337
Preliminary Communications HEALING OF GASTRIC ULCER DURING TREATMENT WITH CIMETIDINE R. E. POUNDER MARTA STEKELMAN
J. J.
R. H. HUNT G. J. MILTON-THOMPSON MISIEWICZ
Medical Research Council Gastroenterology Unit, Central Middlesex Hospital, London, and Royal Naval Hospital,
Haslar, Hampshire with benign gastric ulcer treated with cimetidine 0·8 or 1·6 g/day for six weeks. Relief of symptoms was rapid. Endoscopy at the end of treatment showed that all the ulcers had healed. Healing was not associated with an improvement of acute atrophic gastritis nor with any change of gastric mucosal potential difference. No untoward clinical or laboratory effects were observed.
Summary
10
patients
were
INTRODUCTION
WEEK OF TRIAL
histamine H2-receptor antagonist, has been shown to be better than placebo for the relief of symptoms1 and the healing of duodenal ulcer,2 but the effect of H2-receptor blockade on the healing of gastric ulcer (G.u.) has not been reported. Cimetidine is a nonthiourea H2-receptor antagonist which decreases all forms of stimulated gastric-acid secretion in man.3-7 We describe the treatment of 10 patients with benign G.u. using oral cimetidine 0.88 or 1 - 6 g daily- for six weeks.
METIAMIDE,
a
of daytime and nocturnal ulcer treated with cimetidine for 6 weeks.
Frequency
in 10
patients with G.U.
visit specific questions were asked about symptoms and possible adverse reactions to the drug, and laboratory safety tests were performed. Each morning and evening the patients completed a diary card, recording daytime and nocturnal ulcer
pain as none, mild, or severe. A second endoscopy was performed
at the end of six weeks of treatment and the result was recorded as ulcer healed or unhealed. Healing of the G.u. was defined as complete epithelialisation of the ulcer site. Additional investigations were performed in five of the patients receiving 0.8 g of cimetidine/day. Biopsy specimens of the antral and greater-curve mucosa were taken at both endoscopies. The severity of acute atrophic gastritis was assessed in haematoxylin and eosin stained sections according to established criteria8 by a histopathologist who examined them in random sequence, unaware of the timing of the biopsy in relation to treatment. Gastric mucosal potential difference (P.D.) and pentagastrin-stimulated acid output were measured in a combined procedure on the morning before cimetidine was started and again six weeks later, fifteen hours after the last dose of the drug. After an overnight fast the tip of a doublelumen nasogastric tube (Salem Sump, 10 FG) was positioned under radiological control in the most dependent part of the stomach. After collecting basal acid secretion for sixty min, 100 ml of 150 mmol/1 sodium chloride was placed in the stomach. The gastric mucosal P.D.9 was measured at one-min intervals of forty-five min using the saline-perfused smaller lumen of the nasogastric tube as the probe electrode. The reference electrode was a saline-perfused cannula placed in a
PATIENTS AND METHODS
The study was performed concurrently at the two hospitals, using an identical protocol. 11 consecutive patients, who were
suitable for medical management of their G.u., were selected for cimetidine therapy. The benign nature of the G.u. was confirmed by multiple direct-vision biopsies of the ulcer edge and floor and by brush cytology in 10 of the 11 patients: 1 patient was withdrawn from the study as these tests suggested malignancy. The 10 were treated as outpatients and for six weeks received cimetidine capsules 200 mg four times a day (6 patients) or 400 mg four times a day (4 patients) taken immediately after the three main meals and at bedtime. All other drugs were discontinued, but a supply of 50 antacid tablets (unmarked ’Rennies’, Nicholas Research Laboratories) was provided each week. No specific dietary advice was given, but the patients were advised to avoid food that upset them. They
told that a new treatment was being tested and the procedures involved were explained: all gave their informed consent to the study which was approved by the appropriate ethical committees. The patients were examined at weekly intervals. At each were
TABLE I--CLINICAL DATA ON
pain
10*
PATIENTS WITH BENIGN G.U.
338 TABLE I!—ANTACID CONSUMPTION PER PATIENT
B.A.o.=basal acid output. M.A.o.=maximum acid output
(pentagastrin stimulated).
forearm ’vein. Both electrodes were connected to syringes, sealed with saturated potassium chloride in 3% agar, containing balanced calomel electrodes (Radiometer, K401) in saturated potassium chloride; the P.D. was measured using a battery-powered millivoltmeter (Searle, High Wycombe) to the nearest 1 mV. At the end Of P.D. measurement the stomach was emptied and a routine pentagastrin (6 Ilg/kg subcutaneously) was performed. Student’s t test for paired data was used for statistical comparisons. RESULTS
10 of the 11
patients (table I) completed
the six weeks of treatment with cimetidine and in all ten the G.u. was healed at the second endoscopy. Treatment with cimetidine was associated with a rapid and striking relief of symptoms (see accompanying figure) and with a decreasing consumption of antacid (table II). 1 patient with an apparently benign G.U. was started on cimetidine, but the histopathological and cytological appearances aroused suspicions of malignancy. His symptoms persisted, and at a repeat endoscopy after two weeks of treatment the ulcer was still present and the pathological appearances unchanged. Examination of the specimen removed at partial gastrectomy one week later showed intramucosal carcinoma with ulceration. In the 5 patients studied in more detail, the severity of acute atrophic gastritis was unaffected by the treatment with cimetidine. Fifteen hours after the last dose of cimetidine there was a small, but significant decrease of mean basal and pentagastrin-stimulated acid secretion, but there was no significant change in the mean gastric mucosal P.D. (table III). No unwanted clinical or laboratory effects of cimetidine were recorded in any patient during the trial. DISCUSSION
This uncontrolled pilot study was performed to provide clinical experience in the use of cimetidine for G.u. and evidence of safety before starting double-blind controlled trials. The complete healing of G.u. in all patients, the rapid and striking relief of symptoms, and the absence of unwanted effects are all encouraging, but these results must be regarded with caution in view of the small numbers and the tendency of G.u. to heal spon-
taneously.
In most previous trials the healing of G.u. was judged radiologically by the disappearance of the ulcer niche, Studies in 122 patients receiving a placebo for threeeight weeks showed complete ulcer healing in 5-50% of the patients. 10-16 Studies in 253 patients treated with various drugs other than H2-receptor antagonists for three-eight weeks showed complete healing in 14-70%.10-’s A study of inpatient intensive medical therapy reported that in 638 G.u. patients, complete healing as determined by X-ray occurred in 46.1% at at six weeks, and 75.9% at twelve trial where both radiology and endoscopy were used, assessment by endoscopy increased the proportion of patients deemed to have healed G.u. from 70-90% in the active group and from 20-30% in the placebo group.’6 The 100% healing-rate with cimetidine in outpatients compares well with all previous trials, but without controls a firm conclusion regarding its efficacy cannot be reached at present. The clinical course of the patient with cancer suggests that when symptoms of G.u. do not respond to Hfreceptor blockade, malignancy should be suspected. However the availability of effective symptomatic treatment must not replace careful histological and cytological examination of every G.u. to exclude possible malignancy. Despite the 100% healing-rate, cimetidine at a dose level of 0.88 g/day for six weeks had no effect on the state of the gastric mucosa as recorded in this investigation. The atrophic gastritis did not improve and the P.D. did not increase. This contrasts with the reported rise of P.D. in response to intravenous cimetidine in healthy subects,* suggesting that any effect on P.D. may be transient. Moreover, the slight decrease in acid output recorded in our 5 patients tested before and after treatment does not suggest improvement in the secretory capacity of, or decreased back-diffusion through, the gastric mucosa. The G.u. patients had their acid secretion measured 15 h after the last dose of cimetidine when blood levels of the drug must have been very 10w.21As acid secretion in patients with G.u. is easily inhibited by H2-receptor blockade,22 it is possible that the smaller acid output was due to persistence of some cimetidine in the circulation. The only well-documented pharmacological effects of " H2-receptor blockade in man are inhibition of acid3-’
three
weeks, 70.4%
weeks.19 In
a
339
pepsin3 2= secretion. Previous therapeutic trials in patients with duodenal ulcer’together with the present and
results suggest that acid and pepsin secretion are important in the maintenance of established gastric or duodenal ulceration. This pilot study should encourage controlled trials of cimetidine in G.u. We thank our colleagues for referring patients and Dr D. Lovell for examing the biopsy material. Cimetidine was generously provided by
South, Kline and French Laboratories. The millivoltmeter by G. D. Searle & Co. Ltd.
was
loaned
Requests for reprints should be addressed to R. E. P., M.R.C. Gastroenterology Unit, Central Middlesex Hospital, London, NW10 7NS. REFERENCES
1. Pounder, R. E., Williams, J. G., Milton-Thompson, J. G., Misiewicz, J. J. Br. med. J. 1975. ii. 307. 2. A. Multicentre Trial. Lancet, 1975, ii, 779. 3. Burland, W. L., Duncan, W. A. M., Hesselbo, T., Mills, J. C., Sharpe, P. C., Haggle, S. J., Wyllie, J. H. Br. J. clin. Pharmac. 1975, 2, 481. 4. Pounder, R. E., Williams, J. G., Russell, R. C. G., Milton-Thompson, G. J., Misiewicz, J. J. Gut, 1975, 16, 397. 5. Richardson, C. T., Fordtran, J. S. Gastroenterology, 1975, 68, 972. 6. Henn, R. M., Isenberg, J. I., Maxwell, V., Sturdevant, R. A. L. New Engl.
J. Med. 1975, 293, 371. 7. Carter, D. C., Forrest, J. A. H., Logan, R., Ansell, I., Lidgard, G., Heading, R. C., Shearman, D. J. C. I. R. C. S. 1975, 3, 377. 8. Whitehead, R., Truelove, S. C., Gear, M. W. L. J. clin. Path. 1972, 25. 9. Geall, M. G., Code, C. F., McIlrath, D. C., Summerskill, W. H. J. Gut,
1970, 11, 34. 10. Doll, R., Hill, I. D., Hutton. C., Underwood. D. I. Lancet, 1962, ii, 793. 11. Doll, R., Hill, I. D., Hutton, C. Gut, 1965, 6, 19. 12. Doll, R., Langman, M. J. S., Shawdon, H. S. ibid. 1968, 9, 46. 13. Turpie, A. G. G., Runcie, J., Thompson, T. J. ibid. 1969, 10, 299. 14. Black, R. B., Rhodes, J., Davies, G. T., Gravelle, H., Sweetnam, P. Gastroenterology, 1971, 61, 821. 15. Baume, P. E., Hunt, J. H., Piper, D. W. ibid. 1972, 63, 399. 16. Boyes, B. E., Woolf, I. L., Wilson, R. Y., Cowley, D. J., Dymock, I. W. Postgrad. med. J. 1975, 51, suppl. 5, 29. 17. Fraser, P. M., Doll, R., Langman, M. J. S., Misiewicz, J. J., Shawdon, H. S. Gut, 1972. 13, 459. 18. Langman, M. J. S., Knapp, D. R., Wakley, E. J. Br. med. J. 1973, iii, 84. 19. Veterans Administration Co-operative Study on Gastric Ulcer. Gastroenterology, 1971, 61, 567. 20. Ivey, K. J., Baskin, W., Jeffrey, G. Lancet, 1975, ii, 1072. 21. Pounder, R. E., Williams, J. G., Milton-Thompson, G. J., Misiewicz, J. J. ibid. p. 1069. 22. Thjodleiffson, B., Wormsley, K. G. Gut, 1975, 16, 501.
NON-SPECIFIC CYTOTOXICITY OF WHEAT GLIADIN COMPONENTS TOWARDS CULTURED HUMAN CELLS D. A. HUDSON D. R. PURDHAM
J. CORNELL* C. J. ROLLES
H.
INTRODUCTION
WHEAT gluten and its ethanol-soluble portion, gliadin, produce intestinal mucosal damage in individuals with coeliac disease. Partial digestion of either of these substances in vitro by some proteolytic enzymes under certain conditions does not destroy this toxicity, though not all of the products seem to be active in this respect.1 Intestinal mucosal biopsy specimens obtained from patients with coeliac disease that are cultured in the presence of such digestion products have altered morphology and enzyme levels.2-4 We report observations which demonstrate that a particular chromatographic fraction of a gliadin digest is toxic under these conditions, not only to intestinal mucosal tissue, but to various other cell types. METHODS
hydrolysate fractions.-The methods of preparing gliadin from gluten and its subsequent digestion by proteolytic enzymes have been described elsewhere.5 Separation of the hydrolysate into 10 major fractions was achieved by Gliadin
on SP-’Sephadex’ C-2S (Pharmacia). Each of these fractions was either lyophilised immediately or desalted on a column of ’Bio-gel’ P-2 (Bio-Rad. Laboratories, 200-400 mesh). The fractions obtained from three such separations were pooled for the present experiments. For comparison the effects of &bgr;-Iactoglobulin, casein, ovalbumin, and the plant lectin, conconavalin A, were investigated in some experiments. Tissue-culture methods.-Human embryonic lung, intestine, adrenal, kidney, and HEp-2 (human carcinoma of the larynx) cells were prepared as primary monolayer cultures by standard procedures.6 The cell maintenance medium was type 199 supplemented with 2 % fetal calf serum (Gibco/Bio-Cult) and for cell growth at 37°C the same medium containing 10 % serum. Toxicity testing.-All tests were performed blind. The cell maintenance medium was discarded and the cell monolayers washed three times in Dulbecco A phosphate-buffered saline. The cells were then exposed to the various chromatographic fractions which were dissolved in serum-free medium 199, the pH of which was adjusted back to normal if necessary. All tests were performed in duplicate on each occasion. Unless otherwise stated, observations were made at 24 h and fractions were present at an initial concentration of 500 (Jt.g/ml. Initially, 1 x 10 cm tubes were used for both culturing and the toxicity assays, but this was later superseded by a microtitre tissue-culture system. Toxicity scoring.-This was based upon microscopic appearances of the cells usually at a magnification of x 100, thus: + occasional "holes" in an otherwise confluent clear evidence of disruption of sheet monolayer; + + integrity; + + + = pronounced disruption with cell aggretotal disruption with the monolayer gation ; + + + + sloughing from the growth supporting surface.
chromatography
=
Institute
of Child Health; and
Virology Department,
Children’s Hospital, Birmingham
=
=
Summary
Chromatographically separated fractions of a proteolytic digest of wheat were gliadin assayed for cytotoxic properties using cultured human embryonic intestinal, lung, kidney, adrenal, and HEp-2 cells. In all cell types noxious effects were observed microscopically over a 24 h period. The most active fraction was that previously shown to produce xylose malabsorption in subjects with cœliac disease, disruption of lysosomes, and inhibition of morphological recovery of cultured mucosa from a patient on a gluten-free diet. *Present address: Royal Children’s
Hospital, Melbourne, Australia.
RESULTS
The method used here for the separation of the products of gliadin proteolysis produces 10 fractions. Observations on the cytotoxic properties of each of these were made with intestinal mucosal cells only. All but two of the 10 fractions induced signs of toxicity. Fraction 9 was the most active, with fraction 5 being only slightly less so. The presence of serum inhibited the induction of cytotoxicity. These results were obtained with three separate specimens of intestinal tissue. Further tests involved the use of chromatographic fractions 1 (non-toxic to intestinal cells) and 9.
Preliminary Communications HEALING OF GASTRIC ULCER DURING TREATMENT WITH CIMETIDINE R. E. POUNDER MARTA STEKELMAN
J. J.
R. H. HUNT G. J. MILTON-THOMPSON MISIEWICZ
Medical Research Council Gastroenterology Unit, Central Middlesex Hospital, London, and Royal Naval Hospital,
Haslar, Hampshire with benign gastric ulcer treated with cimetidine 0·8 or 1·6 g/day for six weeks. Relief of symptoms was rapid. Endoscopy at the end of treatment showed that all the ulcers had healed. Healing was not associated with an improvement of acute atrophic gastritis nor with any change of gastric mucosal potential difference. No untoward clinical or laboratory effects were observed.
Summary
10
patients
were
INTRODUCTION
WEEK OF TRIAL
histamine H2-receptor antagonist, has been shown to be better than placebo for the relief of symptoms1 and the healing of duodenal ulcer,2 but the effect of H2-receptor blockade on the healing of gastric ulcer (G.u.) has not been reported. Cimetidine is a nonthiourea H2-receptor antagonist which decreases all forms of stimulated gastric-acid secretion in man.3-7 We describe the treatment of 10 patients with benign G.u. using oral cimetidine 0.88 or 1 - 6 g daily- for six weeks.
METIAMIDE,
a
of daytime and nocturnal ulcer treated with cimetidine for 6 weeks.
Frequency
in 10
patients with G.U.
visit specific questions were asked about symptoms and possible adverse reactions to the drug, and laboratory safety tests were performed. Each morning and evening the patients completed a diary card, recording daytime and nocturnal ulcer
pain as none, mild, or severe. A second endoscopy was performed
at the end of six weeks of treatment and the result was recorded as ulcer healed or unhealed. Healing of the G.u. was defined as complete epithelialisation of the ulcer site. Additional investigations were performed in five of the patients receiving 0.8 g of cimetidine/day. Biopsy specimens of the antral and greater-curve mucosa were taken at both endoscopies. The severity of acute atrophic gastritis was assessed in haematoxylin and eosin stained sections according to established criteria8 by a histopathologist who examined them in random sequence, unaware of the timing of the biopsy in relation to treatment. Gastric mucosal potential difference (P.D.) and pentagastrin-stimulated acid output were measured in a combined procedure on the morning before cimetidine was started and again six weeks later, fifteen hours after the last dose of the drug. After an overnight fast the tip of a doublelumen nasogastric tube (Salem Sump, 10 FG) was positioned under radiological control in the most dependent part of the stomach. After collecting basal acid secretion for sixty min, 100 ml of 150 mmol/1 sodium chloride was placed in the stomach. The gastric mucosal P.D.9 was measured at one-min intervals of forty-five min using the saline-perfused smaller lumen of the nasogastric tube as the probe electrode. The reference electrode was a saline-perfused cannula placed in a
PATIENTS AND METHODS
The study was performed concurrently at the two hospitals, using an identical protocol. 11 consecutive patients, who were
suitable for medical management of their G.u., were selected for cimetidine therapy. The benign nature of the G.u. was confirmed by multiple direct-vision biopsies of the ulcer edge and floor and by brush cytology in 10 of the 11 patients: 1 patient was withdrawn from the study as these tests suggested malignancy. The 10 were treated as outpatients and for six weeks received cimetidine capsules 200 mg four times a day (6 patients) or 400 mg four times a day (4 patients) taken immediately after the three main meals and at bedtime. All other drugs were discontinued, but a supply of 50 antacid tablets (unmarked ’Rennies’, Nicholas Research Laboratories) was provided each week. No specific dietary advice was given, but the patients were advised to avoid food that upset them. They
told that a new treatment was being tested and the procedures involved were explained: all gave their informed consent to the study which was approved by the appropriate ethical committees. The patients were examined at weekly intervals. At each were
TABLE I--CLINICAL DATA ON
pain
10*
PATIENTS WITH BENIGN G.U.
338 TABLE I!—ANTACID CONSUMPTION PER PATIENT
B.A.o.=basal acid output. M.A.o.=maximum acid output
(pentagastrin stimulated).
forearm ’vein. Both electrodes were connected to syringes, sealed with saturated potassium chloride in 3% agar, containing balanced calomel electrodes (Radiometer, K401) in saturated potassium chloride; the P.D. was measured using a battery-powered millivoltmeter (Searle, High Wycombe) to the nearest 1 mV. At the end Of P.D. measurement the stomach was emptied and a routine pentagastrin (6 Ilg/kg subcutaneously) was performed. Student’s t test for paired data was used for statistical comparisons. RESULTS
10 of the 11
patients (table I) completed
the six weeks of treatment with cimetidine and in all ten the G.u. was healed at the second endoscopy. Treatment with cimetidine was associated with a rapid and striking relief of symptoms (see accompanying figure) and with a decreasing consumption of antacid (table II). 1 patient with an apparently benign G.U. was started on cimetidine, but the histopathological and cytological appearances aroused suspicions of malignancy. His symptoms persisted, and at a repeat endoscopy after two weeks of treatment the ulcer was still present and the pathological appearances unchanged. Examination of the specimen removed at partial gastrectomy one week later showed intramucosal carcinoma with ulceration. In the 5 patients studied in more detail, the severity of acute atrophic gastritis was unaffected by the treatment with cimetidine. Fifteen hours after the last dose of cimetidine there was a small, but significant decrease of mean basal and pentagastrin-stimulated acid secretion, but there was no significant change in the mean gastric mucosal P.D. (table III). No unwanted clinical or laboratory effects of cimetidine were recorded in any patient during the trial. DISCUSSION
This uncontrolled pilot study was performed to provide clinical experience in the use of cimetidine for G.u. and evidence of safety before starting double-blind controlled trials. The complete healing of G.u. in all patients, the rapid and striking relief of symptoms, and the absence of unwanted effects are all encouraging, but these results must be regarded with caution in view of the small numbers and the tendency of G.u. to heal spon-
taneously.
In most previous trials the healing of G.u. was judged radiologically by the disappearance of the ulcer niche, Studies in 122 patients receiving a placebo for threeeight weeks showed complete ulcer healing in 5-50% of the patients. 10-16 Studies in 253 patients treated with various drugs other than H2-receptor antagonists for three-eight weeks showed complete healing in 14-70%.10-’s A study of inpatient intensive medical therapy reported that in 638 G.u. patients, complete healing as determined by X-ray occurred in 46.1% at at six weeks, and 75.9% at twelve trial where both radiology and endoscopy were used, assessment by endoscopy increased the proportion of patients deemed to have healed G.u. from 70-90% in the active group and from 20-30% in the placebo group.’6 The 100% healing-rate with cimetidine in outpatients compares well with all previous trials, but without controls a firm conclusion regarding its efficacy cannot be reached at present. The clinical course of the patient with cancer suggests that when symptoms of G.u. do not respond to Hfreceptor blockade, malignancy should be suspected. However the availability of effective symptomatic treatment must not replace careful histological and cytological examination of every G.u. to exclude possible malignancy. Despite the 100% healing-rate, cimetidine at a dose level of 0.88 g/day for six weeks had no effect on the state of the gastric mucosa as recorded in this investigation. The atrophic gastritis did not improve and the P.D. did not increase. This contrasts with the reported rise of P.D. in response to intravenous cimetidine in healthy subects,* suggesting that any effect on P.D. may be transient. Moreover, the slight decrease in acid output recorded in our 5 patients tested before and after treatment does not suggest improvement in the secretory capacity of, or decreased back-diffusion through, the gastric mucosa. The G.u. patients had their acid secretion measured 15 h after the last dose of cimetidine when blood levels of the drug must have been very 10w.21As acid secretion in patients with G.u. is easily inhibited by H2-receptor blockade,22 it is possible that the smaller acid output was due to persistence of some cimetidine in the circulation. The only well-documented pharmacological effects of " H2-receptor blockade in man are inhibition of acid3-’
three
weeks, 70.4%
weeks.19 In
a
339
pepsin3 2= secretion. Previous therapeutic trials in patients with duodenal ulcer’together with the present and
results suggest that acid and pepsin secretion are important in the maintenance of established gastric or duodenal ulceration. This pilot study should encourage controlled trials of cimetidine in G.u. We thank our colleagues for referring patients and Dr D. Lovell for examing the biopsy material. Cimetidine was generously provided by
South, Kline and French Laboratories. The millivoltmeter by G. D. Searle & Co. Ltd.
was
loaned
Requests for reprints should be addressed to R. E. P., M.R.C. Gastroenterology Unit, Central Middlesex Hospital, London, NW10 7NS. REFERENCES
1. Pounder, R. E., Williams, J. G., Milton-Thompson, J. G., Misiewicz, J. J. Br. med. J. 1975. ii. 307. 2. A. Multicentre Trial. Lancet, 1975, ii, 779. 3. Burland, W. L., Duncan, W. A. M., Hesselbo, T., Mills, J. C., Sharpe, P. C., Haggle, S. J., Wyllie, J. H. Br. J. clin. Pharmac. 1975, 2, 481. 4. Pounder, R. E., Williams, J. G., Russell, R. C. G., Milton-Thompson, G. J., Misiewicz, J. J. Gut, 1975, 16, 397. 5. Richardson, C. T., Fordtran, J. S. Gastroenterology, 1975, 68, 972. 6. Henn, R. M., Isenberg, J. I., Maxwell, V., Sturdevant, R. A. L. New Engl.
J. Med. 1975, 293, 371. 7. Carter, D. C., Forrest, J. A. H., Logan, R., Ansell, I., Lidgard, G., Heading, R. C., Shearman, D. J. C. I. R. C. S. 1975, 3, 377. 8. Whitehead, R., Truelove, S. C., Gear, M. W. L. J. clin. Path. 1972, 25. 9. Geall, M. G., Code, C. F., McIlrath, D. C., Summerskill, W. H. J. Gut,
1970, 11, 34. 10. Doll, R., Hill, I. D., Hutton. C., Underwood. D. I. Lancet, 1962, ii, 793. 11. Doll, R., Hill, I. D., Hutton, C. Gut, 1965, 6, 19. 12. Doll, R., Langman, M. J. S., Shawdon, H. S. ibid. 1968, 9, 46. 13. Turpie, A. G. G., Runcie, J., Thompson, T. J. ibid. 1969, 10, 299. 14. Black, R. B., Rhodes, J., Davies, G. T., Gravelle, H., Sweetnam, P. Gastroenterology, 1971, 61, 821. 15. Baume, P. E., Hunt, J. H., Piper, D. W. ibid. 1972, 63, 399. 16. Boyes, B. E., Woolf, I. L., Wilson, R. Y., Cowley, D. J., Dymock, I. W. Postgrad. med. J. 1975, 51, suppl. 5, 29. 17. Fraser, P. M., Doll, R., Langman, M. J. S., Misiewicz, J. J., Shawdon, H. S. Gut, 1972. 13, 459. 18. Langman, M. J. S., Knapp, D. R., Wakley, E. J. Br. med. J. 1973, iii, 84. 19. Veterans Administration Co-operative Study on Gastric Ulcer. Gastroenterology, 1971, 61, 567. 20. Ivey, K. J., Baskin, W., Jeffrey, G. Lancet, 1975, ii, 1072. 21. Pounder, R. E., Williams, J. G., Milton-Thompson, G. J., Misiewicz, J. J. ibid. p. 1069. 22. Thjodleiffson, B., Wormsley, K. G. Gut, 1975, 16, 501.
NON-SPECIFIC CYTOTOXICITY OF WHEAT GLIADIN COMPONENTS TOWARDS CULTURED HUMAN CELLS D. A. HUDSON D. R. PURDHAM
J. CORNELL* C. J. ROLLES
H.
INTRODUCTION
WHEAT gluten and its ethanol-soluble portion, gliadin, produce intestinal mucosal damage in individuals with coeliac disease. Partial digestion of either of these substances in vitro by some proteolytic enzymes under certain conditions does not destroy this toxicity, though not all of the products seem to be active in this respect.1 Intestinal mucosal biopsy specimens obtained from patients with coeliac disease that are cultured in the presence of such digestion products have altered morphology and enzyme levels.2-4 We report observations which demonstrate that a particular chromatographic fraction of a gliadin digest is toxic under these conditions, not only to intestinal mucosal tissue, but to various other cell types. METHODS
hydrolysate fractions.-The methods of preparing gliadin from gluten and its subsequent digestion by proteolytic enzymes have been described elsewhere.5 Separation of the hydrolysate into 10 major fractions was achieved by Gliadin
on SP-’Sephadex’ C-2S (Pharmacia). Each of these fractions was either lyophilised immediately or desalted on a column of ’Bio-gel’ P-2 (Bio-Rad. Laboratories, 200-400 mesh). The fractions obtained from three such separations were pooled for the present experiments. For comparison the effects of &bgr;-Iactoglobulin, casein, ovalbumin, and the plant lectin, conconavalin A, were investigated in some experiments. Tissue-culture methods.-Human embryonic lung, intestine, adrenal, kidney, and HEp-2 (human carcinoma of the larynx) cells were prepared as primary monolayer cultures by standard procedures.6 The cell maintenance medium was type 199 supplemented with 2 % fetal calf serum (Gibco/Bio-Cult) and for cell growth at 37°C the same medium containing 10 % serum. Toxicity testing.-All tests were performed blind. The cell maintenance medium was discarded and the cell monolayers washed three times in Dulbecco A phosphate-buffered saline. The cells were then exposed to the various chromatographic fractions which were dissolved in serum-free medium 199, the pH of which was adjusted back to normal if necessary. All tests were performed in duplicate on each occasion. Unless otherwise stated, observations were made at 24 h and fractions were present at an initial concentration of 500 (Jt.g/ml. Initially, 1 x 10 cm tubes were used for both culturing and the toxicity assays, but this was later superseded by a microtitre tissue-culture system. Toxicity scoring.-This was based upon microscopic appearances of the cells usually at a magnification of x 100, thus: + occasional "holes" in an otherwise confluent clear evidence of disruption of sheet monolayer; + + integrity; + + + = pronounced disruption with cell aggretotal disruption with the monolayer gation ; + + + + sloughing from the growth supporting surface.
chromatography
=
Institute
of Child Health; and
Virology Department,
Children’s Hospital, Birmingham
=
=
Summary
Chromatographically separated fractions of a proteolytic digest of wheat were gliadin assayed for cytotoxic properties using cultured human embryonic intestinal, lung, kidney, adrenal, and HEp-2 cells. In all cell types noxious effects were observed microscopically over a 24 h period. The most active fraction was that previously shown to produce xylose malabsorption in subjects with cœliac disease, disruption of lysosomes, and inhibition of morphological recovery of cultured mucosa from a patient on a gluten-free diet. *Present address: Royal Children’s
Hospital, Melbourne, Australia.
RESULTS
The method used here for the separation of the products of gliadin proteolysis produces 10 fractions. Observations on the cytotoxic properties of each of these were made with intestinal mucosal cells only. All but two of the 10 fractions induced signs of toxicity. Fraction 9 was the most active, with fraction 5 being only slightly less so. The presence of serum inhibited the induction of cytotoxicity. These results were obtained with three separate specimens of intestinal tissue. Further tests involved the use of chromatographic fractions 1 (non-toxic to intestinal cells) and 9.
