whether the toxin removed from the adsorbant is as toxic as the original material. Discussion
With the definitive identification of the V. cholerae enterotoxin as the most important determinant in the pathogenesis of cholera and the finding of a similar enterotoxin produced by E. coli, efforts directed against enterotoxicity have come to the fore. Active and/or passive immunization would be in accord with the classic approaches for the prevention of diseases such as diphtheria and tetanus. Local immunity with particular reference to IgA m a y play a major role. If it were possible to prevent the attachment to and entry of the enterotoxin into the intestinal cells by non-specific means, the same aim may be accomplished. Two decades ago it was shown in our laboratory that activated attapulgite prevents toxicity of endotoxin produced by gram--negative bacteria, presumably by adsorption (7). Since the present experiments were initiated, Ericcson et al. (10) presented evidence that pepto-bismol neutralizes enterotoxins. The present study has revealed that attapulgite also prevents the toxicity of V. cholerae and E. coli enterotoxins in the rabbit ileum, provided that attapulgite is injected together with enterotoxin. Since supernates of enterotoxin-attapulgite mixtures do not produce the characteristic fluid accumulation, it is likely that adsorption of the enterotoxin by attapulgite accounts for the finding. In addition, it is of interest to note that the attached enterotoxin is no longer toxic, possibly because it ,does not get to the cell membranes to initiate the pathogenic mechanism. A decade ago, Finkelstein (8) reported similar observations with charcoal, and his findings have been confirmed and extended in the present investigation. It is unlikely that these adsorbents will prove to be effective clinically, since neither prevents enterotoxicity when enterotoxin-producing E. coli cells are injected, as shown here, and since charcoal was reported to be a failure in the treatment of cholera and severe diarrhea (11).
** 0.06 mg of enterotoxin/loop to Dr. Samuel B. Formal, Walter Reed Army Medical Center, Washington, D. C., for crude V. choIerae enterotoxin; to Dr. Robert J. Hosley, Lilly Research Laboratories, Indianapolis, Indiana, for attapulgite; and to Mr. B. J. Viszt for excellent technical assistance. The study was supported by a grant AI 00658 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service.
Literature 1. Finkelstein, R. A.: Cholera enterotoxin. In: MicrobioIogy t975 (Ed.: D. Schlessinger), p. 236-241. American Society for Microbiology, Washington D. C. 1975. 2. Finkelstein, R. A,: Progress in the study of cholera and related enterotoxins. In: Mechanisms in bacterial toxinology (Ed.: A. W. Bernheimer), p. 54-84. John W. Wiley & Sons, New York 1976. 3. Gyles, C. L.: Immunological study of the heat-labile enterotoxins of Escherichia coli and Vibrio choterae. Infect. Immun. 9 (1974) 564-570. 4. Evans, D. 1., Chen, L. C., CurIin, G. T., Evans, D. G.: Stimulation of adenyl cyclase by Escherichia coti enterotoxin. Nature (New Biol.) 236 (1972) 137-138. 5. Guerrant, R. L., Brunton, L. L., Schnaitman, T. C., Rebhun, L. 1., Gilman, A. G.: Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coil. Infect. Immun. 10 (1974) 320-327. 6. HewIett, E. L., Guerrant, R. L., Evans, D. J.: Toxins of V. cholerae and E. coli stimulate adenyl cyclaserin-rat fat cells. Nature 249 (1974) 371-373. 7. Neter, E., Gorzynski, E. A.: The adsorption of enterobacterial endotoxins by activated attapulgite. J. Am. Pharm. Assoc. Scientific Edition, 47 (1958) 651-653. 8. Finkelstein, R. A.: Observations on the nature and mode of action of the choleragenic product(s) of cholera vibrios. In: Proceedings of the cholera research symposium, Hawaii (Ed.: O. A. Bushnell), p. 264-270. U. S. Government Printing Office, Washington 1965. 9. De, S. N., Chatterie, D N.: An experimental study of the mechanism of action of Vibrio c~olerae on the intestinal mucous membrane. J. Path. Bact. 66 (1953) 559-562. 10. Ericcson, C. D., Evans, D. G., DuPont H. L , Pickering, L. K., Evans, D. J.: Neutralization of cholera and E. coli enterotoxin by pepto-bismol (PB). Abstracts, 16th Intersc. Conf. Antimicrob. Agents Chemother., Washington D. C. 1976. 11. Sack, R. B., Cassells, l., Mitra, R., Merritt, C., Butler, T., Acknowledgements Thomas l., Jacob& B., Chaudhuri, A., Mondal, A.: The use The authors are indebted to Dr. Carlton L. Gyles, University of Guelph, Ontario, Canada, for heat-labile enterotoxin prep- of oral replacement soIutions in the treatment of cholera and aration from E. coli strain 7 t l (P307); to the N a t i o n a l other severe diarrhoeaI disorders. Bull. W . H . O . 43 (1970) Institute of Allergy and Infectious Diseases, National Insti- 351-360. 12. Sack, R. B.: Human diarrheal disease caused by enterotutes of Health, Bethesda, Maryland, for cholera toxin Lot toxigenic E. coll. Ann. Rev. Microbiol. 29 (1975) 333-353. No. 002, prepared by Wyeth Laboratories, Inc., Marietta, Pa.;
Infection 5 (1977) Nr. 4
213
The Effect of Attapulgite and Charcoal on Enterotoxicity of Vibrio cholerae and Escherichia coli Enterotoxins in Rabbits Summary: Vibrio cholerae and certain strains of Escherichia coli produce heat-labile enterotoxins which play a significant role in the pathogenesis of the intestinal disease. Activated attapulgite, a heated magnesium aluminum silicate, was previously shown to prevent the toxic effects of endotoxin. The present study has revealed that this drug inhibits the toxic effects of cholera and E. coli enterotoxins in the intestinal loop of rabbits, when toxin and attapulgite are pre-incubated prior to injection. Up to 50 to 100 minimal effective doses are inhibited. Attapulgite is effective also when injected separately, albeit simultaneously, into the intestinal loops, but not when administered after the toxin. Since supernates of toxinattapulgite mixtures are non-toxic, it is postulated that attapulgite acts by adsorption and that the attached enterotoxin is no longer toxic to the rabbit intestine. The previously reported effect of charcoal on V. cholerae enterotoxin, paralleling that of attapulgite, was confirmed. In contrast to the effects of these adsorbents on isolated toxin, both failed to prevent enterotoxicity in the rabbit model of an enterotoxin-producing strain of E. coll.
Zusammenfassung: Die Wirkung yon AttapuIgit und KohIe auJ die Enterotoxittit yon Vibrio choleme- und Escherichia eoIi-Enterotoxine bei Kaninchen. Vibrio cholerae und bestimmte Stiimme yon Escherichia coli produzieren ein hitzeempfindliches Enterotoxin, das eine bedeutende Rolle in der Pathogenese der Darmerkrankungen spielt. Aktiviertes Attapulgit, ein erhitztes Magnesium-Aluminium-Silikat, inhibiert die toxische Wirkung des Endotoxins. Mindestens 100 Dosen yon Cholera-Enterotoxin werden neutralisiert. Attapulgit ist ebenso wirksam, wenn es getrennt, jedoch simultan verabreicht wird, jedoch nicht, wenn es 10 Minuten nach dem Toxin injiziert wird. Da die Oberfliiche der Toxin-AttapulgitMischung unschiidlich ist, kann man annehmen, da6 Attapulgit durch Adsorption wirkt und das adsorbierte Enterotoxin nicht mehr toxisch ist. Die bereits friiher bekanntgegebene Wirkung yon aktivierter Kohle auf das V. choleraeEnterotoxin, welche mit der yon Attapulgit parallel geht, wurde bestiitigt. Im Gegensatz zu der Wirkung dieser adsorbierenden Drogen auf isoliertes Toxin waren beide unwirksam gegen einen Enterotoxin-produzierenden Stamm yon E. coti in diesem Modell.
Introduction
fasted for 48 hr prior to surgery; water was given ad lib. The animals were operated under pemothal anaesthesia and infused intravenously with 100-200 ml of physiologic saline solution; 6 to 9 segments of small intestine, measuring approximately 6-12 cm each, were ligated; separated from each other by approximately 3 cm of intestine. Unless stated otherwise, toxin was mixed with equal amounts of saline solution or 10% attapulgite and incubated for 1 hr at room temperature; the mixtures in 2 ml amounts were injected into alternate segments. In additional experiments enterotoxin and drugs were injected simultaneously, albeit separately, into the loops or the adsorbents were injected either 10 min prior to or following the enterotoxin. Further studies were carried out with an enterotoxin-producing strain of E. coli. For each experiment two rabbits were used and the samples injected in reverse order in the second rabbit. Following closure of the abdomen, the animals were kept in a fasting state and were sacrified approximately 18 hr following surgery. In addition to gross inspection, length and volume of the segments were determined. The ratio of volume to length was defined as the index of fluid accumulation (V/L). Figure 1 shows the striking fluid accumulation caused by cholera enterotoxin (A, C, E) and partial (B) as well as complete (D, F) inhibition by attapulgite. E. coli strain H339 (O I5 : K?), isolated from an adult human subject was kindly obtained from Dr. C. Gyles, University of
Intensive research carried out during the past few years has resulted in the identification of heat-labile enterotoxins of Vibrio cholerae and Escherichia coli as the most important factors of pathogenicffy. These enterotoxins are heat-labile, antigenically similar and have a similar or identical mode of action (1, 2, 3, 12). The physiologic alterations caused by these toxins are due to their effect on cyclic A M P (4-6). Some years ago, it was reported from this laboratory that activated attapulgite, a heated magnesium a l u m i n u m silicate, inhibits the lethal effects of endotoxin in mice, presumably by adsorption (7). I n the present investigation it is shown that this material Mso prevents the characteristic intestinal lesions produced by V. choIerae and E. coli ente.rotoxins in the rabbit. This effect parallels that of charcoal as reported several years ago by Finkelstein (8). As shown by De (9), injection of V. cholerae and its filtrates results in fluid accumulation in the ligated intestinal loop of rabbits. This method has been widely used for the study of enterotoxins of this microorganism and of E. coli as well (12). The technique was employed to study the effect of attapulgite on enterotoxins produced by both V. cholerae and E. coll.
Materials and Methods Two V. cholerae preparations and one E. coli enterotoxin preparation were used in the present study. Adult albino New Zealand rabbits, weighing approximately 2-2.5 kg, were
Received: 1 June 1977 Dr. M. M. Drucker, Dr. ar. Goldhar, Dr. P. L. Ogra, Dr. E. Neter, Departments of Pediatrics and Microbiology, State University of New York at Buffalo and Children's Hospital, 219 Bryant Street, Buffalo, New York 14222, USA. Reprints: Dr. E. Neter
Infection 5 (t977) Nr. 4
211
M. M, Drucker, ]. GoIdhar, P. L. Ogra, E. Neter: Enterotoxicity of Vibrio cholerae and Escherichia coli in Rabbits
Figure 1: Fluid accumulation caused by cholera enterotoxin (A, C, E), and partial (B) as well as complete (D, F) inhibition by attapulgite. Guelph, Guelph, Ontario, Canada. The strain produces adequate amounts of heat-labile enterotoxin to yield positive reactions in rabbit loops. The strain was maintained on Kauffmann's egg medium. For the experiment it was grown in brain heart infusion broth for 6 hr at 37 °C and then transferred to trypticase soy broth; the latter was incubated for 18 hr on a rotator at 37 °C; it was used in a dilution of 1:100 in physiologic saline solution for the injection of the intestinal loops.
Results The ileal loop model was used to determine whether the adsorbents attapulgite and charcoal affect the toxicity of V. cholerae and E. coli enterotoxins. The results are summarized in Table 1. It is evident that significant protection was afforded when the drugs were pre-incubated with all three enterotoxins used, two obtained from V. cholerae and one from E. coll. Protection was afforded even when up to 50 to 100 effective doses of V. cholerae toxin were
used. Although attapulgite did not prevent the toxic effects in all animals, it is evident from the data shown on Table 1 that it materially reduced fluid accumulation (V/L) when comparison is made with the appropriate controls. Additional experiments were carried out to determine whether the adsorbents are effective also when injected separately from the toxin. The results are summarized in Table 2. It is evident that both adsorbents are effective when given simultaneously with, albeit separately from, the enterotoxin. They are essentially ineffective when given after toxin injection and of moderate effectiveness when given prior to enterotoxin. Whether the failure of attapulgite and charcoal to protect more effectively when given prior to enterotoxin is due to blockage of the inhibitors by materials in the lumen remains to be determined. A limited number of experiments were carried out with viable E. coil producing heat-labile enterotoxin. When used in a dilution of 1 : 100 in physiologic saline solution it produced positive loops in 7 out of 10 instances; when mixed with attapulgite in 4 out of 6 instances and when mixed with charcoal in 3 out of 6. There were no substantial differences in the indices of fluid accumulation, the figures being 1.3, 1.4, and 1.0, respectively. It is evident that neither attapulgite nor charcoal prevented the effects of enterotoxin under these conditions. To provide evidence that, as postulated, attapulgite and charcoal inhibit the effects of enterotoxin by adsorption, supernates of enterotoxin-adsorbent mixtures were injected into intestinal loops. It was shown that these superhates did not produce fluid accumulation in contrast to the toxin given alone. Thus, it is also evident that the enterotoxin attached to the adsorbent is no longer toxic to the rabbit intestine. It will be of interest to learn from
Table 1 : Effect of attapulgite and charcoal on toxicity of V. cholerae and E. coli enterotoxins Toxin and saline
Enterotoxins (rag per loop)
Mean V/L* Cholera (NIH)
Toxin and attapulgite (1070)
No. of pos. loops/total
1.5-3.0 0,6 0.12-0.3 0.06 0.01-0.03 0.007
2.40 2/2 } 2.51 II/I1 2.58 6/6 2.13 13/13 1.52 17/18 0.280/3
Cholera (Formal)
1.0 0.5 0.1
1.8 1.5 1.6
5/5 7[7 4/4
E. coli
8.0 4.0 2.0 1.0 0.5
1.9 1.7 0.95 0.97 0.84
414 } 6/7 5/9 4/8 4/9
(Gylies)
* Volume (ml)/length (em) of loops
212
Infection 5 (1977) Nr. 4
49/50
} 16/16
15/20
- ~ not done
Mean V/L*
Toxin and charcoal (10 ~)
No. of pos. loops/total
0.93 3/6] 0.84 5/10 0.53 1/8 0.56 1/7 0.33 2/14 0.240/2 }
0.45 0.55 0.32 . .
}
. .
No. of pos. loops/total
0.87 0.67 0.48 0.29 -
3/6} 2/t0 1/6 0/7 -
4/16
-
-
2/20
-
-
12/45
0.8 2/5 0.5 2/7 0.250/4 0/4 2/7 0/9
Mean V/L*
. .
. .
6/29
M. M. Drucker, I. Goldhar, P. L. Ogra, E. Neter: Enterotoxicity of Vibrio cholerae and Escherichia coli in Rabbits Table 2: Effect on toxicity of attapulgite m~d chargoal given prior to, simultaneous with, or after V. Choterae enterotoxin. Drugs Attapulgite
Charcoal
Saline
Injection order
Enterotoxin** simultaneous with adsorbent Enterotoxin followed by adsorbent Absorbent followed by enterotoxin
Mean V/L*
No. of pos. loops/total
Mean V/L*
No. of pos. loops/total
Mean V/L*
No. of pos. loops/total
0.51
2/13 6/8 2/5
0.50 1.60 0.77
0/6 4/5 2/5
1.47
7/7
-
-
-
-
1.56
1.15
* Volume (ml/length (cm) of loops future experiments whether the toxin removed from the adsorbant is as toxic as the original material. Discussion
With the definitive identification of the V. cholerae enterotoxin as the most important determinant in the pathogenesis of cholera and the finding of a similar enterotoxin produced by E. coli, efforts directed against enterotoxicity have come to the fore. Active and/or passive immunization would be in accord with the classic approaches for the prevention of diseases such as diphtheria and tetanus. Local immunity with particular reference to IgA m a y play a major role. If it were possible to prevent the attachment to and entry of the enterotoxin into the intestinal cells by non-specific means, the same aim may be accomplished. Two decades ago it was shown in our laboratory that activated attapulgite prevents toxicity of endotoxin produced by gram--negative bacteria, presumably by adsorption (7). Since the present experiments were initiated, Ericcson et al. (10) presented evidence that pepto-bismol neutralizes enterotoxins. The present study has revealed that attapulgite also prevents the toxicity of V. cholerae and E. coli enterotoxins in the rabbit ileum, provided that attapulgite is injected together with enterotoxin. Since supernates of enterotoxin-attapulgite mixtures do not produce the characteristic fluid accumulation, it is likely that adsorption of the enterotoxin by attapulgite accounts for the finding. In addition, it is of interest to note that the attached enterotoxin is no longer toxic, possibly because it ,does not get to the cell membranes to initiate the pathogenic mechanism. A decade ago, Finkelstein (8) reported similar observations with charcoal, and his findings have been confirmed and extended in the present investigation. It is unlikely that these adsorbents will prove to be effective clinically, since neither prevents enterotoxicity when enterotoxin-producing E. coli cells are injected, as shown here, and since charcoal was reported to be a failure in the treatment of cholera and severe diarrhea (11).
** 0.06 mg of enterotoxin/loop to Dr. Samuel B. Formal, Walter Reed Army Medical Center, Washington, D. C., for crude V. choIerae enterotoxin; to Dr. Robert J. Hosley, Lilly Research Laboratories, Indianapolis, Indiana, for attapulgite; and to Mr. B. J. Viszt for excellent technical assistance. The study was supported by a grant AI 00658 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service.
Literature 1. Finkelstein, R. A.: Cholera enterotoxin. In: MicrobioIogy t975 (Ed.: D. Schlessinger), p. 236-241. American Society for Microbiology, Washington D. C. 1975. 2. Finkelstein, R. A,: Progress in the study of cholera and related enterotoxins. In: Mechanisms in bacterial toxinology (Ed.: A. W. Bernheimer), p. 54-84. John W. Wiley & Sons, New York 1976. 3. Gyles, C. L.: Immunological study of the heat-labile enterotoxins of Escherichia coli and Vibrio choterae. Infect. Immun. 9 (1974) 564-570. 4. Evans, D. 1., Chen, L. C., CurIin, G. T., Evans, D. G.: Stimulation of adenyl cyclase by Escherichia coti enterotoxin. Nature (New Biol.) 236 (1972) 137-138. 5. Guerrant, R. L., Brunton, L. L., Schnaitman, T. C., Rebhun, L. 1., Gilman, A. G.: Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coil. Infect. Immun. 10 (1974) 320-327. 6. HewIett, E. L., Guerrant, R. L., Evans, D. J.: Toxins of V. cholerae and E. coli stimulate adenyl cyclaserin-rat fat cells. Nature 249 (1974) 371-373. 7. Neter, E., Gorzynski, E. A.: The adsorption of enterobacterial endotoxins by activated attapulgite. J. Am. Pharm. Assoc. Scientific Edition, 47 (1958) 651-653. 8. Finkelstein, R. A.: Observations on the nature and mode of action of the choleragenic product(s) of cholera vibrios. In: Proceedings of the cholera research symposium, Hawaii (Ed.: O. A. Bushnell), p. 264-270. U. S. Government Printing Office, Washington 1965. 9. De, S. N., Chatterie, D N.: An experimental study of the mechanism of action of Vibrio c~olerae on the intestinal mucous membrane. J. Path. Bact. 66 (1953) 559-562. 10. Ericcson, C. D., Evans, D. G., DuPont H. L , Pickering, L. K., Evans, D. J.: Neutralization of cholera and E. coli enterotoxin by pepto-bismol (PB). Abstracts, 16th Intersc. Conf. Antimicrob. Agents Chemother., Washington D. C. 1976. 11. Sack, R. B., Cassells, l., Mitra, R., Merritt, C., Butler, T., Acknowledgements Thomas l., Jacob& B., Chaudhuri, A., Mondal, A.: The use The authors are indebted to Dr. Carlton L. Gyles, University of Guelph, Ontario, Canada, for heat-labile enterotoxin prep- of oral replacement soIutions in the treatment of cholera and aration from E. coli strain 7 t l (P307); to the N a t i o n a l other severe diarrhoeaI disorders. Bull. W . H . O . 43 (1970) Institute of Allergy and Infectious Diseases, National Insti- 351-360. 12. Sack, R. B.: Human diarrheal disease caused by enterotutes of Health, Bethesda, Maryland, for cholera toxin Lot toxigenic E. coll. Ann. Rev. Microbiol. 29 (1975) 333-353. No. 002, prepared by Wyeth Laboratories, Inc., Marietta, Pa.;
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