John F. Prudden

Lysozyme activity in ulcerative alimentary disease: II. Lysozyme activity in chronic ulcerative colitis

Abstract 1.1. The stools of C.U.C. patients have a mean lysozyme content 27 times that of normal stools. 2.2. The mean twenty-four-hour output of lysozyme in C.U.C. stools is 168 times that of normal stools. 3.3. Upon purging the twenty-four-hour output of lysozyme in normal individuals is 6.7 times that of normal stools while the concentration of the enzyme decreases by about one-third. 4.4. The lysozyme content of regional enteritis stools is 6.1 times that of normals. 5.5. With clinical improvement, the lysozyme titers and daily outputs fall. 6.6. The colonic mucosa of individuals with acute C.U.C. shows an 8.5-fold increase in lysozyme content over that of normal colons. 7.7. These data, together with the experimental production by lysozyme of ulcerations in the canine alimentary tract, indicate that lysozyme is an etiologic agent which locally initiates the lesions of chronic ulcerative colitis.

Lysozyme content of the stomach and its possible relationship to peptic ulcer.

The bacteriolytic enzyme lysozyme has been shown to depolymerize and hydrolyze a mucopolysaccharide obtained from lysozyme-susceptible organisms. 1 The depolymerization of this substrate as measured viscosimetrically has been used as an accurate and speedy test for the assay of the enzyme. Acetone desiccated mucosa of horse and hog stomachs were found by this method to contain 15.2 and 714 units of lysozyme per gram respectively (samples were obtained through the courtesy of Dr. R. H. Barnes of Sharp and Dohme, Philadelphia). Mucosa of the fundus, antrum, pylorus and duodenum of human stomachs resected for peptic ulcer were found to contain mean lysozyme titers of 12, 120, 316, and 213 units per gram of wet weight respectively. One case of ulcer of the duodenum showed the following lysozyme titers: fundus: 2, antrum: 200, pylorus: 224, and duodenum: 400 units per gram. Assays of the gastric juice of 30 normal individuals and 29 unoperated ulcer cases showed mean lysozyme values of 7.69 and 14.3 units per cc respectively. The difference of the means is statistically significant. All cases with obstruction, as evidenced by intractable vomiting, had a mean lysozyme titer of 1.7 units per cc. This low value is probably explained by peptic destruction of lysozyme. Approximately 50 γ of crystalline pepsin inactivated (at 37°C) 81% of an equivalent amount of lysozyme in one hour and 92% in 2 hours. The mean lysozyme titers of the unoperated ulcer cases without the 7 obstructed individuals was 18.3 units

Lysozyme activity in ulcerative alimentary disease: 1. Lysozyme in peptic ulcer

Abstract 1.1. Lysozyme, a mucolytic enzyme, has been found in the gastric mucosa of animals and man in a relatively high concentration. 2.2. The enzyme is present in normal human gastric juice. 3.3. In man the mucosal concentration of lysozyme is very low in the fundus and increases to a maximum in the first portion of the duodenum. 4.4. The mean lysozyme titer of the gastric juice of ulcer patients is increased over that of normal individuals. 5.5. Frankly obstructed ulcer cases showed a uniformly low lysozyme titer, due either to dilution or destruction of lysozyme by pepsin. This destruction was demonstrated in vitro. 6.6. Five ulcer subjects studied before and after vagectomy showed a mean fall in lysozyme titer of 44 per cent. 7.7. In one Pavloc pouch dog a superficial antral ulceration and complete removal of the surface mucus of the pouch was produced in four hours by the instillation of crystalline egg-white lysozyme in pooled human gastric juice. 8.8. Upon instillation of egg-white lysozyme in saline into the intact stomach of a dog for eight hours complete removal of the surface mucus without ulceration was produced. 9.9. In three normal dogs oral administration of lysozyme in enteric-coated capsules or in cherry syrup produced a pyloric, antral and fundal ulceration in one dog and a fundal lesion in another. All dogs showed multiple ulcerations, hyperemia and edema of the entire small intestine. In addition, one dog showed ulceration, hyperemia and edema of the colon. 10.10. The probable etiologic role of lysozyme in peptic ulcer is discussed.

Lysozyme content of granulation tissue.

The highest lysozyme concentrations in the mammalian body occur in tears and in the mucosa of the antrum, pllorus, and duodenu1n. 1 The origin of these high local concentrations presumably is epithelium, i.e., the tear glands and as yet undetermined cell types in the gastrointestinal mucosa. In contrast, the Iysozyme titer in mesodermal tissue is low; for example, serum averages 1 unit/ccl, and human leucocytes (from the buffy coat of normal blood) contain only 1.8 units per 5,000,000 cells2 However, human cartilage averages about 40 units/g, although this value is without doubt too low because of the difficulty in extracting this tissue. Normal human skin (including a considerable quantity of fibrous tissue) was found to have less than 1 unit/g. The finding of high lysozyme titres in granulation tissue was)therefore unexpected. The lysozyme assays on granulation tissue of man and dog are shown in the accompanying table. The assays were done by a viscosimetric method 4 on extracts prepared as previously described. 3 As a result of these observations it is now apparent that high lysozyme concentrations are associated with some mesodermal cell types as well as with epithelium. Therefore, further study is warranted with regard to the role of this tissue in the production of lysozyme in ulcerative alimentary disease. The deleterious effect of egg white lysozyme on the gastrointestinal mucosa 1 3 has been confirmed in our own 5 as well as other laboratories. 6 7 Furthermore, high stool titres in the absence of occult blood in the feces and with sigmoidoscopically non-ulcerated mucosa are frequently observed in chronic ulcerative colitis. These two considerations render less likely the possibility that granulation tissue is the source of the major fraction of the lysozyme titre in ulcerative alimentary disease.

Lysozyme in chronic ulcerative colitis.

It has been previously shown in experimental animals that there is a low lysozyme content of the colonic mucosa in contrast to that of the stomach and in assays of apparently normal segments of 3 human large intestines, surgically removed for carcinoma, the lysozyme concentration was similarly low (mean = 3.5 units/g tissue). However, following the observation that lysozyme was able to remove the surface mucus from the dog stomach, 1 the present investigation was undertaken to determine whether abnormal concentrations of this mucolytic enzyme were present in the feces of patients with chronic nonspecific ulcerative colitis. Table I summarizes the results of the stool lysozyme determinations. It is to be noted that the concentration of the enzyme in the feces of the control individuals was low whether the determinations were made on specimens obtained following a normal bowel movement or after purging with magnesium sulfate or castor oil. Similarly, in 3 chronic ulcerative colitis patients whose disease necessitated ileostomy and colectomy, the lysozyme concentration of the ileal stools was uniformly low. Also, in the single patient with idiopathic diarrhea who failed to show any organic change in the mucosa of the gastro-intestinal tract, there was little lysozyme present in the stool. In marked contrast to the above noted findings was the elevated lysozyme concentrations in the fecal excretions of 12 patients in whom the diagnosis of nonspecific chronic ulcerative colitis was established by roentgen and proctoscopic examination and in whom exhaustive search for pathogenic bacteria and/or parasites was negative. Even more striking was the high titer of lysozyme found in specimens of mucus obtained from the rectosigmoid region of patients with this disease.

Acceleration of wound healing with heterologous cartilage. Immunological considerations.

Summary A bovine cartilage preparation which has demonstrated therapeutic efficacy in accelerating wound healing in experimental animals and humans has been investigated for its potential antigenicity of the immediate type. Rabbits injected with an emulsion of this material with complete Freunds adjuvant did show the presence of precipitating antibodies while rabbits with implanted subcutaneous pellets showed no such activity. The precipitating antibodies produced did not cross react with human cartilage nor with other bovine tissues. Four groups of human subjects were tested intracutaneously with an aqueous extract of bovine cartilage. One laboratory worker with a history of clinical allergy and voluminous inhalation exposure of the material over a period of 8 years developed skin sensitizing antibody activity. Mild activity was demonstrated by 2 subjects who had received the cartilage therapeutically by topical application. These 3 subjects had no symptoms that could be attributed to sensitivity reactions to the cartilage preparation and demonstrated negative skin reactions when tested with purer cartilage material prepared more recently. There was no cross reactivity with other bovine material tested. No activity was found in 24 subjects who had received the cartilage topically and in 4 laboratory workers who had long term inhalation exposure despite the fact that some of these subjects presented histories of previous clinical allergies. The sera from 12 of the subjects were examined for precipitating antibody to bovine cartilage and none was demonstrable. It is concluded that while the cartilage preparation does have some capacity for inducing antibody formation of the immediate type, on the basis of the above experiments this capacity appears relatively weak. Further purification of the active principle to eliminate all antigenic potential should be possible.