Increased protein tyrosine kinase activity of the colonic mucosa in ulcerative colitis.

The protein tyrosine kinase (PTK) activity was measured in the inflamed colonic mucosa of 12 patients with ulcerative colitis and in the normal colonic mucosa of 12 control patients with colon cancer. The specific PTK activity in the particulate fraction obtained from ulcerative colitis mucosa was significantly increased compared with that of normal mucosa (5.10 +/- 0.60 pmol/min/mg versus 2.12 +/- 0.44 pmol/min/mg protein; p less than 0.05). Inflamed ulcerative colitis mucosa also showed a significantly higher total PTK activity in the particulate fraction than normal mucosa (2.60 +/- 0.42 pmol/min/g versus 0.91 +/- 0.16 pmol/min/g tissue; p less than 0.05). Mucosal samples from ulcerative colitis patients were divided into those with mild and those with severe inflammation on histologic examination (n = 6 each). The particulate PTK activity of severely inflamed mucosa was significantly higher than that of mildly inflamed mucosa (p less than 0.05). These results suggest that colonic inflammation in ulcerative colitis is associated with alterations in cellular PTK activity.     

Phospholipase A2 activity of colonic mucosa in patients with ulcerative colitis.

Activation of enzyme phospholipase A2 (EC 3.1.1.4), the major liberator of arachidonic acid, has been proposed as a component in the pathogenesis of inflammatory bowel disease. To test the hypothesis that phospholipase A2 activity is increased in ulcerative colitis, enzyme activity was investigated in colonic biopsies using radiolabelled Escherichia coli as substrate. The study comprised patients with ulcerative colitis (n = 19) and controls without inflammatory bowel disease (n = 7). Ulcerative colitis patients were grouped into those with active disease (n = 8) and those in remission (n = 11) at the time of colonoscopy. No differences were found in mucosal phospholipase A2 activity between patients with active disease, patients in remission and controls. With the present assay we were unable to demonstrate activation of phospholipase A2 as a mechanism in the inflammatory process of ulcerative colitis.     

Glucosamine synthetase activity of the colonic mucosa in ulcerative colitis and Crohn's disease.

Glucosamine synthetase is the first enzyme in glycoprotein biosynthesis, catalysing the formation of glucosamine-6-phosphate, from which N-acetylglucosamine is formed. The levels of this enzyme in normal human colonic mucosa (in colectomy specimens and rectal biopsies) were found to be 13-8 +/- 4-0 micron mol glucosamine synthesised/h/g wet wt. In the colonic mucosa in ulcerative colitis and Crohn's colitis the enzyme level was diminished when there was loss of epithelial cells in the mucosa, although not when there was just loss of goblet cells. In patients recovering from an acute attack of ulcerative colitis, the enzyme levels rose to a peak above the normal range, an effect which did not occur in patients who did not recover promptly. This recovery peak may be related to the synthesis of gastrointestinal mucus, or immunoglobulin, or the secretory component of IgA, all of which contain large amounts of N-acetylglucosamine.     

Glycoprotein composition of colonic mucosa. Specific alterations in ulcerative colitis

A micromethod has been developed to permit determination of human colonic mucin glycoprotein heterogeneity in biopsy specimens of colonic mucosa. Sialic acid, galactose, and galactosamine residues of oligosaccharide side chains from colonic glycoproteins were radiolabeled by combined metaperiodate and galactose oxidase treatment followed by sodium borotritide reduction. Mucin glycoproteins were separated from nonmucin components by mini-Sepharose 4B column chromatography. Subsequent chromatography of labeled mucin of normal controls (n = 15) on diethylaminoethyl-cellulose demonstrated at least six labeled mucin species. Labeled mucin species I-VI were found to cochromatograph with corresponding unlabeled mucin species prepared from large surgical specimens. An identical mucin profile was observed in normal biopsy specimens from rectum (n = 5), sigmoid (n = 10), transverse (n = 5), and ascending colon (n = 4). However, mucin profiles from sigmoid mucosa of patients with ulcerative colitis (n = 14) demonstrated a selective decrease in mucin species IV, which was also present in specimens from uninvolved proximal colon (n = 7). This finding persisted in patients who had a subsequent biopsy at times of clinical and histologic remission (n = 8). In addition, colonic mucin from samples of ulcerative colitis patients in remission had a relative decrease in mucin fraction III and an increase in fraction V when compared to patients with active disease. Normal mucin profiles were found in a variety of colonic disease controls including Crohn's (n = 9), ischemic (n = 4), infectious (n = 8), and radiation (n = 3) colitis. These observations indicate the presence of a relatively uniform mucin profile throughout the normal colon and substantiate the association of specific alterations in colonic mucin with ulcerative colitis.     

Bactericidal/permeability-increasing protein in colonic mucosa in ulcerative colitis.

BACKGROUND/AIMS: Increased mucosal concentration of bactericidal/permeability-increasing protein (BPI) has been shown in inflammatory bowel diseases. The purpose of the present study was to investigate the relationship between the mucosal concentration of BPI and the grade of mucosal inflammation in ulcerative colitis. METHODOLOGY: Samples of colonic mucosa from 12 patients with ulcerative colitis and from 8 control patients were studied. The concentration of BPI in tissue extracts was measured by a time-resolved fluoroimmunoassay. The concentration of BPI was compared between samples with histological inflammatory changes of different severity. BPI was localized in tissue sections by immunohistochemistry. RESULTS: The concentration of BPI was higher (p < 0.001) in samples of colonic mucosa from patients with ulcerative colitis (median: 3.2 micrograms/g, range: 0.3-22.6 micrograms/g) than in control samples (0.4 microgram/g, 0.1-0.6 microgram/g,). Moreover, the concentration of BPI was higher (p = 0.015) in samples with severe inflammation (2.5 mu/g, 0.3-22.6 micrograms/g) than in those with mild inflammation (0.5 mu/g, 0.3-2.5 micrograms/g). The concentration of BPI in mucosal samples correlated well with the degree of histological inflammation (Spearman R = 0.70, p = 0.01). BPI was localized in polymorphonuclear leukocytes in the mucosa and stroma of the colonic wall. CONCLUSIONS: The concentration of BPI is increased in the colonic mucosa of patients with ulcerative colitis. The increase in the concentration of BPI in colonic mucosa seems to be closely associated with the inflammatory activity of ulcerative colitis.     

Tissue-type plasminogen activator of colonic mucosa in ulcerative colitis

Microvascular endothelial alterations are thought to be a crucial step for development of hemorrhagic changes in various pathological states. In this study, we determined the activity and amount of tissue-type plasminogen activator (t-PA) in the biopsy specimens from sigmoid colon of patients with ulcerative colitis to evaluate endothelial alterations and vascular changes of permeability. The results of this investigation revealed that mucosal amount of t-PA in the active stage of ulcerative colitis was two- to threefold higher than in healthy controls, while t-PA levels in plasma samples showed no remarkable differences among the groups. Increased t-PA activity appeared to correlate well to the degree of inflammation of colonic mucosa, and t-PA amount was still increased in the inactive stage. The present study indicates that t-PA determination in colonic biopsy specimens may be useful for the evaluation of clinical activity of ulcerative colitis in terms of the mucosal microvascular endothelial changes.This work was supported by the grant from the Japanese Ministry of Education No. 63440033 and by the grant from Keio University, School of Medicine.     

Lymphatic vessels in the colonic mucosa in ulcerative colitis

In the normal colonic mucosa, lymphatics are found only in a narrow band associated with the muscularis mucosae and are absent from the rest of the mucosa. This study examined whether this arrangement of lymphatics is also valid in ulcerative colitis. Histological sections of colon from 15 long-standing cases were investigated with antibodies against CD 34 (negative for lymphatics; positive for blood vessel endothelium) and, in selected cases, podoplanin (positive for lymphatic endothelium; negative for blood vessel endothelium). Whereas inflammation of the mucosa was not associated with changes in lymphatics, an increase in intramucosal lymphatics was seen when the pathological changes included widening of the muscularis mucosae or penetration of the mucosa by muscle fibers, filiform changes in the mucosa, and hyperplasia of the mucosa-associated lymphoid tissue (MALT). In specimens with epithelial dysplasia, an association between the dysplastic epithelium and ectatic and quantitatively increased lymphatics was observed. With superimposed carcinoma, no relationship between the malignant tumor and lymphatics was identifiable. Nevertheless, pre-existing lymphatics in the muscularis mucosae were involved in lymphatic tumor spread. The immunohistochemical findings demonstrated that lymphatics occurred in all areas of the mucosa in ulcerative colitis (or, in effect, at sites which were not normally found under physiological conditions) and in regions that favored lymphatic tumor dissemination. Whether these lymphatics were actually involved in metastasis remains to be defined.     

Bacterial invasion into the colonic mucosa in ulcerative colitis

Abstract This study investigated interactions between mucosal lesions and bacterial invasion in ulcerative colitis using the acridine-orange staining method. In all 16 cases of ulcerative colitis, the mucosa was found to be invaded by small rods and cocci. In five of 10 controls, bacteria were seen only adhering to the mucosa and no bacteria were detected in the five remaining cases. It is suggested that the presence of bacteria in the colonic mucosa may be a factor responsible for the persistence or aggravation of ulcerative colitis.     

Corticotropin releasing hormone in colonic mucosa in patients with ulcerative colitis.

Corticotropin releasing hormone (CRH) is a key hormone in integrated response to stress, acting as the major regulator of the hypothalamic-pituitary-adrenal axis. Recently, local production of CRH has been detected in normal human colonic enterochromaffin cells. CRH is locally secreted in granulomatous and arthritic tissues in rats and humans, where it seems to act as a local proinflammatory agent. To find out if CRH is present in colonic tissues of patients with ulcerative colitis, this study examined the expression of this peptide in the large bowel of patients with ulcerative colitis. Colonic tissues of patients with ulcerative colitis obtained by endoscopic biopsy were immunostained with anti-CRH antibody. CRH messenger (m) RNA was also examined in biopsy specimens of ulcerative colitis by the reverse transcribed polymerase chain reaction method and by in situ hybridisation. Considerably enhanced expression of immunoreactive CRH was found in mucosal inflammatory cells. Intense staining with anti-CRH antibody was also shown in mucosal macrophages. CRH mRNA was expressed in mucosal epithelial cells. The expression of immunoreactive CRH in colonic mucosal epithelial cells of ulcerative colitis slightly increased, but not significantly, compared with normal colonic mucosal epithelial cells. These results suggest that CRH may play a part in the modulation of intestinal immune and inflammatory system, and as a modulator in the pathogenesis of ulcerative colitis.     

Urocortin 1 in colonic mucosa in patients with ulcerative colitis

Ulcerative colitis (UC) is characterized by a long-standing chronic inflammation of the bowel with intermittent periods of exacerbation and remission. Its acute exacerbation appears to be related to various stresses. Urocortin 1 (Ucn1) may play important roles in integrated local responses to stress. We therefore examined local production of Ucn1 in patients with UC by immunohistochemistry and mRNA in situ hybridization. Ucn1 immunoreactivity was predominantly detected in lamina propria plasma cells and enterochromaffin cells. In UC patients without glucocorticoid treatment, Ucn1-positive cells and plasma cells increased in proportion to the severity of inflammation (P < 0.0001). Ucn1-positive cells significantly increased in UC patients with advanced inflammatory grades, compared with a control group (P < 0.0001) and nonspecific colitis group (P < 0.0001). In glucocorticoid-treated patients, Ucn1-positive cells were significantly lower in number, compared with the nonglucocorticoid-treated group. Ucn1 mRNA was expressed in lamina propria plasma cells, and both corticotropin-releasing factor(1) and corticotropin-releasing factor(2(a)) mRNAs were also partially coexpressed in these cells and macrophages. The present study showed that Ucn1-positive cells were correlated with the severity of inflammation in colonic mucosa with UC, and glucocorticoid treatment decreased these cells. Ucn1 therefore may act as a possible local immune-inflammatory mediator in UC.     

Effect of butyrate enemas on the colonic mucosa in distal ulcerative colitis.

Short-chain fatty acid irrigation has been shown to ameliorate inflammation in diversion colitis. In this study the effect of butyrate enemas was tested in 10 patients with distal ulcerative colitis who had been unresponsive to or intolerant of standard therapy for 8 weeks. They were treated for 2 weeks with sodium butyrate (100 mmol/L) and 2 weeks with placebo in random order (single-blind trial). Before and after treatment, clinical symptoms were noted and the degree of inflammation was graded endoscopically and histologically. Rectal proliferation was assessed by autoradiography. After butyrate irrigation, stool frequency (n/day) decreased from 4.7 +/- 0.5 to 2.1 +/- 0.4 (P less than 0.01) and discharge of blood ceased in 9 of 10 patients. The endoscopic score fell from 6.5 +/- 0.4 to 3.8 +/- 0.8 (P less than 0.01). The histological degree of inflammation decreased from 2.4 +/- 0.3 to 1.5 +/- 0.3 (P less than 0.02). Overall crypt proliferation was unchanged, but the upper crypt-labeling index fell from 0.086 +/- 0.019 to 0.032 +/- 0.003 (P less than 0.03). On placebo, all of these parameters were unchanged. These data support the view that butyrate deficiency may play a role in the pathogenesis of distal ulcerative colitis and that butyrate irrigation ameliorates this condition.     

Telomere shortening in the colonic mucosa of patients with ulcerative colitis

Telomere length in human somatic cells gradually decreases with the number of cell divisions and is regarded as a marker of somatic cell turnover. Mucosal cells of the affected colon show rapid turnover in individuals with active ulcerative colitis (UC). Telomere length was determined by Southern blot analysis of terminal restriction fragments (TRFs) from the colonic mucosa of 17 patients with UC in remission, two of whom showed dysplasia, and 17 control subjects without colitis. For each individual, mean TRF length was compared between rectal mucosa and unaffected cecal mucosa. The mean TRF length of the rectal mucosa was significantly less than that of cecal mucosa in UC patients (7.87 ± 0.36 kb versus 8.77 ± 0.21 kb; P = 0.0015, Wilcoxon signed rank test), whereas no significant difference was detected in the control subjects. The extent of telomere shortening was 10.6 ± 3.35% in UC patients, compared with 0.8 ± 0.64% in noncolitis controls (P = 0.0024, Mann-Whitney U-test). Four UC patients, two of whom had dysplasia, showed telomere shortening of more than 20% in the rectal mucosa. These observations suggest that telomere shortening in the colonic mucosa of individuals with UC may represent the history of mucosal inflammation during disease of long duration, and that it may contribute to aneuploidy in UC. (Received May 6, 1997; accepted Sept. 26, 1997)     

Impairment of mitochondrial acetoacetyl CoA thiolase activity in the colonic mucosa of patients with ulcerative colitis

Background and aims

Butyrate oxidation by colonocytes is impaired in ulcerative colitis. This study examined the activity of enzymes involved in butyrate oxidation in ulcerative colitis.

Methods

Activities of mitochondrial acetoacetyl coenzyme A (CoA) thiolase, crotonase and β‐hydroxy butyryl CoA dehydrogenase were estimated spectrophotometrically in rectosigmoid mucosal biopsies from patients with ulcerative colitis and Crohn''s colitis, and control subjects undergoing colonoscopy for colon cancer or rectal bleeding.

Results

The activity of mitochondrial acetoacetyl CoA thiolase was decreased by 80% in ulcerative colitis (3.4 (0.58) μmol/min/g wet weight, n = 30) compared with control (16.9 (3.5), n = 18) and with Crohn''s colitis (17.6 (3.1), n = 12) (p<0.0001). The activity of two other mitochondrial butyrate oxidation enzymes—crotonase and β‐hydroxy butyryl CoA dehydrogenase—as well as of cytoplasmic thiolase was normal in ulcerative colitis. Mitochondrial thiolase activity in ulcerative colitis did not correlate with clinical, endoscopic or histological indices of disease severity. Mitochondrial thiolase activity was reduced in the normal right colon mucosa of patients with left‐sided ulcerative colitis. Enzyme kinetic studies revealed a lowered V_max, suggesting inhibition at a site distinct from the catalytic site. Reduced thiolase activity in ulcerative colitis was returned to normal by exposure to 0.3 mM β‐mercaptoethanol, a reductant. Using normal colon mucosal biopsies, redox modulation of thiolase activity by hydrogen peroxide, a mitochondrial oxidant, could be shown. A significant increase in hydrogen peroxide formation was observed in ulcerative colitis biopsies.

Conclusion

A defect of mitochondrial acetoacetyl CoA thiolase occurs in ulcerative colitis. Increased reactive oxygen species generation in mitochondria of epithelial cells in ulcerative colitis may underlie this defect.Despite considerable advances in our understanding of the inflammatory bowel diseases, the aetiology of ulcerative colitis remains unclear. Crohn''s disease, which is characterised by transmural inflammation involving the small bowel and/or colon, is believed to result from defective innate immune recognition of luminal bacteria in a genetically predisposed individual.¹ In ulcerative colitis, where inflammation is restricted to the mucosa of the colon, it has been suggested that a primary defect in the colonic epithelium resulting in the increased absorption of pro‐inflammatory molecules from the colonic lumen may underlie the development of disease.^2,3 Defective colonic epithelial oxidation of butyrate in ulcerative colitis, first demonstrated by Roediger⁴ and confirmed by other investigators,^5,6 has been implicated in the pathogenesis of ulcerative colitis. Butyrate, a short‐chain fatty acid, is formed from the bacterial fermentation of unabsorbed carbohydrates in the colonic lumen. It undergoes oxidative metabolism in the mitochondria of colonic epithelial cells to CO₂ and ketone bodies and is the major source of energy, providing >70% of the energy requirements⁷ for the colonic epithelium, which cannot effectively utilise glucose as energy substrate.⁴ Colonocyte butyrate metabolism was defective in an animal model of colitis.⁸ Inhibition of butyrate oxidation in experimental animals resulted in mucosal inflammation similar to ulcerative colitis.⁹ Decreased ATP levels are evident in mucosa from ulcerative colitis patients.¹⁰ Diversion colitis occurring in the colon surgically excluded from the faecal stream, and therefore a model of butyrate deficiency, is successfully treated by the rectal infusion of butyrate and other short‐chain fatty acids.¹¹ Thus, impaired butyrate oxidation is associated with the development of colitis in a variety of situations.Oxidation of butyrate is carried out in mitochondria by a series of five enzymes (fig 1​1).). An earlier study, which examined all these enzymes, failed to demonstrate any significant alteration in these enzymes in quiescent ulcerative colitis.¹² Mitochondrial acetoacetyl coenzyme A (CoA) thiolase (EC 2.3.1.9) catalyses the last step in butyrate oxidation to acetyl CoA. In addition to being present in mitochondria, there is a second isoform of this enzyme in the cytosol, which is principally involved in cholesterol biosynthesis as well as ketone body synthesis.¹³ The two isoforms can be distinguished biochemically by the fact that the mitochondrial isoform is K⁺ sensitive while the cytoplasmic isoform is K⁺ insensitive. The earlier study of butyrate‐oxidising enzymes assayed acetoacetyl CoA thiolase, but did not specifically examine the mitochondrial isoform of the enzyme—that is, the study used the appropriate substrate but did not measure potassium‐sensitive thiolase activity. This differentiation has been shown to be important, as for example in children with repeated ketoacidotic episodes, in whom mitochondrial acetoacetyl CoA thiolase deficiency was not picked up by the normal assay, but a specific assay of potassium ion‐sensitive enzyme activity diagnosed the deficiency.¹⁴ The present study specifically examined the activity of mitochondrial potassium‐sensitive acetoacetyl CoA thiolase in mucosal biopsies from the colon of patients with active and quiescent ulcerative colitis and compared it with that in control subjects. In addition, studies were undertaken to determine the genesis of decreased enzyme activity in ulcerative colitis.Open in a separate windowFigure 1 Schematic illustrating the function of acetoacetyl coenzyme A (CoA) thiolase: butyrate is oxidised in the mitochondria by a series of five different enzymes. The mitochondrial isoform of acetoacetyl CoA thiolase catalyses the last step of the reaction, converting acetoacetyl CoA to two molecules of acetyl CoA. In contrast, the cytosolic form of acetoacetyl CoA thiolase functions predominantly in cholesterol metabolism, and is involved in the synthesis of acetoacetyl CoA from two molecules of acetyl CoA.     

Glucosamine synthetase activity of the colonic mucosa in membranous colitis.

Glucosamine synthetase, the first enzyme in glycoprotein synthesis, has been measured in serial rectal biopsies in four patients with membranous colitis. In two of the patients the levels of the enzyme were very high initially and in the other two patients the enzyme levels rose to a peak above the normal range after a delay of up to 10 days. These high levels may be related to the mucus hypersecretion which is a feature of membranous colitis but it seems more likely that they represent the healing of the mucosa.